The Prion Protein is Embedded in a Molecular Environment ... · The Prion Protein: What We Do and...

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The Prion Protein is Embedded in a Molecular Environment that Modulates Transforming Growth Factor β and Integrin Signaling by Farinaz Ghodrati A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Farinaz Ghodrati 2018

Transcript of The Prion Protein is Embedded in a Molecular Environment ... · The Prion Protein: What We Do and...

Page 1: The Prion Protein is Embedded in a Molecular Environment ... · The Prion Protein: What We Do and Don’t Know 1.1 Introduction The prion protein (PrP) is central to the pathogenesis

The Prion Protein is Embedded in a Molecular Environment that Modulates Transforming Growth Factor

β and Integrin Signaling

by

Farinaz Ghodrati

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Laboratory Medicine and Pathobiology University of Toronto

© Copyright by Farinaz Ghodrati 2018

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The Prion Protein is Embedded in a Molecular Environment that Modulates Transforming Growth Factor β and Integrin Signaling

Farinaz Ghodrati

Master of Science

Department of Laboratory Medicine and Pathobiology University of Toronto

2018

Abstract

The prion protein (PrP) is known for its fundamental role in a group of neurodegenerative

disorders, aptly called prion diseases. The function of the normal cellular prion protein (PrPC) as

well as the underlying molecular mechanisms that lead to neurotoxicity in disease are still

unresolved. The previously discovered evolutionary relationship between PrP and ZIP zinc

transporters implicated PrPC in morphogenetic reprogramming events but also uncovered

surprising differences when comparing signaling pathways downstream of PrPC in separate

cellular paradigms. To extend this line of research, we combined CRISPR-Cas9-based genetic

engineering to generate four relevant murine PrP-deficient cell models, with quantitative mass

spectrometry-based analyses to compare the molecular environment of PrP in these models.

Interestingly, a unified theme emerged from these studies that placed PrPC in a specialized

membrane domain that modulates TGF beta and integrin signaling.

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Acknowledgments

First and foremost, I would like to wholeheartedly thank my supervisor, Dr. Gerold Schmitt-Ulms

for his exceptional mentorship and unwavering support throughout my studies. I credit his genuine

passion and dedication to research and his keen scientific mind with helping me grow both

personally and professionally over these last two years. Though my time under his mentorship has

now ended, I will take the lessons I’ve learned here with me onwards through my career.

I would also like to extend my thanks to the members of my advisory committee – Dr. Joel Watts

and Dr. Sunit Das. Their advice and guidance have helped me immensely in my research.

Additionally, many thanks to the tireless efforts of the Department of Laboratory Medicine and

Pathobiology’s Graduate Coordinator, Dr. Harry P. Elsholtz, and the Graduate Administrator,

Rama Ponda.

I am also tremendously grateful for the pleasure and privilege of collaborating with past and

present members of the Schmitt-Ulms lab – Dr. Hansen Wang, Louisa Wang, and Michael

Solarski. My time here would not have been as productive or enjoyable without them. Special

thanks to Dr. Declan Williams, who helped move this project leaps and bounds with his invaluable

guidance and expertise in the field of mass spectrometry. And finally, this entire project would not

have been possible without the accomplishments of Dr. Mohadeseh Mehrabian. To her, I express

my immense gratitude for her untiring mentorship, guidance and, above all, friendship.

Lastly, I would like to thank my friends and family for their love, patience, and support over the

years, specifically, AS, NS, NR, NM and ABZ, for your unique sense of humour. I would also

want to express my thanks to the colleagues at the Tanz CRND who provided me with tremendous

scientific and personal support during my degree. And above all, my utmost appreciation to my

parents for being such amazing sources of strength and support.

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Table of Contents Acknowledgments..................................................................................................................iii

TableofContents...................................................................................................................iv

ListofTables.........................................................................................................................vii

ListofFigures.......................................................................................................................viii

ListofAppendices..................................................................................................................ix

Abbreviations..........................................................................................................................x

Chapter1...............................................................................................................................1

ThePrionProtein:WhatWeDoandDon’tKnow............................................................1

1.1 Introduction.........................................................................................................................1

1.2 Priondiseases......................................................................................................................1

1.2.1 Overviewofpriondiseases.....................................................................................................1

1.2.2 Inheritedhumanpriondiseases.............................................................................................2

1.2.3 Acquiredhumanpriondiseases.............................................................................................3

1.2.4 Sporadichumanpriondiseases..............................................................................................5

1.3 Prionpropagation................................................................................................................7

1.3.1 Howtheprionconceptcametobe........................................................................................7

1.3.2 PrPCexpressionisessentialforprioninfection.......................................................................8

1.3.3 Priontoxicity...........................................................................................................................9

1.4 Structureoftheprionprotein............................................................................................10

1.4.1 OverviewofthestructuralfeaturesofPrP...........................................................................10

1.4.2 ProteolyticprocessingofPrP................................................................................................11

1.5 Evolutionaryoriginoftheprionprotein.............................................................................12

1.5.1 DiscoveryofthePrP-ZIPconnection....................................................................................12

1.5.2 HowPrPevolvedfromZIPancestors....................................................................................13

1.5.3 FurtherproofonthefamilyreunionofPrPandZIPs............................................................14

1.6 Proposedfunctionsoftheprionprotein.............................................................................15

1.6.1 OverviewoftherolesascribedtoPrPC.................................................................................15

1.6.2 Epithelial-to-mesenchymaltransition..................................................................................16

1.6.3 AdditionalPrP-relatedphenotypes......................................................................................17

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1.7 Methodologybackground..................................................................................................18

1.7.1 Overviewoftheexperimentalworkflow..............................................................................18

1.7.2 Cellmodels...........................................................................................................................19

1.7.3 CRISPR-Cas9knockouttechnology.......................................................................................20

1.7.4 Affinitycapturematrix..........................................................................................................22

1.7.5 Massspectrometry...............................................................................................................22

Chapter2..............................................................................................................................25

Rationale,HypothesisandObjectives............................................................................25

2.1 RationalefortheZIPstudy.................................................................................................25

2.2 RationaleforthePrPinteractomestudy............................................................................26

Chapter3..............................................................................................................................28

ZIP6-mediatedNCAM1Phosphorylation........................................................................28

3.1 Introduction.......................................................................................................................29

3.2 Results...............................................................................................................................30

3.2.1 InvitrophosphorylationofthelongestNCAM1isoformbyGSK3B.....................................30

3.3 Discussion..........................................................................................................................32

3.4 Methods............................................................................................................................34

3.4.1 Samplepreparationforimmunoprecipitation.....................................................................34

3.4.2 Proteinimmunoprecipitation...............................................................................................34

3.4.3 Activekinaseassay...............................................................................................................35

Chapter4..............................................................................................................................36

ThePrionProteinisEmbeddedinaMolecularEnvironmentthatModulates

TransformingGrowthFactorβandIntegrinSignaling............................................................36

4.1 Introduction.......................................................................................................................37

4.2 Results...............................................................................................................................38

4.2.1 DesignofcomparativePrPinteractomeanalysisinfourmousecellmodels.......................38

4.2.2 ComparisonofPrPinteractomeanalysesacrossmodels.....................................................41

4.2.3 Celltype-specificeffectsofPrPknockoutontheglobalproteomereflectitsmolecular

interactions........................................................................................................................................48

4.2.4 PrPselectivelyinteractswithEce1andTfrcdimers.............................................................51

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4.2.5 CandidatePrPinteractorsexistinthesecretorypathwayoratthecellularmembrane.....55

4.2.6 TGFβ1profoundlyaffectssteady-statelevelsofseveralPrPinteractorsbutdepletionofPrP

onlyreducesNCAM1.........................................................................................................................58

4.3 Discussion..........................................................................................................................61

4.4 Conclusion.........................................................................................................................65

4.5 Methods............................................................................................................................65

4.5.1 Westernblotanalyses..........................................................................................................65

4.5.2 Cellcultureandtransfection................................................................................................66

4.5.3 Samplepreparationforimmunoprecipitationanalyses.......................................................66

4.5.4 Proteinimmunoprecipitationworkflow...............................................................................67

4.5.5 NanoscaleHPLC-ESItandemmassspectrometry.................................................................67

4.5.6 Proteinidentificationandquantification.............................................................................68

4.5.7 Dataavailability....................................................................................................................68

Chapter5..............................................................................................................................70

ConclusionandFutureDirections...................................................................................70

References............................................................................................................................76

Appendix...............................................................................................................................87

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List of Tables Table 4.1: Comparison of PrP interactome in four mouse cell models (list of non-specific

interactors truncated, see also Supplementary Table S1)

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List of Figures Figure 3.1: In vitro phosphorylation of longest NCAM1 isoform by GSK3B.

Figure 4.1: Design of comparative PrP interactome study.

Figure 4.2: Validation of successful technical execution of quantitative interactome analysis.

Figure 4.3: The molecular environment of PrP is cell model-specific and comprises several novel

candidate interactors.

Figure 4.4: PrP interacts selectively with the Ece1 dimer, not its more abundant monomer.

Figure 4.5: PrP’s molecular environment is enriched for proteins with known roles in TGFβ1 and

integrin signaling.

Figure 4.6: Whereas TGFβ1 treatment causes divergent shifts in steady-state levels of a subset of

PrP interactors, PrP-depletion in the same paradigm only affected NCAM1 protein levels.

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List of Appendices Supplementary Figure S4.1: Consistent and selective enrichment of PrP contrasted to non-specific

binding of Gapdh.

Supplementary Figure S4.2: Selective PrP co-enrichment of Cd109 and Tmem206.

Supplementary Figure S4.3: Evidence that Ece1 is not expressed in CAD5 cells at levels detectable

by western blot analysis.

Supplementary Figure S4.4: PrP co-immunoprecipitates Tfrc from wild-type but not PrP knockout

NMuMG cell lysates.

Supplementary Table S4.1: Comparison of the PrP interactome in four mouse cell models.

Supplementary Table S4.2: Global proteome analysis of NMuMG cells -/+ TGFB1 (datset I) in

PrP-deficient and wildtype cells (dataset II).

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Abbreviations AA amino acid

AD Alzheimer’s disease

BCA bicinchoninic acid

BSE bovine spongiform encephalopathy

CFC cysteine-flanked core

CID collision induced dissociation

CJD Creutzfeldt-Jakob disease

CNS central nervous system

CRISPR clustered regularly interspaced short palindromic repeats

CWD chronic wasting disease

DSB double-stranded break

Ece1 endothelin converting enzyme 1

ECL enhanced chemiluminescence

EMT epithelial-to-mesenchymal transition

ESI electrospray ionization

fCJD familial Creutzfeldt-Jakob disease

FOI Fear of Intimacy

FFI fatal familial insomnia

GAPDH glyceraldehyde 3-phosphate dehydrogenase

GPI glycosylphosphatidylinositol

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GSK3 glycogen synthase kinase 3

GSS Gerstmann-Straussler-Scheinker

HCD higher energy collisional dissociation

HDR homology-directed repair

HPLC high performance liquid chromatography

iCJD iatrogenic Creutzfeldt-Jakob disease

iTRAQ isobaric tag for relative and absolute quantitation

IP immunprecipitation

KD knockdown

kDa kilo Dalton

KO knockout

Met Methionine

Mo mouse

MS2 tandem MS

MW molecular weight

NCAM1 neural cell adhesion molecule 1

NHEJ nonhomologous end joining

PIPLC phosphatidylinositol-specific phospholipase C

PK proteinase K

PL PrP-like

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PTM post-translational modification

PrP prion protein

PrPC cellular isoform of the prion protein

PrPSc pathogenic isoform of the prion protein

PSM peptide-to-spectrum match

RNAi RNA interference

sCJD sporadic Creutzfeldt-Jakob disease

SDS sodium dodecyl sulfate

sFI sporadic fatal insomnia

SLC39 solute carrier 39

TGFβ1 transforming growth factor beta 1

TM transmembrane

TSE transmissible spongiform encephalopathy

Val Valine

vCJD variant Creutzfeldt-Jakob disease

VPSPr variably protease-sensitive prionopathy

UK United Kingdom

ZIP Zrt-, Irt-like protein

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Chapter 1

The Prion Protein: What We Do and Don’t Know

1.1 Introduction The prion protein (PrP) is central to the pathogenesis of prion diseases [1], also known as

transmissible spongiform encephalopathies (TSEs). These fatal neurodegenerative disorders that

can affect humans and other mammals [2] manifest with clinical symptoms of motor and cognitive

dysfunction, often accompanied by the generation of PrP-specific deposits that can be stained with

amyloid dyes [2]. Prions, the transmissible agents in prion diseases, possess unique characteristics

that set them apart from other infectious pathogens, such as their lack of an informational nucleic

acid genome [2,3]. The cellular isoform of PrP (PrPC) is widely expressed in healthy vertebrate

cells of diverse lineages and has been linked to a wide range of cellular activities including, but

not limited to, cell adhesion, neuritogenesis, circadian cycle regulation and ion transport [3,4]. In

the diseased state, the central pathogenesis event occurs when PrPC undergoes a conformational

change, leading to the alteration of its physiochemical properties and conversion into its

pathogenic isoform, the scrapie prion (PrPSc) [2,3]. It is well documented that PrPC is not only the

substrate of prion replication but also plays a key role in the prion-induced neurodegeneration that

follows [2].

1.2 Prion diseases

1.2.1 Overview of prion diseases

The classic prion diseases affecting humans include Creutzfeldt-Jakob disease (CJD), Kuru, fatal

familial insomnia (FFI) and Gerstmann-Straussler-Scheinker (GSS) disease [2]. Variant CJD

(vCJD), sporadic fatal insomnia (sFI) and variably protease-sensitive prionopathy (VPSPr) have

more recently been identified as new forms of these disorders [5]. Clinical symptoms of human

prion diseases include rapidly progressive dementia, visual or cerebellar impairments, myoclonus,

akinetic mutism and pyramidal as well as extrapyramidal signs [2]. The most common prion

diseases affecting other mammals include bovine spongiform encephalopathy (BSE) also known

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as “mad cow” disease, scrapie, which affects sheep and goat, and chronic wasting disease (CWD)

affecting elk and deer [2]. Among the more recently recognized animal prion diseases are feline

spongiform encephalopathy and TSE in non-human primates, affecting domestic cats and lemurs

respectively [6,7]. The histopathological features of prion diseases observed in the central nervous

system (CNS) consist of neuronal loss, astrogliosis as well as spongiform change, which is defined

as the vacuolation of the neuropil [1,8]. These phenotypes are observed to variable degrees in

different forms of the disease [8].

Worldwide, prion diseases were thought to affect about one person in a million every year [6] but

numbers are trending upward with closer to 1 in 500,000 new cases tallied in more recent

epidemiological surveys [9]. In spite of this low incidence rate, prion diseases have provoked

remarkable interest both within the scientific community as well as the general public [2,6]. This

is due to the unique pathobiology of these rare neurodegenerative diseases, owing to their initially

unprecedented transmissible agents, or prions, as well as the public health threat that they could

pose to the human population with the possibility of transspecies transmission from zoonotic

sources, with BSE being the best known example of this risk [6,10].

1.2.2 Inherited human prion diseases

Human prion diseases are categorized as inherited, infectious or sporadic; this is based on the

genetic, neuropathological, and clinical profiles associated with each case [1,2]. Patients with

inherited forms of the disease, also called familial prion diseases, carry a germline mutation in the

PRNP gene and follow an autosomal dominant inheritance pattern [1,2,11]. Pathogenic PRNP

mutations can fall into three main categories: point mutations causing an amino acid (AA)

substitution, point mutations leading to a premature stop codon, and additional octapeptide repeat

insertions [10]. To date more than 30 different mutations of PRNP have been reported [10].

Familial prion diseases account for 10-20% of all cases and include FFI, GSS and familial CJD

(fCJD) [1,2].

FFI has a mean age of onset of 51 years and a clinical duration of about 18 months [12]. FFI

patients initially present with insomnia or disrupted sleep, with additional symptoms, including

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hallucinations, autonomic hyperactivation as well as motor abnormalities, such as ataxia and

myoclonus [5,10,12], presenting at some point. Interestingly, the disease was originally called

thalamic dementia due to the severe degeneration of the thalamus that was observed in these

patients [5,12,13]. FFI is genetically characterized by the D178N mutation in the PRNP gene,

coupled with a methionine (Met) at the M129V human PRNP polymorphism [5,10,12]. The same

D178N mutation is linked to fCJD when a Val is present at codon 129; however, recent data

suggests that this association might not be as concrete as previously assumed [5,10]. This AA

polymorphism, presenting with either a Met at codon 129 of PrP or a valine (Val) has also been

shown to influence the etiology of sporadic and iatrogenic forms of the disease, with individuals

homozygous at codon 129 being relatively more susceptible to prion diseases [2,14]. PrP codon

129 heterozygosity (Met/Val) provides relative protection against the disease and is associated

with a lower risk and prolonged incubation time in various prion disorders [2,14].

GSS presents with slowly progressive ataxia followed by later onset dementia and has been linked

to a number of PRNP mutations, including P102L [5,10,15]. GSS patients are usually 30 to 60

years of age at the onset of the disease, which has a course of 5 years on average [5,6]. Familial

forms of CJD account for about 5-15% of all CJD cases [5]. Clinical symptoms of the disease

include the rapid progression of dementia and myoclonus [5,10]. The E200K mutation, which is

the most common mutation leading to fCJD, is also the most common cause of inherited prion

disease worldwide [10]. Although the clinical expression of these disorders varies, it should be

noted that in reality these conditions are symptomatically overlapping, with the above

characteristic features recognized as extremes within a spectrum of clinical presentations [2,5].

1.2.3 Acquired human prion diseases

Acquired prion diseases, which include iatrogenic CJD (iCJD), Kuru and vCJD, are transmitted to

patients as a result of environmental exposure to prions and, therefore, rely on the infectious nature

of prion agents [5,6]. A feature of these disorders is the long silent incubation period following

exposure to the transmissible agent, that precedes clinical manifestation [2]. However, it has been

shown that different routes of transmission could lead to contrasting incubation periods with

intracerebral cases exhibiting incubation periods ranging about 2 to 4 years, as opposed to

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peripheral cases, displaying an average of approximately 15 years [6]. The iatrogenic form of CJD

is transmitted to patients during surgical or medical procedures [2]. Documented cases of iCJD

have occurred following corneal transplantation of a graft from a sporadic CJD (sCJD) carrier, as

a result of surgical operations with contaminated equipment, and after neurosurgical implantation

of contaminated EEG electrodes [2]. In addition, possible vCJD transmission following blood

transfusion from incubating asymptomatic donors has been observed in a few cases [16]. CJD

transmission has also been reported in patients that received human growth hormone preparations

harvested from prion disease contaminated human pituitary [2]; notably, the majority of these

patients that developed iCJD were homozygous at the 129th codon of PRNP for either Val or Met

[2].

Because prions are unusually resistant to traditional sterilization and disinfection procedures and

have long silent incubation times during which further transmission can occur, it is of great

importance to find effective decontamination techniques [2,17]. Therefore, ongoing research in

the field is in pursuit of prion inactivation methods that could be broadly and cost-effectively

applied to medical instruments [17].

Kuru is another acquired prion disease that originated in the Fore linguistic population of Papua

New Guinea Eastern Highlands [5,14]. The Kuru epidemic is thought to have arisen due to

ritualistic endocannibalism of a carcass from a human sCJD patient [5,18]. Symptoms of Kuru

include tremor, in addition to progressive cerebellar ataxia, which consequently led to this

horizontally transmitted prion disease being named ‘Kuru’ meaning ‘to shiver’ in the Fore

language [5,18]. At its peak, the Kuru epidemic killed up to 2% of the village populations annually

and imposed a strong selection pressure on the PRNP gene in the Fore groups affected by the

disease [5,18]. Similar to other prion diseases, heterozygosity at codon 129 of PRNP has been

associated with a lower risk of Kuru and longer symptom-free disease incubations. This is

supported by the high number of survivors carrying this polymorphism as well as longer than

average -as long as 56 years- incubation times in M129V Kuru patients [2,5,14]. Intriguingly, this

positive evolutionary pressure has led to development of a novel protective G127V PrP variant

during the Kuru epidemic [14]. Remarkably, further investigation on this polymorphism has shown

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that transgenic mice expressing human PrP V127 are as resistant to prion disease as PrP knockout

(KO) mice, demonstrating complete resistance to sCJD, Kuru and vCJD prions [14]. However,

transgenic mice that expressed both wild-type and variant human PrP only displayed resistance to

sCJD and Kuru prions, which are very similar; these mice did not exhibit vCJD prion resistance,

likely due to the fact that the Fore people were not previously exposed to this disease variant [14].

More insights into the mechanism was gained by studying transgenic mice expressing different

ratios of wild-type to variant PrP, showing that V127 can inhibit wild-type prion propagation in a

dose-dependent fashion [14].

In contrast to the classical (sporadic) form of CJD, vCJD presents clinicopathologically with

psychiatric and behavioural symptoms accompanied by sensory disturbances in some patients

[5,6]. As the disease progresses, patients exhibit ataxia and late-onset dementia, in addition to other

typical sCJD symptoms, such as myoclonus [2,6]. The distinguishing pathological marks of vCJD

include an abundance of a daisy-like vacuolation pattern, called “florid plaques”, made up of

amyloidotropic fibrils [2]. It also has a relatively younger patient pool, with a mean age of onset

of 29 years, and on average a longer clinical duration of 18 months [5,6]. This new variant of CJD,

which was first reported in 1996 in the United Kingdom (UK), has been linked to the BSE epidemic

of the mid-1980s and early 1990s that affected the UK, and to a lesser extent other European

countries [2,5]. More specifically, it has been proposed that BSE prions infected the human

population via dietary exposure to prion-contaminated tissue, leading to the emergence of this

novel form of acquired human prion disease through zoonosis [2,6,10]. Transmission studies also

support the notion that vCJD is caused by the same prion strain that leads to BSE, which is indeed

distinct from the sCJD strain [19].

1.2.4 Sporadic human prion diseases

Sporadic prion diseases include sCJD, sFI and VPSPr. Given that these patients lack a germline

PRNP mutation as well as exposure to a TSE agent, the underlying cause of sporadic prion diseases

remains unknown [2,5]. However, it has been hypothesized that the sporadic forms of the disease

could be due to somatic mutations in the PRNP gene as well as a spontaneous conversion of PrPC

into PrPSc [5]. Accounting for 85% of cases, sCJD is the most common human prion disease [2]

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with clinical and neuropathological features similar to those of the genetic forms of the disease

[10]. The onset of sCJD typically occurs in patients aged 45 to 75 years old with a peak at the 60

to 65 age group [6]. The disease usually has a short clinical duration with about 70% of the subjects

succumbing to illness in under 6 months [6].

In 1999 a sporadic form of fatal insomnia with clinical symptoms similar to FFI was reported in

patients lacking PRNP mutations as well as a family history of the disease [5,12]. The

neuropathological features of sFI also resemble those of the familiar form of the disease [5,12]. In

line with the role of M129V polymorphism, all the patients diagnosed with sFI have thus far been

Met homozygotes at PrP codon 129 [5].

A new type of atypical dementia was reported in 2008 in eleven 129VV homozygous patients with

no other mutation in the PRNP gene [20]. The disease had characteristic neuropathological features

of TSEs; however, a notable feature of the pathology was the reduced resistance of the insoluble

PrPSc isoform to proteinase K (PK) digestion; as such, this variant of sporadic prion disease was

named “protease-sensitive prionopathy” (PSPr) [20]. In the following years new cases of the

disease with 129MV and 129MM genotypes were found [21]. Interestingly, the sensitivity to PK

proteolysis is reduced in patients exhibiting a Met variant at the 129 codon, which led to the

renaming of the disease to “variably protease-sensitive prionopathy” [21]. The patients present

with aphasia, Parkinsonian signs as well as ataxia [21]. The 129MV heterozygotes have a later

average age of onset of 72 years compared to 64 and 65 years in 129MM and 129VV subjects

respectively. For a prion disease, VPSPr has a long clinical duration; cases involving the 129VV

variant exhibit an average clinical duration of 2 years compared to approximately 3.5 years

observed in the heterozygote and Met homozygote genotype groups [21]. Lastly, clinicians have

noted that, thus far, the majority of affected individuals exhibit the 129VV genotype. As such, it

has been proposed that the 129MM genotype could play a protective role in VPSPr disease

formation [22].

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1.3 Prion propagation

1.3.1 How the prion concept came to be

Prions are unlike any other infectious agent due to their unique propagation mechanism [23]. The

transmissibility of the scrapie agent was accidentally discovered in 1937 in a now seminal study

in which a population of sheep were exposed to scrapie during a vaccination, using formalin

extracted sheep brain tissue, which was intended to immunize the sheep against a common virus

[24]. In the following years, studies in the field went on to also illustrate the infectious nature of

human prion diseases such as Kuru and CJD by demonstrating their transmissibility to animals

[24]. A pivotal study by Alper and colleagues in 1967 showed that the TSE agent is highly resistant

to treatments such as ultra violet and ionizing radiation, which are typically used to inactivate

nucleic acids [25]. This finding prompted a number of hypotheses regarding the nature of the agent

responsible for TSEs. One of the most remarkable conjectures was the ‘protein-only’ hypothesis,

launched in 1967 by Griffith, which speculated that a self-replicating protein could be the

responsible agent for TSEs [2,26]. Future work by Prusiner and colleagues provided further

support for this hypothesis and the notion that TSE agents are free of nucleic acids [23]. Prusiner

went on to coin the term ‘prion’ for these novel proteinaceous infectious particles [23].

During the same year in 1982, PrP was purified as the protein constituent of these infectious prions

[27]. The protease resistant core of PrPSc that was isolated from infectious material had a molecular

weight (MW) of about 27 to 30 kilo Daltons (kDa) and was therefore named PrP 27-30 [27]. This

led to the identification of PRNP as the gene that encodes for PrP [28]. However, a surprising

finding at the time was the constitutive expression of PRNP as well as its corresponding mRNA

in healthy animals; this led to the idea that PrP must be present in alternative forms of PrPC and

PrPSc [29]. Given that PrPC and PrPSc have identical AA sequences, it seemed plausible that there

must be a post-translational modification (PTM) distinguishing these two PrP isoforms [29].

However, it was found that rather than a chemical modification, PrPC and PrPSc differ by their

conformation [30]. The two PrP isoforms have vastly different secondary structures; while PrPC is

predominantly an alpha helical molecule, PrPSc loses its alpha helical content and gains a more

beta-sheet-rich structure [30]. Future studies solidified the notion that PrPSc is generated by a

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template-driven conformational change of PrPC and propagates by triggering the misfolding of

endogenous PrPC molecules [1–3].

1.3.2 PrPC expression is essential for prion infection

Although TSE agents are made up exclusively of PrPSc [1], it is important to recognize the pivotal

role of PrPC in prion replication, as it is the required substrate for the propagation of prion

infections [31–38]. The critical importance of PrPC expression in prion replication was

documented by studies which highlighted that the presence of PrPSc alone fails to cause disease

manifestation in mice lacking the Prnp gene [31,35]. It was also found that the relative

susceptibility to prion diseases is determined by PrPC expression levels [31,32], again indicating

that cellular PrPC is the limiting factor in the infection paradigm. Moreover it has been shown that

disease progression and incubation time are inversely proportional to PrPC levels [31,32]. RNA

interference (RNAi)-based knockdown (KD) of PrPC expression, or ablation of PrPC in the neurons

of transgenic mice resulted in recovery from early neuronal dysfunction and extended the survival

of mice, regardless of extraneuronal PrPSc accumulation [33]. In addition, a clever study design

utilizing intraocular prion inoculation into PrP-deficient mice that contain PrPC only in a neural

graft tissue of embryonic neuroectodermal origin, highlighted that prion spread within the CNS is

dependent on PrPC expression, as the planted neural grafts remained uninfected and failed to show

prion pathology [34].

Further support for the requirement of PrPC in infection was provided in two 2001 studies,

demonstrating prevention of prion propagation via anti-PrP antibodies with selective affinity for

PrPC and slight or no binding to the pathogenic isoform of the protein [36,37]. In these studies

scrapie infection of mouse neuroblastoma Neuro2a (N2a) cells, which are one of the few cell lines

susceptible to prion infection, was prevented using anti-PrP monoclonal antibodies or via

phosphatidylinositol-specific phospholipase C (PIPLC), which cleaves cell surface-bound PrPC

[36,37]. Treatment of infected cells with PIPLC or the antibodies resulted in rapid loss and

degradation of PrPSc in the cultures [36,37]. PrPSc levels are therefore regulated by its rate of

formation from PrPC as well as its degradation process [37]. The results of these in vitro studies

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were replicated in vivo in a scrapie mouse model where it was shown that administration of anti-

PrP monoclonal antibodies also inhibited prion replication [38].

1.3.3 Prion toxicity

Despite the prion field’s continuous interest in the precise nature of conditions that promote prion

infection, the underlying molecular mechanisms leading to CNS pathology in these disorders are

yet to be fully established [2,39]. Three different hypotheses have been put forth regarding the

mechanism behind the neurodegeneration associated with the conversion of cellular PrPC into its

pathogenic isoform:

1. The most widely accepted hypothesis proposes a toxic gain-of-function for PrP; more

specifically, it implies that upon conversion to PrPSc, the conformational change brings about toxic

properties in the protein unrelated to the usual physiological functions of PrPC [39]. For instance,

PrPSc aggregates could trigger neurodegeneration by interfering with synaptic function, blocking

axonal transport or activating apoptotic pathways [39].

2. The second hypothesis states that there is a biological function of PrP that is lost when the

cellular isoform is converted to PrPSc and the presumed loss-of-function in turn leads to the prion-

induced neurodegeneration [39]. Given that one of the many functions attributed to PrPC is its anti-

apoptotic role, it is conceivable that a loss-of-function mechanism could at least contribute to

neuronal death [39]. However, since prenatal or postnatal depletion of PrP expression causes no

significant phenotypic change, rather than giving rise to prion disease symptoms, PrPC loss-of-

function cannot solely explain the neurodegeneration that occurs in prion diseases [31,40].

Nevertheless, an aspect of PrP’s biology such as its cytoprotective function, which might be

replaceable under normal cellular conditions, can possibly become indispensable under stressful

conditions in the disease state [39].

3. A third hypothesis postulates that PrP’s normal cellular function is subverted upon interaction

with the pathogenic PrPSc isoform [39]. Essentially, the third hypothesis proposes a paradigm in

which the conversion into PrPSc transforms PrPC from a neuroprotective molecule to a neurotoxic

agent [39]. It is suggested that this neurotoxicity then leads to neurodegeneration [39]. Given that

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PrPC is a glycosylphosphatidylinositol (GPI)-linked cell surface glycoprotein and many GPI-

anchored proteins serve as signal transducers, it could be hypothesized that misfolded PrP alters

this signal transduction machinery, resulting in prion toxicity [39]. It has been shown that

transgenic mice expressing GPI-negative PrP only show minimal neurological dysfunction and

brain pathology despite PrPSc amyloid plaque accumulation [41,42]. Remarkably, in a more recent

study, neuronal overexpression of anchorless PrP in wild-type mice induced late-onset

neuropathology presenting with CNS amyloid depositions resembling that of GSS patients;

however, this study further substantiated the crucial role of membrane-anchored PrPC in disease

pathogenesis [42]. These findings point to the possible role of the GPI anchor in prion replication

as well as prion toxicity, since both were reduced in GPI-negative PrPC-expressing mice [41,42].

The mechanism by which the interaction between PrPC and PrPSc culminates in neurotoxicity could

involve aggregation of the cell surface PrPC, which blocks specific regions of the protein [39]. The

overall effect of these events could then be the hindrance of the biological function of PrP through

blockade of its protein interactions and signaling transduction [39].

1.4 Structure of the prion protein

1.4.1 Overview of the structural features of PrP

The PRNP gene is located on chromosome 20 in humans and Prnp on chromosome 2 in mice [43].

Both prion genes possess three exons with exon three encompassing the entire open reading frame

[43]. The prion protein is 208 amino acids in length and has an approximate molecular weight of

35 kDa in mice and humans [3]. PrPC is located in lipid rafts, which are sphingolipid and

cholesterol-rich microdomains, and attaches to the cell membrane’s outer leaflet via a GPI anchor

[3]. GPI anchors are glycolipid structures that are attached to the C-terminus of eukaryotic proteins

during PTM [44,45]. There are also two potential N-glycosylation sites located at the C-terminal

of PrPC that could be glycosylated post-translationally, with un-, mono- and diglycosylated forms

of PrPC present in the cell [3,43]. The prion protein structure is fairly conserved among different

species presenting with an unstructured N-terminal domain and a C-terminus consisting of three

alpha helices, two short beta strands and loop domains [3,43]. The C-terminal of PrPC contains a

disulfide bond between two cysteine residues of helices B and C [43].

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1.4.2 Proteolytic processing of PrP

As a protein’s function is dependent on its structure, this section will provide an overview of the

functional relevance of the different domains of PrP as well as its proteolytic processing. The

flexible N-terminal of the protein, among other features, is comprised of a neurotoxic domain, an

octameric repeat region and a hydrophobic core [3]. The histidine residues present in the octarepeat

region harbour a high affinity for binding copper and to a lower extent zinc [43,46]. The neurotoxic

domain, located between AA 105 to 125 of mouse PrPC, has proven essential for the

conformational conversion of PrPC into PrPSc [3].

PrPC can undergo three pivotal cleavage events: alpha, beta and ectodomain shedding [3]. Alpha

cleavage occurs directly N-terminal of the hydrophobic core of mouse PrPC (AA 111-134)

generating a soluble N1 fragment (~11 kDa) and a membrane-bound C1 fragment (~18 kDa) [3].

Since the N-terminal domain of PrPC possesses ligand binding properties, the alpha cleavage

serves as a negative regulator of these functions [3]. The resulting N1 and C1 segments have

displayed distinct physiological functions [3]. N1 has been implicated in neuroprotection and

intercellular signalling [3]. As for C1, its role has proven controversial with some studies reporting

a protective and myelinotrophic role while others have linked it to neurotoxic effects [3]. Overall,

alpha cleavage serves a protective role by preventing prion propagation, as the cleavage site resides

within the neurotoxic domain of PrPC [3]. Alpha cleavage has also been found to be impaired upon

conversion of PrPC to PrPSc [3]. Since an intact neurotoxic domain is required for the misfolding

of PrPC, transgenic mice expressing only C1 displayed no markers of neurodegeneration or PrPSc

accumulation [3]. In addition, transgenic mice expressing both the C1 fragment and PrPC exhibited

prolonged disease incubation and decreased production of PrPSc compared to wild-type mice [3].

The alpha cleavage event also inhibits the binding of PrPC N terminus to amyloid beta oligomers,

which are the toxic species in Alzheimer’s disease (AD) [3]. The oligomer-induced neurotoxicity

is further blocked by the binding of the N1 fragment to these toxic elements [3].

The beta cleavage event, which occurs at the end of the octapeptide repeat region (AA 51-90),

results in the formation of N2 and C2 fragments with respective MWs of approximately 9 kDa and

20 kDa [3]. This cleavage event is less prominent under physiological conditions in comparison to

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the alpha cleavage [3]. Unlike the alpha cleavage event, which is prevented in misfolded PrP, beta

cleavage is actually the result of proteolytic processing during disease states [3]. The C2 fragment

has been postulated to serve a pathophysiological role since it is the major PrP cleavage product

in brains of CJD patients as well as prion infected neuroblastoma cells [3]. C2 also shares

resemblance with the protease-resistant core of PrPSc, and as a result, has been called the in vivo

homologue of PrP 27-30 by prion researchers [3]. Intriguingly, the beta site cleavage can also take

place under oxidative stress conditions, linking it to the suggested protective role of PrPC against

oxidative stress [3].

Lastly, ectodomain shedding takes place near the C-terminal GPI-anchor of the protein, giving rise

to the release of a nearly full-length PrP from the cell membrane [3]. Notably this cleavage event

differs from the phospholipase-induced cleavage of PrP’s GPI anchor [3]. It has been indicated

that shedding of PrP occurs in neurons as well as lymphoid cells, as observed by the presence of

soluble shed PrPC in human cerebrospinal fluid and blood [3]. As GPI anchors are employed in

signal transduction [39], shedding of PrPC can affect its receptor properties by interfering with the

signalling cascades involving PrPC. Similar to the N1 fragment, shed PrPC can also bind amyloid

beta oligomers in AD and suppress their toxicity [3]. Prion disease-associated neurodegeneration

seems to require membrane-anchored forms of PrPC, as models expressing anchorless PrPC present

with longer incubation periods [3,42]. However, it should be noted that shed PrPC is capable of

misfolding; and, as a matter of fact, GSS has been linked to PRNP mutations giving rise to

anchorless forms of PrPC [3,42].

1.5 Evolutionary origin of the prion protein

1.5.1 Discovery of the PrP-ZIP connection

An interactome investigation of PrPC in N2a cells by Watts et al., documented two members of the

Zrt- Irt-like protein (ZIP) family of metal ion transporters, ZIP6 and ZIP10, as novel interactors

for PrP [47]. This finding provoked a deeper comparison of ZIP and PrP folds and sequences as

well as examination of their membrane topology and other functional and phylogenetic

characteristics [48] that culminated in the surprising discovery that the prion gene family has

evolved from the ZIP family of zinc transporters [49]. It is worth mentioning that the evolutionary

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origins of the prion protein were a mystery to the field until the aforementioned studies revealed

the shared phylogenetic origin of ZIPs and PrP [48], a remarkable leap forward in the quest to find

the function of PrP.

ZIPs are a family of Type-III transmembrane (TM) proteins that are involved in the import of zinc

and other divalent cations into the cytosol and are encoded by an ancient gene family, called the

solute carrier 39 (SLC39) family [49]. The SLC39 gene family is universally present in all

organisms [50] and plays a major role in controlling the cellular zinc homeostasis [51]. The LIV-

1 subfamily of ZIP transporters is one of the four branches within this protein family that is

distinguishable from other ZIPs by a conserved intramembrane metalloprotease motif [51] and

includes ZIP5, ZIP6 and ZIP10 [49]. These three ZIP ion transporters form a distinct subbranch of

the human ZIP protein family based on AA sequence similarities [48]. More specifically they

contain an ectodomain similar to that of PrPC, which is accordingly termed the PrP-like (PL)

domain [48] by our group. This PL domain contains a conserved AA sequence flanked by two

cysteine residues, which is appropriately named the cysteine-flanked core (CFC) domain [48].

These two cysteine residues are ubiquitously present in all prion and PL domains and form a

disulfide bridge in the prion family of proteins.

1.5.2 How PrP evolved from ZIP ancestors

Prion protein and its paralogs including Doppel and Shadoo are only present in vertebrates [48]

but genes encoding ZIPs are present in all kingdoms of life. The Schmitt-Ulms group have

performed a systematic bioinformatics investigation into the underlying mechanism with which

the prion founder gene has evolved from ZIPs [48]. These findings pointed to two key genomic

rearrangement events that took place half-billion years apart: the first event being the rise of the

first ZIP gene encoding a PL ectodomain containing a CFC, and the second being the emergence

of the prion founder gene itself [48]. To further elaborate, during the emergence of early metazoan,

a CFC domain was inserted into an ancient ZIP transporter gene or was evolved de novo [48].

Then, in the course of early vertebrate speciation, a descendant of that ZIP transporter, which

harboured a PL ectodomain resembling that of ZIP5, ZIP6 and ZIP10, produced a processed

mRNA which was subsequently reverse transcribed and inserted into a region of the genome with

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no synteny relationship with the parent gene [48]. The said retrocopy is thought to have become a

functional retrogene by employing its nearby 5′ promoter element, giving rise to the prion

subfamily founder gene [48].

1.5.3 Further proof on the family reunion of PrP and ZIPs

Other orthogonal pieces of evidence pointing towards the shared origin of the prion gene family

and the LIV-1 branch of the ZIP gene family could be found by probing the function and structure

of these proteins. Various studies in the literature have linked PrP and its closest molecular cousins

within the ZIP transporter family to a morphogenetic reprogramming event called epithelial-to-

mesenchymal transition (EMT). Independent lines of research have displayed converging results

that point to both ZIP6 [52] and PrP [53] playing a role in cell migration during zebrafish

gastrulation events. Particularly, developmental studies have shown that zebrafish embryos

lacking ZIP6 display a gastrulation defect that resembles that of PrP1-deficient zebrafish [52]. This

phenotype was later shown to also be mimicked by knocking down ZIP10 [54]. Interestingly, the

aforementioned morpholino-based KD of PrP1, one of the two orthologs of PrP in zebrafish, leads

to the stop of zebrafish embryogenesis at the gastrula stage, which involves cellular

rearrangements relying on EMT [53]. Furthermore, the ZIP ortholog Fear of Intimacy (FOI) has

also been implicated in EMT-like morphogenetic cell movements involved in trachea and gonad

formation in Drosophila [55]. Mammalian cousins of these proteins have also been associated with

EMT, as showcased by the upregulation of both ZIP6 and ZIP10 in particular cancers, and their

involvement in the EMT process during certain human carcinomas [50]. It should then be apparent

that there is ample evidence supporting the involvement of ZIP descendent proteins in EMT.

Independent lines of investigation have also indicated the critical role that PrPC plays during EMT

[55], which points to the functional overlap observed between PrP and its evolutionary cousins.

Metal ion binding and transport is another example of the functional similarity between PrP and

its relatives in the ZIP family. ZIPs are known to play a significant role in cellular zinc homeostasis

and the mammalian prion proteins have also been documented to have an affinity for binding

divalent cations such as copper and zinc. Therefore, it is not surprising that both PrP and

ZIPs5/6/10 are capable of transporting zinc ions across the plasma membrane [49].

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The protein structure and amino acid sequence of PrPC also shares resemblance with the ZIP family

of metal ion transporters [49]. PrPC has a disordered N-terminal and a globular PL domain, which

is similar to that of ZIPs5/6/10 [49]. In addition, the octarepeats present in the prion sequence

resemble the histidine-rich repeat motifs of ZIPs and the GPI anchor attachment sequence of PrPC

is reminiscent of the first TM domain of the ZIP proteins [49]. This structural overlap can in turn

explain the recorded protein-protein interactions between PrPC and both ZIP6 and ZIP10 [47]. It

could be that their similar PL ectodomain provides an affinity for the binding of these

phylogenetically related proteins [49]. Given that the sole mechanism of prion replication pertains

to the binding of PrPSc to PrPC, and PrPC dimers have been observed previously [50], it is indeed

possible for proteins with PrP-like folds to be capable of interacting with each other. Other

commonalities such as the shared localization of PrP and ZIPs5/6/10 to the plasma membrane [49]

only further substantiate the evolutionary link between the two gene families.

Taken together, it is evident that the extent of functional and structural overlap observed between

the prion and ZIP protein families as well as the phylogenetic evidence of their shared ancestry

suggest the emergence of the prion founder gene from the ZIP family of metal ion transporters.

1.6 Proposed functions of the prion protein

1.6.1 Overview of the roles ascribed to PrPC

PrPC is ubiquitously expressed in all vertebrates and is highly conserved in mammals [6]. More

specifically, PrPC is highly expressed in the CNS, neuromuscular junctions, as well as in

lymphoreticular tissue [6]. An assortment of functions has been linked to PrPC and many

hypotheses have been put forth regarding the role of this protein; however, it is conceivable that

each of these reports represent a specific aspect of PrPC’s biology. Since PrP KO mice undergo

gross normal development and only exhibit more subtle abnormalities under certain conditions,

this line of investigation has not succeeded in shedding conclusive light on the molecular function

of PrPC [2]. The most dramatic phenotype observed in PrP KO mice is their resistance to prion

infection [2]. It is however puzzling for a protein that is highly expressed among various species,

including those not known to be susceptible to prion diseases, to have no function other than

determining the organisms’ susceptibility to prion diseases [2].

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1.6.2 Epithelial-to-mesenchymal transition

As briefly mentioned above, EMT occurs during the developmental stages of organisms,

organogenesis and wound healing [55]. EMT is also one of the main cellular events that take place

during disease states, including cancer and fibrosis [55]. During these cellular reprogramming

events, the cell-to-cell contacts of epithelial cells are replaced with cell-to-matrix connections

present in mesenchymal cells [55]. In other words, as adherens junctions are dissociated, the cells

gain a more fibroblastic morphology and focal adhesion complexes are formed, which facilitate

the transition of cells with epithelial identity to a more dynamic network of mesenchymal cells.

Focal adhesion complexes are integrin-mediated points of contact between the cells and their

surrounding extracellular matrix that maintain the structure of mesenchymal tissues [56]. The prion

protein had previously been linked to cell adhesion biology [3], with several independent

observations implicating PrPC in diverse aspects of EMT [55]. Early downregulation of PrP in

zebrafish embryos using morpholino technology led to gastrulation arrest due to a defect in the

migration of cells [53], the most significant phenotype linked to PrP deficiency. Additionally, PrPC

levels were correlated with metastasis and cell invasion propensities in the field of cancer research

[55], which further supports a role of PrP in EMT, given that it is one of the main steps in tumour

progression [56]. It is also interesting to note that proteins belonging to the ZIP family of metal

ion transporters, from which the prion protein descended from, also play roles in EMT [49,50].

These findings prompted research in our group that investigated whether the reported role of PrP

in EMT is conserved in mammalian paradigms [4]. These investigations made use of a well-known

model of mammary gland epithelial cells that robustly undergoes EMT upon addition of

transforming growth factor beta 1 (TGFβ1) [4]. Since TGFβ1 signaling is implicated in EMT [56],

it is regularly used to induce EMT in cultured epithelial cells in various research paradigms. It was

found that PrP transcript levels are more than tenfold upregulated during EMT and cells lacking

PrP fail to complete this morphogenetic reprogramming [4]. Moreover, it was discovered that PrP

controls the levels of its major interactor, neural cell adhesion molecule 1 (NCAM1) [57] as well

as a specialized PTM, called polysialylation [4,57] that predominantly exists on NCAM1.

Intriguingly, NCAM1 is a crucial regulator of EMT, with an increase in NCAM1 levels, followed

by its recruitment to lipid rafts, constituting one of the initial steps of this cellular rearrangement

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program [58]. This, in turn, activates a signalling cascade facilitating the formation of focal

adhesion complexes [59] and promotes EMT. Remarkably, in neurons PrPC binds NCAM1 and

recruits it to lipid rafts, inducing a similar downstream signalling pathway to drive neurite

outgrowth [60]. The additional importance of NCAM1 polysialylation in EMT is highlighted with

its critical influence on interactions at the cell surface, which in turn affect the balance between

cell-matrix and cell-cell adhesion, as well as related processes, such as cell differentiation and

migration [58]. Given the close next-neighbour relationship between PrPC and NCAM1, one would

expect their functions to be intertwined. PrP’s role in NCAM1 polysialylation and EMT

underscores this point and serves as one of the most striking phenotypes associated with PrP to

date [4,58]. In fact, the role of PrP in cell adhesion and EMT is supported through independent

avenues of research considering PrP’s evolutionary origin, protein interactors, as well as PrP KO

studies.

1.6.3 Additional PrP-related phenotypes

The following section will briefly summarize a number of cellular mechanisms that PrPC has been

linked to during the years as the prion research community pursued their quest of uncovering the

enigma that is the function of the cellular prion protein. In a 1996 study, PrP KO mice were first

shown to exhibit circadian activity alterations and changes in sleep patterns [61]. PrP’s potential

role in circadian rhythmicity regulation is also consistent with the clinical symptoms of FFI,

suggesting a loss-of-function mechanism might contribute to this prion disease [61]. Some other

phenotypic abnormalities observed following deletion of the Prnp gene include a peripheral

myelin maintenance deficiency [62], altered iron metabolism [63], deficits in long-term

potentiation and synaptic function [64], as well as an infrapyramidal mossy fiber development

defect [65]. There has also been evidence of PrP exhibiting cytoprotective activities with multiple

reports demonstrating its inhibition of Bax-mediated apoptosis in neuronal cultures [39]. Other

reports have pointed to a potential role of PrPC in the protection of cells from oxidative stress

[39,66] . Given PrP’s evolutionary relationship to ZIPs, which are involved in divalent metal ion

transport [49,50], it may not surprise that PrPC possesses the ability to bind copper. In fact, the

octarepeat region within the PrPC amino acid sequence harbours four copper-binding sites [43].

However, this domain within PrPC is actually relatively poorly conserved, and merely shares with

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ancestral ZIP transporters its disordered fold and an enrichment of pseudo-repeat motifs.

Moreover, repeated attempts to makes sense of this ability of PrP to bind copper (and zinc) have

been largely unsuccessful, with several studies failing to show a major involvement of PrP in

mediating cellular copper trafficking (for an example, see [39]). Other studies have found PrPC to

promote neurogenesis and neuronal differentiation [67]. In addition, PrPC has been implicated in

the amyloid beta-induced synaptic plasticity impairment observed in AD [68]. More specifically,

PrPC was found to act as a receptor of amyloid beta 42 oligomers, mediating their detrimental

effects [68].

Although some of these PrP-related phenotypes have held up under close scrutiny, no consistent

model that encompasses several of these observations has emerged from them to date. In addition,

discrepancies observed by separate investigators and in distinct experimental paradigms have cast

uncertainty on the degree to which differences in observations reflect biology versus methodology.

1.7 Methodology background

1.7.1 Overview of the experimental workflow

This chapter’s last section will provide essential background on the methodology of our

comparative interactome analyses [69]. It is meant to initially provide a broad overview of the

experimental workflow, followed by a more in-depth account of our methodological steps. Our

study aimed to establish a database for the binding partners of PrP in different cellular paradigms

in order to detect commonalities as well as cell-type specific patterns. It should be noted that

comparative nature of the study dictated that we adhered to the same systematic experimental

protocol for the different cell lines. Each of the interactome datasets were generated by a D18

antibody-driven immunoprecipitation (IP) of PrP as the bait protein, with PrP-deficient cells

available for each of the four models serving as negative controls. Affinity-capture eluates were

then prepared for mass spectrometry, ran on an Orbitrap mass spectrometer and analyzed further

using sophisticated bioinformatics and statistical algorithms that could uncover and compare the

proteins present in the vicinity of PrP across the four cell models of interest.

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The following subsections will more deeply go over topics including the nature of the four cells

types, the CRISPR-Cas9 knockout technology, affinity capture sample preparation, the

characteristics of the D18 PrP-specific antibody and, finally, the mass spectrometry procedures

used in our study.

1.7.2 Cell models

The PrP interactome was compared across four relevant mouse cell models in our study: NMuMG,

C2C12, N2a and CAD5. The NMuMG cell line is a mammary gland epithelial cell population,

which is a widely used mammalian model for studying EMT [4,70]. This murine-derived cell line

was previously used by our group to establish the role of PrP in EMT [58]. NMuMG cells respond

robustly to TGFβ1 exposure by transition from epithelial cells with a cobblestone morphology to

more spindle-like mesenchymal cells [4]. PrP levels were reported to increase in a time-course

treatment of TGFβ1 in NMuMG cells and we have therefore performed this treatment before the

PrP capture was done. Although not exhaustively explored, NMuMG cells have so far not been

successfully infected with PrPSc inoculations (unpublished results, Schmitt-Ulms laboratory).

C2C12 cells are myoblasts that are capable of undergoing differentiation into post-mitotic

myotubes with serum removal from their culture media [71]. These non-proliferative myotubes

have high PrP expression and can replicate prions in culture to high titer [71], hence our use of the

differentiated myotubes in our analyses. N2a neuroblastoma cells have been used for the past 20

years in the prion research field and can be host to many strains of PrPSc. Interestingly, CAD5 cells

are known for their even higher permissibility to infection by a wider range of different prion

strains and higher titer of infectious material [72].

Considering that all of our chosen cell lines share murine origins and have been explored side by

side using an optimized methodology for PrP capture, elution and MS analysis paradigms, our

study facilitates one of the deepest comparative analyses across different cell types that are

performed so far in the field. It is hoped that these findings allow for further analyses relevant to

the disease models and the toxicity that is observed downstream.

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1.7.3 CRISPR-Cas9 knockout technology

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated

protein) machinery is an adaptive immune system found in various bacteria and archaea [73]. This

ability was evolved in these microbial organisms in order to fight off repeated invasions by the

same virus [73]. This defense mechanism first recognizes foreign DNA, originating from viruses

or plasmids, as non-self and goes on to integrate short segments of the said foreign genetic material

in designated locations within CRISPR loci in the host’s genome [73]. These virus-derived DNA

fragments, called spacers, in addition to the host Cas proteins provide surveillance such that if a

bacterium is consecutively invaded with the same bacteriophage, it can get recognized and

disarmed by cleaving its genome [73,74]. More specifically, these incorporated spacer fragments

undergo transcription and are processed into small noncoding RNAs, called guide RNAs (gRNAs)

[73]. These gRNAs in conjunction with Cas protein complexes can base-pair with incoming

foreign nucleic acids that are complementary to the gRNA sequence [73]. This binding in turn

initiates the cleavage of the gRNA-foreign nucleic acid complex [73].

There have been three CRISPR systems (Type I, II, and III) identified in archaeal and bacterial

hosts with Type II being the one utilizing Cas9 [75]. Type II Cas9 system is comprised of proto-

spacer adjacent motifs (PAMs) flanking the 3′ end of the target DNA sequence, which direct the

Cas9 cleavage process [76]. Cas9 nuclease has two nuclease domains and is able to make double-

stranded breaks (DSBs) at specific loci within the genome, guided by small gRNAs that bind the

target DNA [75]. Since the CRISPR mechanism is naturally serving as an antiviral defense, the

sequence of the gRNA is usually corresponding to the phage genome sequences; however, Cas9

can easily be retargeted by substituting the gRNA with a different sequence of interest [76].

Following the Cas9 cleavage, one of the two main DNA damage repair pathways has to be

activated in order to repair the target genomic locus. If a repair template is not present, the

nonhomologous end joining (NHEJ) process is harnessed in order to re-ligate the DSBs [75]. NHEJ

is an error-prone mechanism giving rise to insertion/deletion (indel) mutations, which can be

exploited in order to produce gene knockouts since occurrence of indel mutations within a coding

exon can induce premature stop codons or frameshift mutations [75]. The second DNA repair

mechanism called the homology-directed repair (HDR) occurs when an externally introduced

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repair template is present and can therefore be harnessed to make accurate modifications at the

target genomic locus [75].

The microbial CRISPR adaptive immunity has now been harnessed to establish a novel genome

editing technology, which provides a highly specific system capable of efficiently editing

eukaryotic cells [75]. This system can be used in mammalian cells by heterologous expression of

codon-optimized Cas9 enzyme in addition to the required RNA components used to guide the Cas9

to genomic loci of interest [75]. The PrP KO clones of our four cell lines were generated using the

said CRISPR-Cas9 knockout technology [74]. The cells were transfected with plasmids expressing

the desired gRNAs as well as the Streptococcus pyrogens Cas9 (SpCas9) nuclease [74]. The

plasmids usually contain antibiotic resistance elements that are used to select for the cells that have

successfully taken up the plasmids. The gRNAs were designed to target the beginning of the coding

sequence on the Prnp gene within the third exon [74]. In this CRISPR-Cas9 system, the SpCas9

enzyme was codon-optimized for mammalian cell expression and harboured a mammalian nuclear

localization sequence [74]. When designing the underlying DNA sequence of an expression vector

one can use codon optimization in order to take advantage of the fact that each amino acid can be

coded by multiple codons and the expression efficiency of a protein of interest can therefore be

increased by avoiding rare codons in its sequence [77]. The nuclear localization sequence ensures

that the SpCas9 enzyme is transported to the nucleus after being expressed in the mammalian cell,

which facilitates eukaryotic gene editing. Our genome editing method was aimed to maximize the

production of indel mutations at CRISPR target sites by a double stranded break followed by the

NHEJ pathway [74]. While HDR is typically only active in diving cells [75], NHEJ is less

dependent on the cell cycle and is also the more efficient of the two DNA damage mechanisms

[74]. These indel mutations at the Prnp coding sequence can lead to the generation of PrP-deficient

cells as the sequencing of genomic DNA has verified.

Following plasmid transfection for gRNA(s) and Cas9 nuclease, cells were diluted to single cells

and expanded for screening to identify successfully engineered clones.

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1.7.4 Affinity capture matrix

Each of the PrP pull down experiments start with in vivo mild formaldehyde crosslinking of wild-

type and PrP deficient cells in order to preserve the protein-protein interactions [78]. After the

crosslinking step, the cells are lysed using detergents. The samples undergo centrifugation steps to

discard the cell debris. A bicinchoninic acid (BCA) colorimetric assay is used for protein level

adjustment in each of the input samples at the beginning of capture. Lysates are then incubated

overnight with the affinity capture matrix, which consists of the D18 anti-PrP antibody bound to

the KappaSelect beads.

The D18 antibody is a recombinant protein produced in E. coli that recognizes mouse PrP (AA

132-158) and is conformation dependent [79]. It is solely comprised of the Fab portion of the

antibody, based on which KappaSelect beads can be used as a resin by binding to the constant

domain of the kappa light chain.

Following the overnight incubation, during which PrP along with its interacting protein partners

bind the affinity capture matrix, the samples are thoroughly washed in order to remove nonspecific

binders. Subsequently, bait-bound proteins are eluted from the resin by an acidic pH drop.

1.7.5 Mass spectrometry

Mass spectrometry (MS) is a powerful technology that can be employed to provide the relative

levels of proteins present in different biological samples [80]. In the interactome analyses

performed for this thesis, the relative quantitative comparison of proteins present in affinity-

capture eluates derived from wild-type and PrP KO cells made it easy to discriminate specific

interactors from non-specific binders to the affinity-matrix.

The primary goal of a mass spectrometer is to measure the mass or, more accurately, the mass-to-

charge ratio of analytes [80,81]. Shotgun proteomics experiments typically begin with complex

protein mixtures, which are then fragmented into peptides via sequence-specific chemical or

enzymatic proteolysis [82]. The mass spectrometer isolates the peptides, breaks them into even

smaller fragments and measures the masses of these fragments, which serve as a ‘fingerprint’ of

the specific amino acid sequence of a given peptide. These tandem mass spectra are then matched

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to the known amino acid sequences of proteins using a matching algorithm [82]. The following

sections will provide a more in-depth description of the above steps leading to protein

quantification in a mass spectrometer.

Prior to the injection of samples into the mass spectrometer, IP eluates are subjected to sample

preparation steps, including denaturation, reduction, alkylation and trypsin digestion [83]. Thus,

samples are first denatured in order to make all aspects of their primary structure equally

accessible. Subsequently the disulfide bonds in the proteins are reduced, which further aids protein

unfolding and facilitates the subsequent protein digestion step, in turn enhancing protein sequence

coverage [83]. The free cysteine residues are alkylated in order to prevent the reformation of the

disulfide bonds [83]. The protein samples can then be digested by a protease, such as trypsin, into

peptides that can be analyzed by the mass spectrometer [83].

The samples have to then undergo a protein labelling procedure, in order for the MS apparatus to

successfully provide simultaneous relative quantifications [80,84]. Our interactome studies took

advantage of multiplex isobaric tags for relative and absolute quantitation (iTRAQ) labelling [80].

In this state-of-the-art technique peptides are covalently conjugated to chemical tags of identical

structure that harbor chemical isotopes and yet are of identical total mass (i.e., isobaric). The latter

contain labile components called reporter ions that can be distinguished by tandem MS (MS2)

[80]. More specifically, each isobaric tag contains a reporter group, a chemically reactive group

and finally a spacer to normalize the mass [80]. The tag works such that each sample’s

differentially labelled proteolytic peptides remain isobaric upon mixing and provide a single peak

in the initial MS1 scans. However, upon collision-induced dissociation (CID) the labelled peptides

release distinct reporter ions, which unlike the overall isobaric tags have different mass-to-charge

ratios and can be detected during MS3 scans [80]. The relative intensities of reporter ions

determine the relative quantification of the component peptides and in turn their parent proteins

within different samples [80].

The isobaric labelled samples are then pooled together and the sample mixtures are purified using

strong cation exchange and reversed phase chromatography clean-up procedures [83]. Purification

steps are important since excess salt and residual detergent can interfere with the subsequent

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ionization step and additionally result in background or chemical noise in the mass spectra [83].

The purified peptides are then separated using high performance liquid chromatography (HPLC),

prior to being introduced into the mass spectrometer.

Mass spectrometers consist of three modules: an ion source, a mass analyzer and a detector [81].

Tandem mass spectrometers are comprised of multiple mass analyzers utilizing back-to-back

fragmentation and mass-to-charge separation steps to uniquely identify proteins [81]. In tandem

MS, the first mass analyzer selects ions of a specific mass-to-charge ratio, the selected ions are

then fragmented by CID and a second mass analyzer is used for the analysis of the resulting product

ions [81].

One of the methods to ionize samples is electrospray ionization (ESI), which utilizes electrical

energy to transfer ions from solution to the gaseous phase [85]. The peptides are required to

undergo ionization since MS analyses necessitate the use of charged particles. After ions enter the

mass spectrometer, mass analyzers construct an MS spectrum based on the measured mass-to-

charge ratio [81]; however, because a given mass is not unique to a single peptide, further analyses

are required to determine the AA sequence [82]. The peptides are fragmented into smaller pieces

by collision with inert gas molecules [82]. The information on the AA sequence of each peptide

can subsequently be inferred from a computational analysis of the MS2 fragment spectrum [82].

More precisely, the peptide fragmentation process generates several mass ladders by randomly

fragmenting a given peptide at several possible positions within its peptide and side-chain bonds

and by retaining the charge either at the front-end or back-end of the resultant fragments. Although

there are many ways to match MS2 spectra to protein sequences, in one approach the collected

tandem spectra are computationally matched to a database of predicted protein MS2 spectra, in

order to initially identify the peptide sequences, and then assign them to their parent proteins [82].

Lastly, the relative quantitation of peptides across several samples will be based on an MS3

reporter ion scan that is populated with isotopically labeled iTRAQ reagent fragments following

high-energy collision of the most intense MS2 fragments [80].

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Chapter 2

Rationale, Hypothesis and Objectives

2.1 Rationale for the ZIP study The most severe phenotype linked to PrP deficiency to date, aside from the inability to infect KO

mice, was reported for one of two PrP orthologs in zebrafish, termed PrP1 [55]. Morpholino-based

knockdown of PrP1 had shown to stop zebrafish embryogenesis at the gastrula stage, which

involves cellular rearrangements relying on EMT [55]. Strikingly a similar phenotype has been

observed in ZIP6-deficient zebrafish embryos [55]. Following up on these independent findings,

we have previously reported a pivotal role of PrP in EMT. More specifically, in the NMuMG

mouse cell model, which is a well-known epithelial cell line that robustly undergoes TGFβ1-

induced EMT, PrP ablation disrupted EMT [4]. We also showed that during EMT PrP expression

levels increased more than tenfold [4]. Our subsequent mechanistic studies revealed a link between

PrP and NCAM1, which is one of the main interactors of PrP. Moreover, global proteome analyses,

following TGFβ1 treatment of wild-type NMuMG cells, demonstrated that NCAM1 was amongst

the proteins whose levels were profoundly increased during EMT and that PrP-deficient cells failed

to show this upregulation to the same extent. A parallel line of study comparing wild-type and PrP

KO NMuMG cells showed that PrP controls polysialylation of NCAM1 during EMT and that

NCAM1 fails to undergo polysialylation in PrP-deficient clones we derived from this cell model.

A subsequent ZIP6 interactome study in TGFβ1-treated NMuMG cells in our lab identified ZIP10,

NCAM1 and calreticulin to be the most enriched protein interactors of ZIP6, with ZIP10 being the

main interactor. It is noteworthy that ZIP6 and ZIP10 had also been previously suggested to

interact and have functional similarities [54,86]. Additional investigation into the relationship of

ZIP6 and ZIP10 using both NMuMG and N2a cell models revealed that the expression of these

two zinc transporters is closely co-regulated. Moreover, we were able to document that they form

a functional heteromer that interacts with NCAM1 [55]. Given the evolutionary and functional

links between the ZIPs and PrP, as well as the independently reported similarities in gastrulation

arrest phenotypes of ZIP6 and PrP1 in zebrafish upon morpholino knockdowns, we investigated

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whether PrP had inherited some of its biological functions from its ZIP ancestors. More

specifically, given that PrP affects NCAM1 polysialylation in NMuMG cells by controlling

polysialyltransferase ST8SIA2 transcript levels, we looked into whether ZIPs could similarly

regulate this PTM in mammalian cell models. Thus, we hypothesized that ZIPs, like PrP, can

influence the NCAM1 polysialylation machinery. Analyzing the levels of NCAM1 and its

polysialylated form by western blotting revealed surprisingly that although ZIP6-deficient

NMuMG cells also exhibit reduced NCAM1 expression, unlike PrP deficiency, ZIP6 ablation did

not prevent NCAM1 polysialylation [55].

When we next compared the NCAM1 interactome in wild-type and ZIP6 KO NMuMG cells, we

observed that ZIP6 ablation caused a reduction in NCAM1 phosphorylation. More specifically,

levels of a specific phosphorylation event within a cluster of potential phosphoacceptor sites in the

cytoplasmic, C-terminal domain of the longest isoform of NCAM1 (AA 945 to 974) were

dramatically underrepresented in ZIP6 KO cells. Closer inspection of the phosphorylation

consensus motif in this region pointed toward glycogen synthase kinase 3 (GSK3) as the most

likely kinases responsible for NCAM1 phosphorylation at this site [55]. The notion that this

phosphorylation event might be attributed to GSK3 gained independent support from a ZIP6

interactome investigation we undertook, which revealed the co-enrichment of both GSK3A and

GSK3B kinase with ZIP6. Taken together, these observations led to the hypothesis that the ZIP6-

ZIP10 heteromer binds and directs GSK3 kinases to NCAM1. Because these developments

coincided with me joining the group, one of my initial tasks became to test this hypothesis by

determining if the ZIP6-dependent NCAM1 phosphorylation is driven by GSK3 kinases on the

basis of functional phosphorylation assays.

2.2 Rationale for the PrP interactome study As mentioned above, previous work in our lab regarding the role of PrPC in EMT had led to the

discovery that PrPC affects polysialylation of NCAM1 by regulating the transcript levels of

ST8SIA2, one of two polysialyltransferases that can attach polysialic acids to N-glycans of

NCAM1. Surprisingly, we observed that PrPC differentially regulates NCAM1 polysialylation in

different cell models; thus, whereas in NMuMG cells ablation of PrPC prevented this PTM during

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EMT, in PrPC-deficient C2C12 cells the opposite trend, namely a massive upregulation of NCAM1

polysialylation was observed [4]. This observation invoked our interest to dissect if cell type-

specific differences in the molecular environment of PrPC might exist that could underlie these

differences. In one scenario, the complete opposite effects of PrPC removal in NMuMG and C2C12

cells, might reflect a reality whereby PrPC could be part of separate microdomains in these cell

models. Alternatively, we may find that PrPC is embedded in identical molecular environments

and that differences in downstream signaling circuitry translates in distinct molecular phenotypes.

Learning about these alternative realities is not merely an academic exercise but it is likely that

cell-type specific differences in the molecular environment of PrPC, if they exist, could contribute

to well-known differential susceptibilities of existing cell lines to prion infection.

Until now, the heterogeneity of the cellular paradigms and protocols used in the prion research

field, precluded to answer to which extent differences in reported PrPC interactions reflected

differences in methodology versus biology. Despite numerous studies that explored the function

of PrPC in a wide range of experimental paradigms, no clear consensus has emerged. Thus, this

thesis project was designed to address this unmet need by carrying out a first in depth comparative

analysis of the PrP interactome based on four cell models of interest to the field. We hypothesized

that insights into the molecular environment of PrPC will provide novel therapeutic angles by

modulating PrPC levels, preventing its conversion to PrPSc, or interfering with PrPC-dependent

toxicity.

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Chapter 3

ZIP6-mediated NCAM1 Phosphorylation Please note that this chapter incorporates material published in the following article [87] :

Brethour D, Mehrabian M, Williams D, Wang X, Ghodrati F, Ehsani S, et al. A ZIP6-ZIP10

heteromer controls NCAM1 phosphorylation and integration into focal adhesion complexes during

epithelial-to-mesenchymal transition. Sci Rep. 2017;7: 1–19. doi:10.1038/srep40313

Candidate’s Role: Performed the NCAM1 immunoprecipitation experiments, analyzed the data

and edited the manuscript.

Summary:

The prion protein evolved from the subbranch of ZIP metal ion transporters comprising ZIPs 5, 6

and 10, raising the prospect that the study of these ZIPs may reveal insights relevant for

understanding the function of PrP. Building on data which suggested PrP and ZIP6 are critical

during EMT, we investigated ZIP6 in an EMT paradigm using CRISPR-generated ZIP6 knockout

cells, mass spectrometry technology and bioinformatic analyses. Reminiscent of PrP, ZIP6 levels

are five-fold upregulated during EMT and the protein forms a complex with NCAM1. ZIP6 also

interacts with ZIP10 and the two ZIP transporters exhibit interdependency during their expression.

Additionally, ZIP6 contributes to the integration of NCAM1 in focal adhesion complexes but,

unlike cells lacking PrP, ZIP6 deficiency does not abolish polysialylation of NCAM1. Instead,

ZIP6 mediates phosphorylation of NCAM1 on a cluster of cytosolic acceptor sites. Substrate

consensus motif features and in vitro phosphorylation data, point toward GSK3 as the kinase

responsible, and interface mapping experiments identified histidine-rich cytoplasmic loops within

the ZIP6/ZIP10 heteromer as a novel scaffold for GSK3 binding. Our data suggests that PrP and

ZIP6 inherited the ability to interact with NCAM1 from their common ZIP ancestors but have

since diverged to control distinct posttranslational modifications of NCAM1.

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3.1 Introduction In this chapter the focus is mostly on a specific portion of a 2017 study by our group, which

examines the relationship between ZIPs and NCAM1 [87]. As discussed previously, PrP evolved

from the ZIP family of ion transporters [49] and is particularly similar to the LIV1 subfamily of

ZIPs, which include ZIPs 5, 6 and 10 [48]. NCAM1 on the other hand, is a close PrP interactor

and both proteins have been shown to be involved in EMT [4,57], with PrP controlling the levels

of polysialylated NCAM1 during EMT and related cellular reprogramming events.

Together, this body of work has raised several interesting questions: (1) Did PrP inherit its intimate

involvement in a biology that modulates the expression of NCAM1 and controls its polysialylation

from its ZIP6-like ancestor? and, if so, (2) are ZIP6 and PrP equivalent in this regard, or have they

acquired specialized roles with respect to their influence on NCAM1?

To address these questions, we undertook a ZIP6 analysis in the EMT paradigm of NMuMG cells

that showed ZIP6 transcript and protein levels are both dramatically upregulated upon treatment

with TGFβ1. Moreover, in a follow-up interactome study, we observed that ZIP6 forms a

heteromeric complex with ZIP10, whose predominant interactor is NCAM1. Subsequent in-depth

analyses of NCAM1 interactors revealed a critical role for ZIP6 in the assembly of NCAM1-bound

focal adhesion complexes. However, we also documented a minor influence of ZIP6 on the levels

of polysialylated NCAM1 and instead uncovered a novel signaling module that has ZIP6/10

collaborate closely with GSK3B in its influence on NCAM1. When I took up my training in the

laboratory, the key question whether the ZIP6/ZIP10 heteromer forms a novel hub that can

sequester GSK3B to NCAM1 and thereby controls its direct phosphorylation by this kinase at a

specific site had not been addressed. I therefore will address in this chapter this question before

moving to the main body of my thesis, which naturally followed from differences in NCAM1

posttranslational effects we observed in separate experimental paradigms.

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3.2 Results

3.2.1 In vitro phosphorylation of the longest NCAM1 isoform by GSK3B

Observations thus far raised the intriguing possibility that the ZIP6-ZIP10 heteromer may serve as

a scaffold for binding and directing GSK3 kinases to their NCAM1 substrate. Note that the higher

the number of peptides quantified and matched to a given protein, the more confidently and with

higher statistical power, the differences in relative abundance levels can be assigned in this

methodology. Thus, despite the enrichment levels of GSK3A and GSK3B in ZIP6-specific co-IP

eluates by ~20% compared to the negative controls (Fig. 3.1a), this difference was significant and

distinct from the peptide distribution of, for example, ATP synthase coupling factor 6 (ATP5J), a

mitochondrial protein, which was observed at equal levels among all samples. This suggested that

its binding to the affinity matrix had exclusively been through non-specific interactions (Fig. 3.1b).

To validate whether the longest NCAM1 isoform is indeed a GSK3 substrate, we examined

whether recombinant GSK3B can phosphorylate immunoprecipitated NCAM1 from NMuMG

cells after 48 h induction of EMT by TGFβ1. This design was selected over an alternative strategy,

whereby both the kinase and putative substrate would be obtained from recombinant sources,

because GSK3 typically requires a priming phosphorylation by casein kinase I, that would

otherwise not be present in recombinant NCAM1. Corroborating the mass spectrometry results,

this experiment revealed that a 180 kDa protein, corresponding in size to the longest isoform of

NCAM1, was indeed the prime phosphorylation target of recombinant GSK3B in the NCAM1-

directed immunoprecipitation eluate (Fig. 3.1c and d). Taken together, these data provided

orthogonal evidence for the notion that GSK3B can preferentially phosphorylate the longest

NCAM1 isoform in biological material relevant to this study.

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Figure 3.1. In vitro phosphorylation of longest NCAM1 isoform by GSK3B. (a and b) Box

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plots showing relative quantitations of glycogen synthase kinase 3 beta (GSK3B) and ATP

synthase coupling factor 6 (ATP5J) in NCAM1 interactomes generated with wild-type or ZIP6

KO cells. Whereas GSK3B protein levels in the NCAM1 interactome correlated directly with the

presence or absence of ZIP6, ASCF6 protein level ratios were similar in all samples, consistent

with unspecific binding of this protein to the affinity capture matrix. (c and d) Evidence of NCAM-

180 in vitro phosphorylation by GSK3B. (c) Immunoprecipitation of NCAM1 from cellular

extracts of NMuMG cells, which had been induced to undergo EMT by 48 h addition of TGFΒ1

to the cell culture medium. The OB11 antibody used in this experiment for immunoprecipitation

and Western blot detection binds to a cytosolic epitope shared by NCAM-140 and NCAM-180,

which gave rise to the characteristic band pattern in Lanes 5 and 6. Note that due to its relatively

higher abundance in eluate fractions, signals for NCAM-140 exceeded maximum intensity levels

in lanes 5 and 6, leading to a partially inverted ‘white’ signal. (d) Autoradiographic analysis of in

vitro GSK3B phosphorylation of NCAM1immunoprecipitation eluates seen in Panel ‘c’. Note that

although the signal detected at 180 kDa, corresponding to NCAM-180, has not been validated to

consist of phosphorylated NCAM1, the Coomassie stain of the NCAM1 IP eluate fractions shown

in Panel ‘c’ revealed no signals in the high mass region of the gel that could not be attributed to

the expected NCAM-180 or NCAM-140 bands.

3.3 Discussion Two independent observations placed a spotlight on GSK3 kinases in this work: First, the ZIP6-

ZIP10 heteromer was observed to co-immunoprecipitate with both GSK3 paralogs. Second, we

discovered a ZIP6-dependent phosphorylation site within the cytoplasmic domain of the longest

isoform of NCAM1 that bears striking similarities to a previously known GSK3 substrate. Our

data place GSK3 paralogs in close proximity to the site of ZIP6-ZIP10-mediated zinc influx—

presumably one of only few cytoplasmic locations at which free zinc ion concentrations may

exceed the generally very low levels of free zinc in the cytoplasm. This observation may be

relevant in light of prior work by others which documented that the ability of GSK3 to

phosphorylate its substrates is inhibited in the presence of free zinc [88]. The authors documented

that there is specificity to this zinc-mediated GSK3 inhibition by showing its reliance on zinc, as

opposed to other divalent cations, and by establishing that the zinc inhibition characteristic does

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not extend to CDK2, a closely related kinase. It has long been known that NCAM1 can be

phosphorylated within its intracellular domain by casein kinase 1 (CK1) and GSK3 but in these

early studies the phospho-acceptor site was not determined [89]. Subsequent attempts to map the

NCAM1 phosphorylation sites highlighted initially a domain in juxtaposition to the inner face of

the plasma membrane but also a putative serine 761 phospho-acceptor site that conforms to the

GSK3 consensus motif [90]. The design of the latter phospho-site mapping experiments precluded

the detection of phosphorylation sites observed in this study. This was because analyses were

limited to a truncated NCAM1 expression construct comprising the transmembrane and

cytoplasmic domains of NCAM1 but lacking the Exon 18 coded alternatively spliced insertion that

is only present in the longest NCAM1 isoform. However, a more recent global phospho-site

analysis of developing mouse brain samples independently mapped amino acids 946 and 958

(NCBI accession number: NP_001106675) as NCAM1 phospho-acceptor sites [91]. These sites

are identical to the first and fourth NCAM1 phosphorylation sites within the phosphorylation

cluster (amino acids 945-974) that was repeatedly sequenced and quantified in this study. We

subsequently validated the preferential GSK3B phosphorylation of NCAM-180 by in vitro

phosphorylation assay, using immunoprecipitated NCAM-140 and NCAM-180 as substrates.

Curiously, CRMP2, a member of the CRMP family, which is known to carry a highly similar

GSK3 phosphorylation motif (Fig. 3.1i), had previously also been shown to be a binder of NCAM1

[92], but was not detected in the interactome dataset produced in this study.

What might be the functional consequences of the GSK3-dependent phosphorylation of NCAM1?

One commonly observed scenario would see phospho-occupancy at this site alter NCAM1

interactions with phospho-serine/threonine (pSer/Thr) binding modules on other proteins. In fact,

binding of NCAM1 to 14-3-3 proteins, the first signaling molecules recognized to engage in

pSer/Thr-dependent interactions with other proteins, was also observed to be ZIP6-dependent in

this study, making them excellent candidates for this scenario. While the broader physiological

consequences are not known, a previous report established that inhibition of GSK3 can prevent

NCAM1-induced neurite outgrowth [93].

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3.4 Methods

3.4.1 Sample preparation for immunoprecipitation

Three biological replicates of NMuMG wild-type and ZIP KO clones, undergoing EMT, were used

for the NCAM1 immunoprecipication. TGFβ1 (240-B; R&D Systems, MN, USA) was added for

48 hrs at a concentration of 6.4 ng/mL and replenished with fresh medium after 24 hours. After 48

hrs of control or treatment conditions, medium was removed, cells were washed with ice cold PBS,

and crosslinking was completed with a 15 min incubation with 2% formaldehyde in PBS. The

formaldehyde was removed, and the reaction was quenched with a 10 min incubation with 125

mM glycine in PBS. Cells were again washed with ice cold PBS before undergoing lysis in an ice

cold buffer consisting of 5 mM EGTA, 10% glycerol, 1% sodium deoxycholate, 1% NP-40, 150

mM HEPES (pH 8.0) and 1× Complete Protease Inhibitor Cocktail (11836170001; Roche).

Insoluble cellular debris was cleared by centrifugation for 30 min at 4000 RPM and 4 °C.

3.4.2 Protein immunoprecipitation

Immunoprecipitation with NCAM1 specific antibody was completed with Protein G sepharose

(17-0618-01; GE Healthcare) beads. Beads were transferred to a microcentrifuge tube and washed

twice with ultrapure water and twice with PBS before the addition of the respective antibody,

which had been diluted in PBS to fill the tube. The smaller-scale NCAM1 immunoprecipitations

for in vitro GSK3B phosphorylation was undertaken with the NCAM1-directed antibody OB11.

The bead/antibody mixture was then gently agitated on a turning wheel for 4 hrs at room

temperature (RT), before being equally divided into fresh tubes for individual samples. Beads were

allowed to settle and the excess liquid was removed before protein samples were added. The

samples were gently agitated on a turning wheel overnight at 4 °C, then washed thrice with 5 mM

EGTA, 10% glycerol, 1% sodium deoxycholate, 1% NP-40, 150 mM HEPES (pH 8.0). Detergents

were removed with two consecutive washes of 10 mM HEPES (pH 8.0), before samples were

transferred to lo-bind 0.5 mL microcentrifuge tubes. Proteins were then eluted by acidification

with 0.2% trifluoroacetic acid, 20% acetonitrile (pH 2.0).

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The mapping of the GSK3 binding site to internal loop domains made use of affinity matrices

based on the monoclonal capture antibody HA.11 (901501; BioLegend, CA, USA) bound to

Protein G-sepharose (17-0618-01; GE Healthcare). The cell lysates were initially incubated with

the antibody overnight and then added to beads for an overnight capture with gentle agitation at

4°C. The proteins were at the end eluted with sample buffer and heating at 45 °C for 10 minutes.

3.4.3 Active kinase assay

NCAM1 protein was immunoprecipitated with the OB11 antibody as described above and was in

vitro phosphorylated with recombinant GSK3B while still attached to Protein G beads. More spe-

cifically, 4 µL (9 ng) of active GSK3B (catalog number 14-306, lot number WAA0024-B,

Millipore, Burlington, Canada), 10 µL of hot ATP buffer, composed of 1 volume of [γ-32P]ATP

(0.01 mCi) (catalog number NEG 502Z, Perkin-Elmer Inc., Woodbridge, ON, Canada) and 9

volumes of 75 mM MgCl2 and 500 µM ATP (catalog number A6559, Sigma-Aldrich, Oakville,

ON, Canada), were added to 15 µL of NCAM1 immunoprecipitate to give rise to a reaction mix

of 40 µL that was buffered by 8 mM MOPS, pH 7.0 and supplemented with 0.2 mM EDTA. After

a 10 min incubation at 30 °C the reaction was stopped by the addition of Laemmli sample buffer.

The analysis was undertaken in triplicate for wild-type and ZIP6 knockout cell extracts. As

negative controls served samples that differed by the omission of GSK3B. Next, Protein G beads

were pelleted by centrifugation, the superna- tant containing the eluted proteins boiled for 15 min

and loaded onto a large format isocratic (7.5%) SDS-PAGE gel that was cast in a Hoefer gel

cassette. Incorporated radioactivity was revealed by overnight exposure of the SDS-PAGE gel to

a double-emulsion X-ray film (catalog number Z363006, Carestream Kodak BioMax MS film,

Sigma-Aldrich).

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Chapter 4

The Prion Protein is Embedded in a Molecular Environment that Modulates Transforming Growth Factor β and Integrin Signaling

Please note that this chapter incorporates material published in the following article [69] :

Ghodrati F, Mehrabian M, Williams D, Halgas O, Bourkas MEC, Watts JC, et al. The prion

protein is embedded in a molecular environment that modulates transforming growth factor β and

integrin signaling. Sci Rep. 2018; 8654. doi:10.1038/s41598-018-26685-x

Candidate’s Role: Conceived, designed and performed the experiments. Analyzed the data, wrote

and edited the manuscript.

Summary:

At times, it can be difficult to discern if a lack of overlap in reported interactions for a protein-of-

interest reflects differences in methodology or biology. In such instances, systematic analyses of

protein-protein networks across diverse paradigms can provide valuable insights. Here, we

interrogated the interactome of the prion protein (PrP), best known for its central role in prion

diseases, in four mouse cell lines. Analyses made use of identical affinity capture and sample

processing workflows. Negative controls were generated from PrP knockout lines of the respective

cell models, and the relative levels of peptides were quantified using isobaric labels. The study

uncovered 26 proteins that reside in proximity to PrP. All of these proteins are predicted to have

access to the outer face of the plasma membrane, and approximately half of them were not reported

to interact with PrP before. Strikingly, although several proteins exhibited profound co-enrichment

with PrP in a given model, except for the neural cell adhesion molecule 1, no protein was highly

enriched in all PrP-specific interactomes. However, Gene Ontology analyses revealed a shared

association of the majority of PrP candidate interactors with cellular events at the intersection of

transforming growth factor β and integrin signaling.

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4.1 Introduction Relatively little is known about how interactions of a given protein differ across cell models.

Although there is no shortage of proteins whose binding partners have been studied in more than

one paradigm, chances are such studies were done by separate investigators with different

methodologies, precluding robust conclusions on the confounding effects of the paradigm itself.

In particular, proteins like the cellular prion protein, which lack catalytic domains and exhibit

widespread expression [11,29,94], are prone to escape robust functional assignments. PrPC is

central to the pathogenesis of prion diseases [23] and has been proposed to also act as a critical

cell surface receptor in AD [68], raising the expectation that insights into the function of PrPC will

provide useful angles for understanding the molecular underpinnings of these diseases.

To this end, the molecular interactions of PrPC have repeatedly been characterized [47,57,95] and

many proteins have been reported to interact with PrPC in separate studies, including the laminin

receptor precursor [96], the neural cell adhesion molecule 1 [57], the amyloid precursor like

protein-1 [97], and the stress-inducible protein 1 [98]. Rather than suggest a common theme, this

line of investigation has led to many hypotheses regarding the role of PrPC. Although it is to be

anticipated that some of the reported interactions will not stand the test of time, other reasons for

the diversity of observations need to be considered, including the likely existence of cell type-

specific interactions.

In one study undertaken with neuroblastoma cells, ZIPs, a family of Type-III transmembrane

proteins known to import zinc and other divalent cations into the cytosol, were initially observed

as PrPC interactors [47]. This work then spurred the discovery that prion genes evolved from an

ancient ZIP transporter and are members of the ZIP gene family, which comprises seventeen genes

in humans [49,99]. Because PrP is homologous to the ectodomain present in a subset of ZIPs,

studying the physiological function of this ectodomain may provide additional hints regarding

PrP’s function. Interestingly, the deficiency of PrP, Zip6 or Zip10 causes a rare and phenotypically

indistinguishable gastrulation arrest phenotype in zebrafish, apparently due to the ablation of a

morphogenetic program known as epithelial-to-mesenchymal transition [52–54].

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We recently documented that the expression levels of the aforementioned ZIPs, PrP, and NCAM1

are several-fold upregulated during EMT in mammalian cells [4], consistent with the interpretation

that the interactions these proteins engage in change over time and depend on the cell lineage

characteristics of the model. A hint that there may be additional differences in PrP interactions

when comparisons are done across models came from an observation of model-dependent

proteome shifts in PrP-deficient cells [100]. Not only did PrP-deficiency in distinct cell models

cause the levels of members of the Marcks protein family to shift in opposite ways but it also

prevented NCAM1 polysialylation in one cell model, yet caused a robust increase of this specific

PTM in another model [4].

To address if these phenotypic differences reflect distinct, immediate PrP interactions or depend

on downstream signaling, we undertook deep, quantitative PrP interactome comparisons in four

cell models that made use of in vivo crosslinking and capitalized on recombinant Fabs for the

selective capture of endogenous PrP-containing protein complexes. We identified cell lineage-

specific sets of PrP interactors, including several novel interactors and a membrane protein of

unknown function, whose PrP-dependent capture was supported by high-confidence peptide-to-

spectrum assignments and quantitation. All of the 26 candidate PrP interactors we identified are

known to be embedded in the plasma membrane or exist in the lumen/extracellular matrix. We

demonstrate that NCAM1 is the only transmembrane protein that is a universal and robust

interactor of PrP across the four models. The data comprise examples of homologous proteins

interacting with PrP in a cell model-specific manner. Finally, we highlight that a majority of PrP

interacting proteins observed in this study are known to play roles in EMT, either by acting as

TGFβ1 signaling modulators, by facilitating the formation of NCAM1-dependent focal adhesion

complexes, or by their association with integrin-mediated downstream cell signaling.

4.2 Results

4.2.1 Design of comparative PrP interactome analysis in four mouse cell models

The study made use of four mouse cell lines from which we had previously derived PrP knockout

clones by CRISPR-Cas9 technology (Fig. 4.1a) [100]. The parental wild-type cell lines are familiar

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to the prion research community due to their distinct properties with regard to PrP: 1) NMuMG

cells exhibit a more than five-fold increase in their PrP protein levels when EMT was induced by

the addition of TGFβ1 (attempts to infect these cells with prions have been unsuccessful but were

also not exhaustive) [58]; 2) C2C12 cells are the only muscle cell model currently known to be

susceptible to prion infection [71,101]; 3) N2a neuroblastoma cells may be the most often used

cell model in prion research and can readily be infected with mouse-adapted Rocky Mountain

Laboratory (RML) prions; and 4) CAD5 catecholaminergic cells exhibit susceptibility to infection

with several prion strains [102]. To stabilize existing protein-protein interactions, cells were

subjected to mild formaldehyde crosslinking prior to cell lysis (Fig. 4.1b). PrP-containing

complexes were affinity-captured using a recombinant anti-PrP antigen binding fragment (Fab),

designated as D18, that is known to bind to a non-linear epitope (comprising mouse PrP residues

133-157) within the globular domain of PrP [79]. Affinity-capture eluates were processed using a

workflow that facilitated the relative quantitation of peptides in three biological replicates and

three control samples obtained from wild-type and PrP knockout cells, respectively. The side-by-

side binning of peptide-to-spectrum matches on the basis of cross-correlation (X corr) values

computed by the SEQUEST score function [103] revealed similar stratifications for the four PrP

interactome analyses, indicating that the respective datasets were comparable in regards to a key

quality control benchmark and their depths of coverage (Fig. 4.1c).

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Figure 4.1 Design of comparative PrP interactome study. (a) Models used in this study. (b)

Flow-chart depicting workflow of quantitative interactome analyses. Note that for the NMuMG

cell model, one additional step was inserted into the workplan, namely a 48-hour addition of

TGFβ1 to the cell culture medium, which causes the cells to acquire mesenchymal morphology.

(c) Similar SEQUEST X corr stratifications of the four cell type-specific PrP interactome datasets

indicated comparable data quality

4.2.2 Comparison of PrP interactome analyses across models

A comparison of PrP-specific western blot signals obtained for cell lysates before (input) and after

(unbound) the affinity capture step established that more than 50% of total PrP was captured (Fig.

4.2a, lanes 1 and 2). As expected, eluate fractions exhibited strong PrP-specific signals whose

distribution matched the anticipated pattern, i.e., were comprised of low mass uncrosslinked bands

characteristic for PrP and high mass crosslinked PrP-containing smears that were particularly

concentrated in the 200-250 kDa range. When the same blot membranes were subsequently stained

with Coomassie, equal total protein levels were detected in ‘input’ and ‘unbound’ samples. Eluate

fractions contained protein levels below the Coomassie detection limit, except for one band whose

apparent MW matched the known D18 mass (Fig. 4.2a, lanes 5-10). The band could be seen at

equal intensity levels in all biological replicates of wild-type and control samples, consistent with

the interpretation that it indeed represented small amounts of the Fab, which had detached during

the pH 1.9 elution step. The subsequent mass spectrometry analysis confirmed the successful

enrichment of PrP (Fig. 4.2b, Supplementary Fig. S4.1).

A total of 26 proteins were shortlisted as PrP candidate interactors. Gene Ontology enrichment

analyses flagged categories within the ‘Molecular Function’, ‘Biological Process’ and ‘Cellular

Component’ classes that made sense for a GPI-anchored molecule like PrP (Fig. 4.2c). For

example, a cell-to-matrix binding category, namely binding to laminin was the most enriched

‘Molecular Function’ subcategory. Similarly, 23 of the candidate interactors had a prior annotation

that identified them as members of the ‘Cellular Component’ subcategory ‘extracellular space’

(see below for details on membrane topology). Finally, ‘Biological Process’ annotations of these

proteins are consistent with the notion that PrP is embedded in a membrane domain that serves as

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a signaling hub, with the subcategory ‘positive regulation of response to stimulus’ being the most

overrepresented annotation.

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Figure 4.2 Validation of successful technical execution of quantitative interactome analysis.

Analyses of benchmarks of PrP co-immunoprecipitation, iTRAQ quantitation and GO enrichment.

(a) Western blot validation of co-immunoprecipitation of endogenous PrPC from NMuMG cell

extracts. Strong depletion of PrP-related signals in the unbound fraction and its robust enrichment

in wild-type eluate fractions. Asterisks denote weakly detected cross-reactive bands. (b) Box plot

depicting selective detection of PrP in wild-type eluate samples of CAD5 cell-derived PrP

immunoaffinity captures but not in negative control PrP knockout eluates (see Supplementary

Figure S4.1. for the respective PrP box plots from NMuMG, C2C12 and N2a cells). The box plot

depicts in log2 space enrichment ratios of individual PrP peptides used for quantitation. The

computed Median peptide ratios and Inter Quartile Ranges (IQR) are shown above the graph. Note

that a subset of PSMs (indicated with red circles) were automatically eliminated from the

quantitation, either because their identification was redundant or as a consequence of mass

spectrometry profiles underlying their identification not passing stringency thresholds. In this and

other box plots in this report relative protein levels are depicted as ratios, with ion intensities of

the heaviest isobaric labels within multiplex analyses (representing one of 3 PrP knockout

biological replicates) serving as the reference (denominator). (c) GO enrichment analyses of the

26 shortlisted PrP candidate interactors identified in this study.

Although each of the four PrP interactome datasets comprised more than 200 proteins which had

passed confidence thresholds for identification, approximately 80% of the proteins in each dataset

were observed at levels that did not differ in wild-type versus PrP knockout samples, thereby

revealing them to be non-specific interactors of the affinity matrix. An example which showcases

proteins in this broad non-specific binder category is glyceraldehyde 3-phosphate dehydrogenase

(Gapdh). This protein was robustly identified in all samples on the basis of dozens of peptide-to-

spectrum matches (PSMs), yet was readily identified as a non-specific binder by its similar

enrichment (< 1.5-fold) in all samples, including in eluates derived from PrP knockout samples

(Supplementary Fig. S4.1). Of note, rather than manifesting as a hindrance, proteins in the non-

specific binder category served as a useful internal control in these analyses that further validated

the existence of comparable capture conditions across all samples. A closer look at the datasets

revealed one additional group of highly abundant proteins, whose enrichment behavior suggested

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them to be non-specific interactors. Like Gapdh, these proteins were observed in all samples, yet

in contrast to Gapdh, they exhibited inconsistent and, when encountered, only modest co-

enrichment with PrP. In this category fell a subset of ribosomal proteins, histones, tubulins and 14-

3-3 proteins. These proteins tended to exhibit the same trend in a given sample. For example, in

the second (wt2) versus third (wt3) biological replicate of the PrP interactome analysis from

NMuMG cells, most of these proteins were seen at levels that were consistently lower or higher,

respectively, than those observed in the corresponding PrP knockout control sample

(Supplementary Table S4.1). The distribution of these proteins suggested them to bind mostly non-

specifically to the affinity matrix but also indicated that their binding was more responsive to

sample-to-sample variations than the aforementioned bona fide non-specific interactors.

Based solely on their iTRAQ-enrichment characteristics, the 26 PrP candidate interactors could be

sorted into three categories (Table 4.1), namely:

I) Proteins observed in all or a subset of the four interactome analyses that exhibited consistent

and intermediate (1.5- to 7.5-fold) co-enrichment with PrP: This category encompassed the largest

number of candidate PrP interactors (18 proteins), including protein disulfide isomerase (P4hb),

galectin-1 (Lgals1), calreticulin (Calr), two gene products of the histocompatibility antigen gene

cluster (H2-K1 and H2-D1), transmembrane emp24 domain-containing protein family members

2, 9 and 10 (Tmed2, 9 and 10), the sodium/potassium-transporting ATPase (Atp1a1) and the

transferrin receptor protein 1 (Tfrc). The intermediate enrichment levels these proteins exhibited

suggested that they bound, in addition to PrP, to the affinity matrix or other proteins that were non-

specifically captured.

II) Proteins observed in a subset of samples that exhibited consistent and profound (> 7.5-fold) co-

enrichment with PrP (7 proteins): This category included the ZIP10 zinc transporter (Zip10), 4F2

cell-surface antigen heavy chain (Slc3a2), the large neutral amino acid transporter small subunit 1

(Slc7a5), basigin (Bsg), endothelin-converting enzyme 1 (Ece1), thrombospondin type-1 domain

containing protein 7A (Thsd7a), and insulin-like growth factor-binding protein 5 (Igfbp5).

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III) Proteins observed in all samples that exhibited consistent and profound co-enrichment with

PrP: The neural cell adhesion molecule 1 (NCAM1) was the only protein in this category.

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Table 4.1. Curated list of PrP candidate interactors organized by cell type (please see

Supplemental Table S4.1 for a complete account of shortlisted candidate interactors).

4.2.3 Cell type-specific effects of PrP knockout on the global proteome reflect its molecular interactions

We next analyzed cell type-specific differences of PrP candidate interactors (Fig. 4.3a), making

use of a coordinate system that captured if PrP interactors were observed in a given cell model and

also considered their cell model-specific levels of co-enrichment with PrP. Viewed in this manner,

it was apparent that not only the identity of PrP interactors but also their levels of enrichment were

most similar in N2a and CAD5 cells, with Calr, Atp1a1, NCAM1, Slc3a2 and Slc7a5 ending up

in proximity to a trend line that bisected the quadrant bounded by the coordinate axes assigned to

these cell models, a finding congruent with the shared neuronal origins of these cell models.

Often, next neighbor relationships of proteins are reflected in mutual effects on their expression

levels. We therefore were curious to understand if and how the presence or absence of PrP might

affect the steady-state expression levels of the proteins it is surrounded by in a given cell model.

To answer this question, we were able to lean on data we had collected in a previous study that

explored how PrP knockout affects the global proteome in an overlapping set of cell models (Fig.

4.3b) [100]. The data from this earlier work were based on three of the four cell models used in

this study (i.e., NMuMG, C2C12 and N2a cells, however, instead of CAD5 it had explored 1C11

neuroectodermal cells) and had led to the relative quantitation of >1,500 proteins, per cell model,

whose identification passed a 95% confidence threshold, including a dozen PrP candidate

interactors revealed in the current study. Naturally, we were particularly interested in the possible

influence of a PrP deficiency on the proteins we had assigned to categories II and III on account

of their PrP-specific co-enrichment. The steady-state levels of four of these proteins, namely

NCAM1, Slc3a2, Slc7a5, and Bsg, were quantified in all cell models on the basis of more than six

independent iTRAQ signature ion ratios (counts >6) (Fig. 4.3b). As reported above, Slc3a2, Slc7a5

and Bsg were observed to co-immunoprecipitate with PrP on the basis of robust quantitations

(counts >6) in N2a cells but not in NMuMG or C2C12 cells (Table 4.1). Interestingly, this cell

model-specific relationship was reflected in the global proteome data, which documented that the

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steady state levels of these proteins were pronouncedly perturbed in response to the PrP knockout

only in the N2a cell model, and were only mildly affected in NMuMG cells or C2C12 cells (Fig.

4.3b). This correlation was observed despite the fact that the steady-state levels of these proteins

across the cell models were similar. Interestingly, the direction of change observed in N2a cells

was not consistent, i.e., whereas Bsg steady-state levels increased in the absence of PrP, levels of

Slc3a2 and Slc7a5 were diminished. Taken together, this result corroborated the notion that the

presence of PrP has a modulating effect on the steady-state levels of proteins in its immediate

proximity

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Figure 4.3. The molecular environment of PrP is cell model-specific and comprises several

novel candidate interactors. (a) Graph depicting relative enrichment levels of candidate PrP

interactors by cell type. The x-coordinate of each protein is its average WT/PrP KO ratio in CAD5

minus its average WT/PrP KO ratio in C2C12. Each PrP interactor is represented by a cross, whose

position in the coordinate system is determined by its relative enrichment in the four cell models,

i.e., the y-coordinate represents the average WT/PrP KO ratios observed in N2a versus NMuMG

cell PrP interactomes. The average WT/PrP KO ratios used were normalized against the average

WT/PrP KO ratio for PrP in the same cell line. The cell lines in which a given protein was

quantified are indicated by shading in the corresponding cross arms, proteins identified in all cell

types are therefore represented by fully shaded crosses while proteins identified in only one cell

type are represented by crosses with one shaded arm. At least one protein was enriched to an

exceptional degree in each cell line. Eng was quantified only in the N2a interactome, and Igfbp5

was similarly quantified only in the C2C12 dataset, each protein being second only to PrP in its

level of enrichment. The figure demonstrates that the two neuron-like cell lines (N2a and CAD5)

share several PrP interactors which were only weakly detected or undetected in NMuMG or C2C12

datasets. Conversely Ece1 and Cd109 were highly co-enriched with PrP in NMuMG and C2C12

datasets but not N2a or CAD5 PrP interactomes. (b) Global proteome analyses of wild-type versus

PrP-deficient cells indicates that PrP depletion has reproducible effects on steady-state protein

levels of shortlisted PrP candidate interactors within a given model but leads to inconsistent

consequences of their relative abundance across cell models.

4.2.4 PrP selectively interacts with Ece1 and Tfrc dimers

In addition to several previously known PrP interactors (NCAM1, Slc3a2, Slc7a5, Bsg, P4hb,

Itgb1, Lgals1, Tmem206, Gpc1, Calr, H2-K1 and H2-D1) [47], about half of the candidate

interactors revealed in this work were not previously proposed to reside in immediate proximity

to PrP. Amongst these were a few proteins that highly selectively co-purified with PrP in a subset

of cell models, including Ece1, Thsd7a and Igfbp5. In particular, Ece1 stood out by a remarkable

127 count of quantified PSMs in PrP-specific immunoprecipitation eluates from NMuMG cells.

The protein was also detected, albeit to a lesser extent, in the PrP interactome derived from C2C12

cells but not from N2a or CAD5 cells.

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Ece1 is a Type II transmembrane protein that is best known for its endoproteolytic conversion of

inactive big endothelin-1 (big ET-1) to active endothelin-1 (ET-1) and belongs to the family of

zinc-dependent neprilysin-related endoproteinases. A western blot analysis of selected

formaldehyde crosslinked cell extracts detected the predominant monomeric Ece1 (isoform D)

signals at their expected apparent MW of near 120 kDa [104] and also revealed an SDS-resistant

dimer band (most likely stabilized by the crosslinking reagent) in NMuMG and C2C12 cells (Fig.

4.4a). Consistent with its absence from the N2a and CAD5 interactome datasets, the Ece1-directed

antibody picked up no signals for this protein in these cell models (see also Supplementary Fig.

S4.3). The box plot of quantified spectra assigned to Ece1 exhibited the expected PrP-dependent

enrichment of this protein in NMuMG- and C2C12-derived datasets (Fig. 4.4b). Strikingly,

western blot results of eluate fractions from the interactome analyses revealed PrP to have

exclusively co-immunoprecipitated the crosslinked Ece1 dimer (not the more abundant monomer)

(Fig. 4.4c). To validate this interpretation, we next boiled aliquots of the eluate fractions for up to

30 minutes in the presence of reducing agents (a method known to revert formaldehyde crosslinks)

[57]. As expected, this treatment reverted the crosslink, thereby leading to the appearance of the

Ece1 monomer band through an intermediate band, which we interpreted to constitute residual

levels of Ece1-monomer crosslinked to PrP (see red arrowhead in Fig. 4.4d). If PrP was indeed

crosslinked to dimeric Ece1 in the PrP affinity capture eluate fractions, we reasoned that it might

be possible to document this by separating Ece1-specific signals at higher resolution, next to Ece1

dimer bands seen in PrP knockout cell extracts. Indeed, this approach confirmed that the PrP

affinity-captured Ece1 dimer migrated at a higher MW than the Ece1 dimer seen in PrP knockout

cells, consistent with the interpretation that PrP had formed an SDS-resistant interaction with the

Ece1 dimer in this cell model (compare levels of black and green arrowheads in the right panel of

Fig. 4.4d).

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Figure 4.4. PrP interacts selectively with the Ece1 dimer, not its more abundant monomer.

(a) Ece1-specific western blot analysis of cellular extracts generated from wild-type and PrP

knockout cell models (each sample shown with two biological replicates). Cells were subjected to

mild formaldehyde crosslinking prior to their harvest. Arrowheads indicate signals derived from

monomeric and SDS-stable dimeric Ece1. Note that consistent with the identification of Ece1 as a

PrP interactor in NMuMG and C2C12 cells, but not in N2a cells, the protein is not observed in the

latter cell model. The bottom panel depicts a Coomassie stain of the western blot membrane. (b)

Box plot depicting relative quantitation of Ece1 in PrP interactome datasets of in vivo

formaldehyde crosslinked wild-type and PrP KO NMuMG and C2C12 cells. Please see legend to

Fig. 4.2b for a detailed description of graph elements. (c) PrP co-immunoprecipitation led to the

co-enrichment of a slow migrating Ece1 antibody-reactive band in wild-type (but not in PrP

knockout cell) eluates, suggestive of a selective interaction of PrP with the Ece1 dimer. The

asterisk denotes a non-specific cross-reactivity of the antibody, most likely toward a component

of the affinity matrix that is indicated with an empty arrowhead in the Coomassie stain depicted in

the bottom panel. (d) Formaldehyde crosslink reversal treatment (90 °C, for 10-30 minutes) causes

high mass Ece1 antibody-reactive band to shift to faster gel migration at level of monomeric Ece1.

Note that the panels on the left (Lanes 1-3) and right (Lanes 4-7) were generated with SDS-PAGE

gel systems of different resolution. Portions of the Ece1 western blots that showed no signals were

cropped at the bottom and the corresponding Coomassie stains in this panel were trimmed

accordingly. Arrowheads provide signal interpretation as follows: black, Ece1 dimer-PrP complex;

green, Ece1 dimer; red, Ece1 monomer-PrP complex; blue, Ece1 monomer.

The transferrin receptor protein 1 (Tfrc) is another protein that according to the mass spectrometry

data presented in this study (Table 4.1) resides in immediate proximity to PrP only in the NMuMG

model. In contrast to Ece1, Tfrc-directed immunoblot analyses revealed robust steady-state

expression levels of this protein in cell lysates of all four models tested. Yet, an intense Tfrc

immunoblot signal was only observed in PrP co-immunoprecipitation eluates from wild-type

NMuMG cells (Supplementary Fig. S4.4). However, weak Tfrc signals were also observed in PrP-

specific eluates from wild-type C2C12 and N2a cells, presumably reflecting a slightly higher

sensitivity of western blot analysis over mass spectrometry-based detection for this protein.

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Taken together, the Ece1 and Tfrc validation experiments indicated that the absence of a PrP-

interacting protein in a cell model-specific interactome data set may reflect dramatic differences

in steady-state levels (Ece1) or more subtle differences in the cellular expression of a PrP binder

(Tfrc) that push the amounts of a given protein residing in immediate proximity to PrP below the

level necessary for its detection.

4.2.5 Candidate PrP interactors exist in the secretory pathway or at the cellular membrane

One other way to validate interactome data is to assess the known or predicted cellular localization

of proposed binders of a protein-of-interest. In light of the known, predominant association of PrP

with raft-like domains at the plasma membrane [105], PrP would be expected to encounter other

proteins during its biogenesis in the secretory pathway and at the plasma membrane. A UniProt-

based survey of cellular sites predicted to accommodate the identified PrP interactors established

all 26 candidates as known or predicted residents of the membrane or lumen (Fig. 4.5), a finding

consistent with the initial GO ‘Cellular Component’ enrichment analysis (Fig. 4.2c). Five of the

candidate interactors (Calr, P4hb, as well as Tmed 2, 9 and 10) are predominantly found in the ER

or Golgi, where they play critical roles for quality control and transport. Two, namely H1-K1 and

H1-D1, play roles in antigen presentation and are therefore, context-dependently found in the

Golgi or at the plasma membrane. Amongst the remaining 19 proteins, there are three secreted

proteins, six Type-I, three Type-II and five Type III transmembrane proteins, as well as two GPI-

anchored proteins. Of the latter, glypican-1 (Gpc1) is a well-known PrP interactor, that has been

shown to promote both PrP’s association with detergent-resistant raft domains and its conversion

to PrPSc [106–108], but CD109 (Supplementary Fig. S4.2) was to our knowledge not previously

reported to reside in proximity to PrP.

One approach to learn about the function of a protein-of-interest is to deduce it from the known

functions of the proteins it partners with. A survey of known key functions of the 19 plasma

membrane-associated PrP candidate interactors revealed their involvement in a multitude of

biological activities, including cell adhesion, zinc import, endothelin-1 cleavage, transport of

neutral amino acids, insulin-growth factor signaling, angiogenesis and iron uptake. Strikingly

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56

however, most of these proteins are known to also play critical roles (see Discussion for details)

in either TGFβ1- or integrin-signaling (Fig. 4.5b).

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57

b

Symbol DescriptionTopology /

gnilangis nirgetni dna β-FGT ot kniLnoitcnuFnoitazilacol

Ncam1 Neural cell adhesion molecule 1 (CD56) Type I TM (GPI) Cell TME gnirud ylbmessa noisehda lacof slortnoCnoisehda

eht otni tropmi noi cniZMT 3 epyT01PIZ retropsnart cniZ01a93clS cytosol Binds Ncam1 and controls its Gsk3-dependent phosphorylation.

id lanimret-C sesaeleRMT 2 epyTemyzne gnitrevnoc-nisnetoignAecA peptide of its substrates Releases GPI-anchored proteins by cleaving mannose linkage

Ece1 Endothelin-converting enzyme 1 Type 2 TM Converts big endothelin-1 to endothelin-1 TGF-β regulates endothelin-1 signaling

Slc3a2 4F2 cell-surface antigen heavy chain (CD98 hc) Type 2 TM Transports large neutral amino acids Binds to and modulates integrin activity

Slc7a5 Large neutral amino acids transporter small subunit 1 (CD98 lc)

Type 3 TM Transports large neutral amino acids Binds to and modulates integrin activity

Thsd7a Thrombospondin type-1 domain-containing protein 7A Type 1 TM Promotes cell migration via FAK-mediated signaling.

Co-localization with integrin complexes in focal adhesions

snoitcaretni xirtam llec setaideMdeterceS1-nidnopsobomorhT1sbhT Binds integrin beta-1, TGF-β, CD47 and heparin.

oisehda llec ni eloRMT 3 epyT74DC negitna ecafrus etycokueL74dC n, memory formation and synaptic plasticity

Integrin co-receptor that interacts with thrombospondin-1

Igfbp5 Insulin-like growth factor-binding protein 5 Secreted Prolongs half-life of insulin growth factor Localizes to and activates β1-containing integrin complexes

hto dna edisotcalag-ateb gnidnib nitceLdeterceS1-nitcelaG1slagL er carbohydrates

Binds integrins and activates focal adhesion complex signaling

ytilitom ,noisehda llec setalugeRMT 1 epyT1-ateb nirgetnI1bgtI and angiogenesis

Controls TGF-β release from LAP complexes

Tmem206 nwonknUnwonknUMT 3 epyT602 nietorp enarbmemsnarT

Gpc1 Glypican-1 GPI Cell surface proteoglycan that bears heparan sulfate

Involved in Wnt and TGF-β receptor family signaling

Atp1a1 Sodium/potassium-transporting ATPase subunit alpha-1 Type 3 TM Exchange of Na+ and K+ across plasma membrane

Inhibition causes downregulation of TGF-β signaling

Cd109 CD109 antigen GPI Modulates TGF-β receptor signaling Modulates TGF-β receptor signaling negatively

ni ,rotpecer-oc β-FGTsisenegoigna setalugeRMT 1 epyTnilgodnEgnE teracts with integrins

sretropsnart etalyxobraconom stegraTMT 1 epyT)741DC( nigisaBgsB to the plasma membrane

Clusters with CD98 and integrin beta-1

ekatpu nori ralulleCMT 2 epyT1 nietorp rotpecer nirrefsnarTcrfT Facilitates TGF-β receptor family signaling signaling

Figure 4.5a

Eng

C

N

Orp

han

ZP-N

ZP-C

CD

CD9 8

N C

N

-SS-

Lat1

4f2hc

C

Gpc1N

sAceN

C

Tfrc

N

C

Prot

ease

-like

Api

cal

Hel

ical

Tmem20 6C

N

Extracellular

Intracellular

Igfbp5

NCTG1

C

N

Atp1a1

S-S

N

PrP C

C

N

S-S

Zip6

C

N

S-S

Zip10

Cd10 9

Thsd7 aN

TSP1

C

TSP1

TSP1

TSP1

TSP1

TSP1

TSP1

TSP1

TSP1

TSP1

TSP1

Lgal1

N

NC

C

CBD

CBD

Cd47

N

C

S-S

C

N

Bsg (Cd147 )

S-S

S-S

S-S IG-like domain C2-type

S-S IG-like domain V-type

GPI anchor

Heparan sulfate (GAG)

N-linked glycosylation

IGFBP-N

Ncam1

S-S

S-S

S-S

S-S

S-S

N

C

FN 3

FN 3

Ece1

N

C

C

Itgb1

βTD

I-EGF3

I-EGF4

I-EGF2

I-EGF1PSI

Hybrid

βI domain

N

N

A2Mr

A2M

A2M

A2M

A2M

C

S-S IG-like domain C1-type

N

S-S

C

α1

α2

H2K1

N CTSP1

TSP3

TSP3

TSP3

TSP3

TSP3

TSP3

TSP3

Thbs1

EGF-

like

EGF-

like

EGF-

like

TSP1

TSP1 TSP CLaminin G-like

VWFC

C C

Pept

idas

e M

13A

ctiv

e si

te

Pept

idas

e M

2A

ctiv

e si

tePe

ptid

ase

M2

Act

ive

site

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Figure 4.5. PrP’s molecular environment is enriched for proteins with known roles in TGFβ1

and integrin signaling. (a) Cartoon depicting domain organization, as well as known or predicted

mode of membrane association of PrP interactors based on UniProt annotations. (b) Functional

annotations of shortlisted PrP interactors.

4.2.6 TGFβ1 profoundly affects steady-state levels of several PrP interactors but depletion of PrP only reduces NCAM1

TGFβ1 had previously come to the fore in relation to PrP, because this cytokine is understood to

be a major inducer of EMT, which leads cells to acquire mesenchymal morphology and to shift

their mode of attachment to focal adhesion complex-based cell-matrix contacts. A link to EMT

was initially suggested by the aforementioned gastrulation arrest phenotype observed in zebra fish

deficient for a PrP ortholog and was recently strengthened, when we observed mammalian PrP to

be dramatically induced during EMT [4,58]. Here, we were interested in how the execution of this

morphogenetic program affects steady-state levels of PrP candidate interactors and in learning to

what extent PrP depletion influences changes in their steady-state levels. To address these

questions, we capitalized on our access to previously reported global proteome data [4] of wild-

type NMuMG cell extracts collected two days following mock- or TGFβ1-treatment (dataset I). A

2nd dataset which compared the global proteomes of TGFβ1-treated wild-type cells versus TGFβ1-

treated PrP-deficient cells (dataset II) [4] could be harnessed to elucidate the effect of PrP on its

nearest neighbors (Fig. 4.6a).

Of the 26 PrP candidate interactors revealed in the current study, 14 had been quantified in dataset

I on the basis of more than three PSMs with associated iTRAQ reporter ion ratios. A closer look

at the relative quantitation results led us to observe that except for Ece1, Cd109 and Tmem206

(Supplementary Fig. S4.2), all NMuMG-cell based PrP-candidate interactors had been quantified

in the global proteome analyses (datasets I and II), revealing a striking dichotomy: Whereas the

steady state levels of PrP candidate interactors with predominant plasma membrane localization

were affected by the TGFβ1 exposure of cells, levels of the five PrP candidate interactors with

predominant residence in the secretory pathway were not (Fig. 4.6b). Even more strikingly, the

orientation of change, i.e., whether TGFβ1 addition resulted in an increase or decrease of a given

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interactor, was profoundly divergent, with NCAM1, Itgb1, Lgals1 and Tfrc levels increased upon

TGFβ1 exposure but levels of Cd47, Atp1a1 and subunits of the large neural amino acid transporter

diminished upon exposure. When comparing PrP-depleted versus wild-type cells (dataset II), a

selective reduction in NCAM1 steady-state levels was observed in contrast to the other PrP

candidate interactors which showed no steady state changes. The results from these global

proteome analyses speak to an intricate level of re-organization of the microenvironment of PrP

during EMT (as opposed to mass effects influencing next neighbors in the same way) but also

established that PrP depletion during this cellular program, except for its destabilizing effect on

NCAM1 steady-state levels, has surprisingly little influence on the levels of other proteins it is

surrounded by.

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Figure 4.6. Whereas TGFβ1 treatment causes divergent shifts in steady-state levels of a

subset of PrP interactors, PrP-depletion in the same paradigm only affected NCAM1 protein

levels. (a) Schematic depicting nature of global proteome datasets mined for this study. A more

complete presentation of these datasets had been published before17. (b) Comparison of steady-

Figure 4.6

b

a

wt (without TGFB1)

wt + TGFB1

dataset I

PrP kdstable

+ TGFB1

dataset II

epithelial

mesenchymal

-TGFB1 -TGFB1 -TGFB1 PrP kd1 PrP kd2 PrP kd33twtnuoC1BFGT+1BFGT+1BFGT+egarevoCnoitpircsed deifidoMnoisseccA wt3 wt3 Count

IPI00230665.3 Neural cell adhesion molecule 1 isoform 3 73.27% 0.367 0.353 0.378 28 0.762 0.757 0.751 57.0400.1220.102416.0766.0726.0%65.571-ateb nirgetnI3.47423100IPI 993 27.1700.1989.03617.0236.0895.0%55.761-nidnopsobmorhT2.31481100IPI 000 10

14651.1331.1740.153697.0208.0628.0%03.691-nitcelaG5.71592200IPI69.0169.0098.0%13.471 nietorp rotpecer nirrefsnarT1.00742100IPI 8 37 0.956 0.962 0.895 3680.1101.1961.1%60.1674DC negitna ecafrus etycokueL4.97030400IPI 2 12 1.051 0.967 1.018 13

IPI00311682.5 Sodium/potassium-transporting ATPase subunit alpha-1 61.68% 1.314 1.358 1.350 89 0.926 0.929 0.939 76IPI00930882.1 4F2 cell-surface antigen heavy chain isoform a 76.46% 1.182 1.209 1.177 23 1.027 0.968 1.063 22IPI00129395.2 Large neutral amino acids transporter small subunit 1 42.58% 1.514 1.438 1.425 11 1.121 1.023 1.165 9

28809.0539.0929.0%68.69esaremosi-ediflusid nietorP2.22533100IPI 0.883 0.898 0.914 104879.0259.0539.025469.0169.0789.0%23.37niluciterlaC1.93632100IPI 34

IPI00127983.1 Transmembrane emp24 domain-containing protein 2 71.14% 0.890 0.867 0.904 13 0.939 0.981 1.010 12IPI00473680.2 Transmembrane emp24 domain-containing protein 9 83.64% 0.944 1.000 0.885 15 0.893 0.980 1.055 16IPI00466570.4 Transmembrane emp24 domain-containing protein 10 62.10% 0.980 0.943 0.934 12 1.004 0.893 0.985 25

Global proteome data / NMuMG cells

dataset I dataset II

Heat map color codemesenchymal

wt cellsepithelial wt cells

mesenchymalstable PrP kd cells

wt

PrP kd +TGFB1

wt + TGFB1

wt + TGFB1

proteins with relatively high levels of expression in:

mesenchymalwt cells

dataset I

dataset II

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state protein levels of a subset of PrP interactors before and after TGFβ1 induced mesenchymal

differentiation. The steady-state levels of proteins with predominant localization in the ER / Golgi

compartments did not change upon two-day exposure of NMuMG cells to TGFβ1 (bottom half of

table). However, proteins PrP is expected to be surrounded by at the plasma membrane underwent

divergent changes in their steady-state levels. The stable PrP knockdown did not affect the protein

levels of PrP candidate interactors, except for NCAM1, whose levels were diminished in PrP-

deficient cells. Please see Supplementary Table S2 for a more complete presentation of underlying

quantitations.

4.3 Discussion We set out to investigate the causes for the notorious lack of overlap in the primary literature

concerned with the identification of PrP interactors. To this end, we made use of four mouse cell

models for which PrP knockout control lines were available, using identical tools and workflows

to capture endogenous PrP and its co-purifying partners. The study uncovered 26 proteins that

exhibit selective or partial co-enrichment with PrP. All of these PrP candidate interactors were

known or predicted to reside in cellular locations PrP is understood to have access to, and about

half of them had no previous occurrence in the PrP literature, including the metalloprotease Ece1

and the GPI-anchored molecule Cd109. Except for NCAM1, we observed none of the most co-

enriched proteins in proximity to PrP in all cell models. Cumulatively, our results provide a

powerful testament to the conclusion that the molecular environment of PrP is to a considerable

degree cell type-specific. Yet, the data also establish that across all four cell models PrP is

embedded in a specialized molecular environment that appears to be tasked with governing the

crosstalk of TGFβ1 and integrin signaling.

To the best of our knowledge, this is the first report that systematically compares the molecular

environment of PrP in more than one cell model. A conceptual advance in this study, relative to

earlier reports, including our own work [47], was the targeting of endogenous PrP—as opposed to

affinity-tagged or overexpressed protein—combined with the harnessing of PrP knockout models

for the generation of negative control samples. On the basis of comparable numbers of PSMs (a

parameter known to correlate with protein quantity) assigned to PrP, as well as comparable PrP

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sequence coverages of biological replicates within and across the four interactome datasets, it can

be concluded that similar amounts of this bait protein were present in the capture eluates of all

cell-type specific interactome analyses. It is therefore remarkable that the levels of protein subsets

which co-purified specifically with PrP differed dramatically across the cell models. A striking

example of a protein that exhibited uneven co-enrichment with PrP in a cell-type specific manner

was Ece1, which was not at all observed in N2a- or CAD5-based PrP interactome datasets, yet was

identified and quantified on the basis of >100 PSMs and a cumulative 63.89% sequence coverage

in the NMuMG cell model. We documented that this high level of Ece1 enrichment in the NMuMG

cell model was not the consequence of an unusually high level of expression in this model, as Ece1

remained below the level of detection in our global NMuMG cell proteome analyses, and as we

observed similar steady-state levels for this protein in the C2C12 model by western blot analysis.

Results from additional validation experiments were consistent with the interpretation that PrP was

directly crosslinked to an Ece1 dimer but did not reveal reciprocal influences of Ece1 or PrP on

their maturation or steady-state expression levels. Although this example validated the conceptual

choice to investigate the PrP interactome in more than one model, it also exemplified the limits of

conclusions that can be drawn from this study in regards to 1) previously reported PrP candidate

interactors, not observed in this work, whose identification was based on other cell models or

paradigms, and 2) the completeness of the current list of PrP interactors. Whenever prior reports

of PrP interactors in the same paradigm were available, our current data corroborated and

complemented their findings. For instance, a prior report, which documented interactors of FLAG-

tagged PrP in formaldehyde crosslinked N2a cells6, had—with the exception of endoglin (Eng)—

identified all of the PrP interactors observed in the N2a cell model in the current study. The

technical improvements underlying the current study, however, helped to flag a subset of

previously reported PrP candidate interactors, including several 14-3-3 proteins, tubulin and

ribosomal subunits, as most likely representing non-specific interactors.

In light of the striking differences in the molecular environments of PrP in the four cell models

employed in this study, it warrants repeating that NCAM1 stood out by being prominent in all PrP

interactome datasets. This unique relationship of PrP and NCAM1 was further emphasized by our

observation that PrP deficiency in TGFβ1 exposed NMuMG cells had no effect on the steady-state

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levels of the proteins found in PrP’s molecular environment, except for causing a relative depletion

of NCAM1 levels. The increasingly special status of Ncam1 amongst PrP interactors corroborates

previous observations: 1) formaldehyde crosslinking gives rise to SDS-stable NCAM1-PrP

complexes that represent a majority of the high molecular mass PrP-crosslink signals in N2a cells

[57]; 2) PrP controls NCAM1 polysialylation [4]; and 3) a recent interactome analysis of the

closest PrP relatives amongst the ZIP transporters that documented a selective interaction with

NCAM1 (and calreticulin) but not with other PrP interactors [87].

Despite the distinctness of PrP interactome data in the four cell models, a Molecular Function GO

term analysis pointed toward an overarching similarity in the molecular environment of PrP,

apparently populated by proteins with known roles in TGFβ1 and integrin signaling. A closer look

at the primary reports underlying the relevant functional annotations, as well as the broader

literature, further strengthened this conclusion. For some PrP interactors, the connection to the

biology of TGFβ1 and integrin signaling seems less than obvious at first. For example, Tfrc is best

known as a cellular importer of transferrin-bound iron. However, robust evidence has also linked

this transporter to TGFβ1 signaling (a finding that is not yet reflected in its current GO

annotations). More specifically, the neural crest cell-specific knockout of Tfrc in mice caused

craniofacial abnormalities, including cleft palate and micrognathia (reduced mandible), and was

associated with a suppression of TGFβ1 signaling [109]. The characteristic craniofacial

abnormalities observed are pathognomonic of a disease known as Pierre Robin Sequence (PRS)

caused by mutations in genes that impair TGFβ1 superfamily signaling. Moreover, Cd109 is a

major negative inhibitor of TGFβ1 signaling [110] and Eng is a TGFβ1 co-receptor [111]. Notably,

although these proteins share unequivocal and direct links to TGFβ1 signaling, this study did not

observe their co-enrichment with PrP in the same cell model. Rather, Cd109 was detected in

NMuMG- and C2C12-derived PrP interactome datasets, and endoglin was only seen in proximity

to PrP in N2a cells.

Similarly, the Cd98 complex, composed of 4f2 (Slc3a2) and Lat1 (Slc7a5) subunits, is best known

for its transport of large neutral amino acids. However, the primary literature and several reviews

have drawn attention to the direct involvement of this complex in integrin signaling [112–114].

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Cd98 exerts this involvement in part by its direct interaction with Itgb1, which is an integral

component of integrin-based focal adhesion complexes and was observed in the N2a interactome

dataset in this study. Additional PrP candidate interactors with connections to integrin biology

were Cd47, which acts as an integrin co-receptor [115] and was named integrin-associated protein

[116] after its discovery as a contaminant in integrin preparations, and Lgals1, which is known to

bind to and activate integrin-dependent signaling of focal adhesion complexes [117–119]. As with

TGFβ1-associated proteins, these integrin interactors were not all identified in the same PrP

interactome dataset, but were revealed in different combinations in one or more of the cell-type

specific datasets we presented. Moreover, it is increasingly understood that TGFβ1 and integrin

signaling activities do not exist irrespective of one another. Rather, these signaling portals are

considered sister complexes bound to each other through several connections. In fact, a subset of

proteins shortlisted in this study as PrP interactors, including Cd98 and Thbs1, are known to

facilitate this crosstalk [120,121].

How does PrP influence TGFβ1 and integrin signaling complexes? By its mere presence, PrP

would be expected to influence the composition and architecture of its molecular environment in

many subtle ways. As PrP is a GPI-anchored molecule with affinity for membrane domains

enriched in cholesterol and sphingolipids, it seems plausible that one of these influences will relate

to its effect on the balance between clathrin-mediated endocytosis and caveolae-based transport.

Such a balance shift is unlikely to be innocuous. For example, TGFβ1 signaling is dependent on

clathrin-mediated endocytosis (references in [109]), and it has been proposed that CD109 and Tfrc

influence the canonical signaling of TGFβ1 in such manner [109,110]. A similar dichotomy of

internalization pathways also exists for integrin complexes and Lgals1 has been proposed to

influence their trafficking [119]. A more direct influence of PrP on the regulation of focal adhesion

complexes may derive from its interaction with NCAM1 and its dramatic influence on NCAM1

polysialylation [4]. Although the details of how NCAM1 promotes the establishment of cell-to-

matrix contacts based on focal adhesions have remained murky, and are likely to be complex, there

is ample evidence for a TGFβ1-dependent expression of NCAM1 [122] and a critical role of this

protein in the molecular rearrangements that govern this cellular program [59,123,124].

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4.4 Conclusion There is a tendency to ascribe differences in interactions of a protein-of-interest reported by

separate investigators mainly to differences in methodology, even when studies are undertaken

with different paradigms. Insights gained in this work caution that the molecular environment of a

given protein-of-interest can be surprisingly diverse when comparing distinct models. The

comparative interactome data we presented placed the evolutionarily conserved interaction

between PrP and NCAM1 in the context of a plasma membrane microdomain tasked with

modulating the crosstalk between TGFβ1 and integrin signaling. We anticipate that further

investigations will substantiate roles of PrP in cellular programs relying on these sister signaling

hubs. The results from this study further suggest that, unless PrP interactome studies are

undertaken with brain cells, next-neighbor relationships central to the cellular toxicity that

manifests in prion disorders may be overlooked.

4.5 Methods

4.5.1 Western blot analyses

Equal amounts of protein were separated on 4–12% Bis-Tris gels (Life Technologies, ON, Canada)

and transferred to a 0.45 micron polyvinylidene fluoride membrane. The membranes were blocked

with 10% skim milk in Tris-buffered saline with Tween 20 (TBST), and incubated overnight at

4°C with the respective primary antibody. Subsequently, the western blots were washed thrice with

TBST and incubated with HRP-conjugated anti-mouse (catalog number 1706516; BioRad,

Mississauga, ON, Canada), anti-rabbit (catalog number 1706515; BioRad), or anti-rat (31476;

Thermo Fisher Scientific, Waltham, MA, USA) secondary antibodies for two hours at room

temperature. Membranes were subjected to three washes with TBST and incubated with the ECL

reagent (catalog number RPN2106; Sigma Aldrich, Oakville, ON, Canada). Signals were then

visualized using either X-ray film or a LI-COR Odyssey Fc digital imaging system (LI-COR

Biosciences, Lincoln, NE, USA). Immunoblotting was undertaken with the Sha31 antibody against

PrP (catalog number A03213; Bertin Bioreagent, Montigny le Bretonneux, France), or antibodies

directed toward Ece1 (catalog number ab71829; Abcam, Cambridge, United Kingdom) or Tfrc

(catalog number ab84036; Abcam).

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4.5.2 Cell culture and transfection

Mouse mammary gland NMuMG cells (catalog number CRL-1636; American Type Culture

Collection (ATCC), Manassas, VA, USA) and Cath.a-differentiated (CAD-2A2D5; CAD5 for

short) cells were a kind gift from Dr. Jeffrey Wrana (University of Toronto, Toronto, ON, Canada)

and Dr. Charles Weissmann (The Scripps Research Institute, Jupiter, FL, USA), respectively.

Mouse myoblast (C2C12) cells (catalog number CRL-1772) and mouse neuroblastoma Neuro-2a

(N2a) cells (catalog number CCL-131) were purchased from ATCC. Cells were cultured in

D(MEM) or opti-MEM supplemented with 10% heat inactivated fetal bovine serum (catalog

number 12484028; Invitrogen Canada, Burlington, ON, Canada), 1% GlutaMAX (catalog number

35050061; Invitrogen Canada), and 1% antibiotic-antimycotic solution (catalog number

15240062; Invitrogen Canada). Human insulin solution (catalog number I9278; Sigma-Aldrich,

Oakville, ON, Canada) was added at a concentration of 10 µg/mL for NMuMG cells.

To induce EMT, NMuMG cells were treated with TGFβ1 (catalog number 240-B; R&D Systems,

Minneapolis, MN, USA) at a concentration of 6.4 ng/mL daily for 48 hours. C2C12 myoblasts

were differentiated to myotubes by replacing the DMEM containing 10% FBS with medium

supplemented with 2% horse serum.

The knockout of Prnp in NMuMG, C2C12, and N2a cells had been achieved using CRISPR-Cas9

genetic engineering and was described previously [74]. The CAD5 PrP knockout line was made

with identical sgRNA reagents as the other PrP knockout lines but had not been previously

described.

4.5.3 Sample preparation for immunoprecipitation analyses

Comparative interactome analyses were performed with three biological replicates for each

condition (unless indicated otherwise), that were side-by-side expanded to large scales (average

yield of 6 x 107 cells). Cells were washed with ice-cold PBS, and mildly crosslinked with 2%

formaldehyde in PBS for 15 minutes at room temperature. Subsequently, residual crosslinking

reagent was quenched during a 10-minute incubation with 125 mM glycine in PBS and lysed in

ice-cold buffer consisting of 150 mM Tris (pH 8.3), 150 mM NaCl, 0.5% NP-40, 0.5% sodium

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deoxycholate supplemented with protease inhibitor cocktail (catalog number 11836170001;

Roche, Mississauga, ON, Canada) and phosphatase inhibitor tablet (catalog number 4906837001;

Roche). Cellular debris were removed by centrifugation at 2,000 RPM for 5 minutes, followed by

30 minutes at 4,000 RPM (4 °C). Centrifugation supernatants were collected and protein levels

were adjusted using a bicinchoninic acid colorimetric assay reagents A (catalog number 23228)

and B (catalog number 1859078) from Thermo Fisher Scientific.

4.5.4 Protein immunoprecipitation workflow

PrP was captured using the D18 antibody, a humanized recombinant Fab developed against PrP

that was provided by the laboratory of Dr. Emil F. Pai. The PrP antibody was conjugated to

KappaSelect beads (catalog number 17-5458-01; GE Healthcare, Oakville, ON, Canada) under

gentle agitation by a turning wheel at 4°C overnight. The affinity capture bead / antibody mixture

was then equally aliquoted for individual samples and adjusted protein samples were added for an

overnight capture at 4°C. The next day, the affinity matrices were stringently washed twice with

lysis buffer and twice with lysis buffer containing 500mM NaCl to remove non-specific binders,

followed by a pre-elution wash of 10mM HEPES, pH 8. Proteins were then eluted by acidification

in 0.2% trifluoroacetic acid, 20% acetonitrile.

4.5.5 Nanoscale HPLC-ESI tandem mass spectrometry

Sample preparation for mass spectrometry was done as described previously [74].

Immunoprecipitation eluates were dried under vacuum then diluted in 9 M deionized urea.

Reduction with tris (2-carboxyethyl) phosphine at 60°C was followed by room temperature

sulfhydryl group alkylation with 4-vinylpyridine. The urea concentration was lowered to 1.25 M

in 500 mM triethylammonium bicarbonate prior to the addition of mass spectrometry-grade trypsin

(catalog number 90057; Thermo Fisher Scientific). Digestion occurred at 37°C overnight. Trypsin

treated samples were covalently modified with 8-plex isobaric tags for relative and absolute

quantitation (iTRAQ) (catalog number 4390811; Sciex, Concord, ON, Canada) according to the

manufacturer’s protocol then mixed. Sample mixtures were purified on reversed phase resin in

Bond Elut OMIX cartridges (catalog number A57003100; Agilent Technologies, Santa Clara, CA,

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USA) alone and in combination with strong cation exchange cartridges (catalog number

A57004100; Agilent Technologies).

All sample mixtures were analyzed over a four-hour reversed phase 300 nL/min gradient on an

EASY-nLC 1000-Orbitrap Fusion Tribrid mass spectrometer platform (Thermo Fisher Scientific).

The analytical column was a 25 cm long Acclaim PepMap RSLC 100 of 75 µm inner diameter

with 2 µm C18 particles having 100 Å pores. Each liquid chromatography-mass spectrometry run

was divided into scan cycles up to 3 seconds long, each including one orbitrap precursor ion MS

scan and as many linear ion trap product ion (MS2) scans and orbitrap MS3 scans as possible

within the 3 second time window. CID and higher energy collisional dissociation (HCD) were

used for MS2 and MS3 respectively. The Orbitrap resolution was set to 60,000 for both MS and

MS3.

4.5.6 Protein identification and quantification

MS2 data were converted to protein sequence information with Proteome Discoverer (version

1.4.0.288; Thermo Fisher Scientific) using the embedded Mascot and Sequest HT search

algorithms with the mouse international protein index database (version 3.87). Up to two missed

cleavages were allowed per peptide. The allowed peptide mass range was 400-6000 Da, with a

precursor ion mass tolerance of 20 ppm and product ion mass tolerance of 0.4 Da. Variable

modifications considered were asparagine and glutamine deamidation, methionine oxidation as

well as serine, threonine and tyrosine phosphorylation. Cysteine pyridylethylation as well as

iTRAQ 8-plex reagent labeling of peptide N-termini and lysines were defined as fixed

modifications. False discovery rate estimation based on q-Value was performed with the Percolator

algorithm. Relative protein quantification was produced from MS3 data by the Reporter Ions

Quantifier built into Proteome Discoverer with the most confident centroid under a mass tolerance

of 20 ppm.

4.5.7 Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium

via the PRIDE partner repository [125] with the dataset identifier PXD008781.

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Chapter 5

Conclusion and Future Directions Following up on the discovery of an ancient ZIP zinc transporter as the evolutionary ancestor of

PrP [49], and the characterization of shared functional and structural similarities [50], an

interactome study of ZIP6 implicated ZIPs in a signaling pathway that regulates the post-

translational modification of NCAM1 by serving as a hub for GSK3B [87]. Specifically, we

established that ZIP6 and ZIP10 form a heteromer that binds to GSK3B kinase and controls the

phosphorylation of the longest isoform of NCAM1 [87]. The ZIP protein family plays a role in

regulating the cellular zinc homeostasis and, more precisely, is involved in the transport of zinc

(and perhaps other divalent cations) into the cell [51]. Intriguingly, cellular zinc levels have

previously been shown to directly affect GSK3 kinase activity, i.e., the presence of free zinc was

shown to inhibit GSK3 phosphorylation activity [88]. This zinc-mediated inhibition is reported to

be specific to GSK3 since its closely related kinase CDK2 does not share this property [88].

Furthermore, the inhibition of GSK3 is solely zinc-dependent and does not extend to other divalent

cations [88]. Moreover, previous reports have singled out GSK3 as one of very few kinases whose

activity might be influenced by zinc [88]. Based on our interactome data, the cellular location at

which ZIP6- and ZIP10-mediated zinc influx occurs is in close proximity to where GSK3 paralogs

are located, presumably leading to a micro-environment characterized by free zinc levels that may

dramatically exceed the normally very low pico- to femtomolar cytoplasmic free zinc levels [87].

These findings could point to a regulatory mechanism in which the ZIP6-ZIP10 heteromer acts as

a regulator of GSK3 kinase activity by controlling free zinc levels in immediate proximity to the

kinase [87]. Thus, cells may have harnessed the ZIP metal ion transport ability to control specific

GSK3-dependent substrate phosphorylation events and, by extension, the downstream signaling

that depends on them. Although the functional relevance of the ZIP6/10 mediated, GSK3-

dependent phosphorylation of NCAM1 is not yet known, there is ample precedent for single

phosphorylation events modulating critical protein interactions. Interestingly, NCAM1’s ability to

bind to a number of proteins, including integrins and 14-3-3 proteins, the latter being notoriously

sequestered to phosphorylated interactors, was hindered in ZIP6-deficient cells [87]. It is therefore

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plausible that the GSK3-dependent phospho-occupancy of NCAM1 could constitute a molecular

switch by which ZIP proteins impact focal adhesion assemblies [87]. Thus, this angle of my work

has generated a specific testable hypothesis. Although emerging from a study undertaken in a

broad neurodegenerative disease research context, this type of focal adhesion biology is of broad

interest in cancer research, where metastatic cells switch from cell-to-cell based adherence to cell-

substrate-based adhesion [56,59]. Several avenues could be pursued to take this research to the

next level. Follow-on experimentation could, for example, as a first step employ an in vitro assay

format (similar to the one described in Chapter 3), to determine if recombinant 14-3-3 proteins can

be co-immunoprecipitated with recombinant or affinity-purified NCAM1 that had been

phosphorylated with recombinant GSK3B in the presence or absence of GSK3 specific inhibitors.

An analogous experiment could be employed to clarify if phosphorylation of NCAM1 is strictly

dependent on GSK3B or can also be mediated by GSK3A, a question our current data did not

explicitly answer, although we observed both paralogs of this kinase in the ZIP6 interactome

dataset.

The study was the first to investigate the relationship between ZIPs and NCAM1, highlighting that

NCAM1 is the key interactor of the ZIP6-ZIP10 heteromeric complex [87]. Considering the

evolutionary link between PrPC and ZIPs [49] and the fact that NCAM1 was already established

to be a prominent molecular interactor of PrPC [4,47,57,60], it is in hindsight not surprising for

NCAM1 biology to also be closely associated with ZIPs. The direct interaction of NCAM1 and

PrP has been documented in a number of reports using different cellular and experimental

paradigms [4,47,57,60]. Previous work by our group [4] had uncovered one physiological

relevance of the interaction between PrPC and NCAM1 by demonstrating that PrPC controls

NCAM1 polysialylation. A surprising finding of this line of work was the differential regulatory

influence of PrPC on NCAM1 polysialylation in different cellular paradigms [4].

Numerous studies have examined the molecular environment of PrPC with the intent to deduce the

biological function of this protein [47,57,95–98]. However, the use of inconsistent cell models and

experimental protocols in these analyses caused different investigators to emphasize the

importance of distinct PrPC interactors, leading to an abundance of hypotheses on the physiological

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role of this protein. To dissect to which extent differences in paradigms and methods versus cell-

type specific differences caused these inconsistencies in the literature, we employed an optimized

pull-down methodology, followed by MS-based protein identification from mildly crosslinked

lysates, to explore side-by-side the protein networks comprising PrPC in multiple cellular models

[69]. CRISPR-generated knockout cells served as negative controls which revealed the identity of

unspecific binders in each dataset [69].

The main theme that emerged from these comparative interactome analyses was that the majority

of PrPC interactors are known to play roles in TGF beta as well as integrin signaling in the context

of focal adhesion complex biology [69]. Strikingly, our results revealed differences between PrPC

interactions in the four mouse cell models utilized in our investigations [69]. However, our

analyses also revealed striking similarities between the PrPC interactome of N2a and CAD5 cells,

which we attributed to their shared neuronal lineage [69]. Knowledge of the signaling pathways

and the molecular context in which PrPC operates can open new rational avenues for intervening

in prion diseases. Additionally, results from this study may be used to generate new hypotheses

regarding the mechanisms that underlie differential susceptibility of distinct cell lines to prion

infection.

More specifically, these findings may inform two approaches for prion disease intervention: the

first of them would aim to take advantage of the known biology of these interactors in order to

reduce PrPC levels. Removal of the PrPC substrate is widely considered a promising route for

interfering with the conversion to PrPSc based on data which documented that there is an inverse

correlation between prion disease incubation period and steady-state PrPC levels in mouse

infections studies [31,33]. Considering that the majority of PrPC interactors are known to be

embedded into the plasma membrane or to have access to it [69], treatments that trigger their

endocytosis may be promising in this regard as it is plausible that PrPC might get passively co-

internalized. A second approach might capitalize on data presented in this study to derive avenues

for rationally blocking PrPSc-induced toxicity. Such a study would be strengthened if data

generated in this work were complemented by results from co-immunoprecipitation analyses that

compare the levels of interactors in proximity to PrPC versus PrPSc. The current data on PrPC

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interactors would guide the selection of antibodies and serve as a reference. With this approach,

differences in the levels of a given interactor in proximity to PrPC versus PrPSc could direct

subsequent efforts to reveal if such a perturbation might underlie aberrant signaling that leads to

toxicity observed in the disease. This knowledge could then suggest therapeutic angles to address

prion disease neurodegeneration by correcting the perturbation or modulating the molecular

circuitries involved.

In conclusion, the study emphasized the power of systematic side-by-side investigations of the

molecular environment of a protein of interest in various cellular paradigms. Although interactome

studies are not rare in the field of proteomics, to the best of our knowledge, a comparative, mass

spectrometry-based analysis of the same protein target in more than two models has not been

reported before. The methodology we employed represents a transferable platform for the

characterization of protein-protein interactions as well as their cell type-specific differences. Had

we restricted analyses to only one cell model, we might have uncovered specific interactions

between PrPC and a subset of proteins that are involved in TGF beta and integrin signaling

pathways but would also have added to a fragmented and highly diverse literature. Only by having

interrogated interactions of PrPC in several cell lines, did a broader theme of PrPC involvement in

focal adhesion complex biology emerge that united data from all cell models we studied. Thus, the

systematic approach we took eliminated potential confounding variables introduced via

methodological differences and allowed for a deeper understanding of the context in which PrPC

operates. We are confident that the broader strokes of the study design and experimental platform

can serve as a template also for the study of other proteins. Knowledge of the cellular context a

protein of unknown function operates in can be a critical step toward elucidating its function. In

particular in instances, when a large number of competing hypotheses cloud vision, this type of

hypothesis-free discovery work can generate a much-needed framework for breaking through an

impasse and for generating easily testable hypotheses that move the field forward.

Although the data generated in this study do not directly offer clinical insights, they serve as a

stepping stone towards more translational research that can leave a mark in the clinic. To harness

their potential diagnostic value, follow-on work could explore if focal adhesion complexes are

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altered in prion diseases. In addition to the aforementioned experiments that aim to reduce PrPC

levels or block toxicity by manipulating the TGFβ1 or integrin signaling hubs, also urgently needed

is a line of investigation that evaluates whether the results observed with murine cells translate to

human paradigms. Further analyses could utilize our optimized experimental workflow in human

cell lines in order to compare the protein interactions of PrPC between the two species. It is

noteworthy to mention due to the transmissible nature of prion diseases, use of human-derived

infected samples brings about significant technical cost and requires more strict biosafety

regulations, which limits the availability of research facilities that can undertake such investigative

work.

While our study aimed at identifying cell type-specific features of the PrP interactome, another

avenue could look into mouse brain tissue for these analyses. The underlying reason for our cell-

based approach goes back to a previous global proteome study by our group, which highlighted

that cell-based rather than tissue-based proteomics investigations can serve as more informative

experimental paradigms when exploring the physiological function of PrP [100]. More

specifically, we observed that proteome shifts detected in distinct cell models can escape detection

in the brain [100], which is indeed plausible when one considers the diversity of cell types present

in a particular tissue. If the expression of a certain protein follows opposite trends in individual

cell models, one can see how the said effect can get cancelled out and therefore masked in a

complex tissue sample [100]. However, this is not to undermine the importance of using more

biologically relevant models, such as brain tissue for studying prion diseases. In fact, a deep

interactome investigation of wild-type vs. prion infected mouse brain, could reveal novel insights

for mechanism-driven drug design.

Our interactome analyses have mainly used dividing cell lines, however, since prion diseases are

disorders of the nervous system, an improved study paradigm for cellular dynamics of prion

biology could involve non-dividing or primary neuronal cultures. In order to look deeply into not

only strong but also weak and even more transient interactions in a pull-down coupled with mass

spectrometry design, a high protein yield and thus highly enriched bait levels are required. This

criterion can be achieved more easily when using mitosis-competent cell lines. Having stated the

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above, with recent advances in the sensitivity of cutting-edge mass spectrometers, in-depth

analyses have become possible with less biological starting material, and an extension to non-

diving cellular models, including primary neuron culture would nicely complement the data gained

in this study.

The epitope of the D18 antibody that was used in our study is localized such that, in addition to

full-length PrP, it can potentially also bind C1, C2 and shed PrP [3,79]. Since the majority of these

PrP cleavage products are physiologically present in the body, binding partners of these fragments

could serve a biological role in the cell or a functional role in the disease state. Additional work is

needed in order to pinpoint which of the interactors, if any, are specific to a single cleavage product

and whether or not there is a functional relevance of said interaction. Given the apparent protective

role of the alpha cleavage and PrP shedding [3,42] as well as the toxic effects associated with beta

cleavage [3], it could be relevant in this context to investigate if these proteolytic processing events

lead to differential interactions within the molecular environment of PrP, which may trigger

distinct downstream signaling pathways.

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Appendix

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Supplementary Figure S4.1. Consistent and selective enrichment of PrP contrasted to non-

specific binding of Gapdh. (a) Box plots of PrP-derived peptides in all four models. Please see

legend to Fig. 4.2b for a detailed description of graph elements. (b) Box plots of Gapdh-derived

peptides in all four models.

Supplementary Figure S4.2. Selective PrP co-enrichment of Cd109 and Tmem206. Box plots

of CD109 and Tmem206 in the subset of datasets, in which these proteins were robustly identified

and quantified. Please see legend to Fig. 4.2b for a detailed description of graph elements.

-4

Supplementary Figure S4.2

-4

-2

0

2

4

6

log 2

Fold

Cha

nge

CD109 TMEM206

NMuMG

-2

0

2

4

6

log 2

Fold

Cha

nge

CD109

C2C12

2.250.61

1.201.42

1.831.28

0.550.93

2.171.17

2.252.01

-0.490.47

2.802.22

1.100.88

2.691.72

MedianIQR

wt1ko3

ko1ko3

wt2ko3

ko2ko3

wt3ko3

wt1ko3

ko1ko3

wt2ko3

ko2ko3

wt3ko3

2.111.54

-0.121.36

2.661.70

0.201.45

2.722.49

wt1ko3

ko1ko3

wt2ko3

ko2ko3

wt3ko3

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Supplementary Figure S4.3. Evidence that Ece1 is not expressed in CAD5 cells at levels

detectable by western blot analysis. Ece1 western blot analysis of formaldehyde crosslinked

lysate and eluate fractions from the PrP-directed co-immunoprecipitation of wild-type and PrP

knockout CAD5 cells (analogous to data shown in Fig. 4.4a). The results validate the PrP

interactome data (Table 4.1) which failed to detect Ece1 in PrP-directed co-immunoprecipitations

from CAD5 cell lysates. NMuMG cell lysates were loaded as a positive control for Ece1 detection,

Supplementary Figure S4.3

MW[kDa]

1 8765432 109

anti-Ece1

Coom

assie

Lysates

wt1

wt1

PrP

ko1

wt2

PrP

ko1

wt1

PrP

ko2

wt2

PrP

ko1

PrP

ko2

NMuMG CAD5

Eluates

51

39

191

97

64

51

39

191

97

64

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and a Coomassie-stain of the western blot documents total protein levels in the respective lysates

and eluates. Arrowheads indicate signals derived from monomeric and SDS-stable crosslinked

dimeric Ece1.

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MW[kDa]

1 8765432 109 11 12 1 8765432 109 11 12

Supplementary Figure S4.4

MW[kDa]

Coom

assie

51

191

64

51

191

64

51

39

191 97

28

64

51

39

191

97

28

64anti-Tfrc

Coom

assie

51

191 97

64

51

39

191 97

64

Lysates

wt1

wt1

PrP

ko1

wt2

PrP

ko1

wt1

PrP

ko2

wt2

PrP

ko1

PrP

ko2

NMuMG CAD5

Eluates

1 8765432 109 28

Coom

assie

Lysateswt

1

PrP

ko1

wt2

PrP

ko2

wt1

PrP

ko1

wt2

PrP

ko2

wt1

PrP

ko1

wt2

PrP

ko2

NMuMG C2C12 N2a

wt1

PrP

ko1

wt2

PrP

ko2

wt1

PrP

ko1

wt2

PrP

ko2

wt1

PrP

ko1

wt2

PrP

ko2

NMuMG C2C12 N2a

Eluates

97 97

anti-Tfrc

anti-Tfrc

MW[kDa]

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Supplementary Figure S4.4 PrP co-immunoprecipitates Tfrc from wild-type but not PrP

knockout NMuMG cell lysates. Validation of the transferrin receptor protein 1 (Tfrc) as a PrP

binder. Consistent with the PrP interactome data, Tfrc is only prominently represented in PrP co-

immunoprecipitation eluate fractions derived from formaldehyde-crosslinked wild-type NMuMG

cells. However, whereas Tfrc escaped detection by mass spectrometry in PrP co-

immunoprecipitation eluates derived from wild-type C2C12 and N2a cells (Table 1), weak Tfrc

signals can be detected in the respective eluates by western blot analysis, presumably reflecting a

slightly higher sensitivity of western blot analysis over mass spectrometry-based detection for this

protein. No Tfrc was observed in PrP co-immunoprecipitation eluates from CAD5 wild-type cells.

Coomassie stains of the western blot membranes are shown underneath the immunoblot panels to

document protein amounts in the respective samples. Blue and green arrowheads point toward Tfrc

monomer and formaldehyde-crosslinked dimer signals, respectively. A higher molecular mass

band (possibly a crosslink of two Tfrc dimers) can also be detected. The empty arrowhead shown

in the Coomassie images indicates the D18 recombinant Fab used for PrP-directed

immunoprecipitation. Note also the subtle differences in Tfrc immunoblot signal intensities when

comparing wild-type and PrP knockout lysates in NMuMG, C2C12 and N2a cell models, which

could be indicative of molecular crosstalk between PrP and Tfrc modulating steady-state levels of

Tfrc.

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Supplementary Table S4.1. Comparison of the PrP interactome in four mouse cell models.

Supp

lemen

tary T

able

4.1. C

ompa

rison

of th

e PrP

inter

actom

e in f

our m

ouse

cell m

odels

(only

a su

bset

of no

n-spe

cific

intera

ctors

show

n)

NMuM

GC2

C12

N2A

CAD5

wt1

ko1

wt2

ko2

wt3

wt1

ko1

wt2

ko2

wt3

wt1

ko1

wt2

ko2

wt3

wt1

ko1

wt2

ko2

Acce

ssion

Prote

in na

meGe

neCo

verag

ePe

ptide

sPS

Msko

3ko

3ko

3ko

3ko

3Co

unt

Scor

eko

3ko

3ko

3ko

3ko

3Co

unt

Scor

eko

3ko

3ko

3ko

3ko

3Co

unt

Scor

eko

3ko

3ko

3ko

3Co

unt

Scor

e

IPI00

1207

93.1

Major

prion

prote

inPr

np62

.99%

1295

712

.750.8

016

.291.1

411

.4349

558.5

22.02

1.59

27.84

2.95

30.59

5551

2.110

.881.3

88.3

51.7

07.6

360

371.9

13.37

17.31

13.89

0.67

3836

5.3IP

I0012

2971

.2Ne

ural c

ell ad

hesio

n mole

cule

1Nc

am1

75.52

%66

852

4.62

0.75

3.97

2.02

4.88

2414

9.610

.261.5

24.7

61.0

16.6

16

49.9

13.72

1.60

17.55

1.18

21.13

8653

6.810

.0513

.2012

.000.8

410

389

7.8IP

I0027

3801

.3Zin

c tran

sport

er ZI

P10

Slc39

a10

24.37

%18

3232

.7744

.8743

.180 .2

32

30.9

IPI00

9308

82.1

4F2 c

ell-su

rface

antig

en he

avy c

hain

Slc3a

261

.77%

3549

52.4

41.1

51.5

20.9

05.2

82

21.0

7.07

1.26

9.69

1.01

10.55

8343

8.812

.5414

.7811

.531.0

843

333.3

IPI00

1293

95.2

Large

neutr

al am

ino ac

ids tr

ansp

orter

small

subu

nit 1

Slc7a

536

.13%

1033

6.88

1.34

7.69

1.35

7.81

935

.218

.7818

.6212

.141.1

83

38.0

IPI00

4084

95.1

Basig

inBs

g56

.04%

1610

83.2

31.7

42.4

91.0

32.4

010

55.5

17.37

14.40

18.57

0.72

1710

0.1IP

I0076

2815

.1En

dothe

lin-co

nvert

ing en

zyme

1Ec

e163

.89%

5353

29.4

30.6

79.1

11.4

38.5

912

765

9.95.3

71.2

16.1

21.1

26.1

28

112.8

IPI00

9476

29.1

Throm

bosp

ondin

type

-1 do

main-

conta

ining

prote

in 7A

Thsd

7a45

.57%

6119

134

.514

.0714

.1414

.440.8

115

152.1

IPI00

1140

22.1

Insuli

n-like

grow

th fac

tor-bi

nding

prote

in 5

Igfbp

540

.59%

1033

9.67

2.42

9.37

3.02

12.96

518

.1IP

I0015

3809

.1CD

109 a

ntige

nCd

109

63.87

%77

535

4.74

0.44

3.56

1.47

4.49

1529

4.04.3

20.9

26.3

21.1

56.6

122

348.1

IPI00

1279

83.1

Tran

smem

brane

emp2

4 dom

ain-co

ntaini

ng pr

otein

2Tm

ed2

40.80

%9

629.1

01.1

67.3

81.1

713

.389

45.9

IPI00

1335

22.2

Prote

in dis

ulfide

-isom

erase

P4hb

65.42

%38

745

1.93

0.78

2.04

1.32

2.20

216

962.0

3.44

1.40

2.89

1.25

3.18

3822

1.81.4

21.0

01.1

51.0

21.0

933

209.5

1.99

2.30

2.55

1.08

1369

.4IP

I0092

9762

.1En

dogli

nEn

g71

.61%

2994

5.72

0.90

7.82

1.31

9.56

378

.2IP

I0046

6570

.4Tr

ansm

embra

ne em

p24 d

omain

-conta

ining

prote

in 10

Tmed

1031

.96%

623

6.34

1.09

6.25

1.03

9.59

1676

.1IP

I0031

1682

.5So

dium/

potas

sium-

trans

portin

g ATP

ase s

ubun

it alph

a-1At

p1a1

51.32

%46

319

34.0

1.35

2.57

0.96

1.27

0.65

231

.62.8

01.2

52.9

00.6

82.5

715

201.7

3.50

3.29

3.65

0.97

2027

4.0IP

I0013

2474

.3Int

egrin

beta-

1Itg

b147

.74%

3060

2.91

1.02

3.25

1.26

3.50

115

.31.0

10.7

81.2

60.9

41.3

91

20.2

1.81

2.77

3.44

0.99

334

.8IP

I0022

9517

.5Ga

lectin

-1Lg

als1

77.78

%11

459

1.38

0.89

1.20

1.33

1.83

126

493.1

1.67

1.06

1.57

1.13

1.46

5827

1.12.0

41.3

91.4

00.7

10.9

031

93.5

1.97

2.85

2.71

0.94

12IP

I0040

3079

.4Le

ukoc

yte su

rface

antig

en C

D47

Cd47

40.26

%15

545.7

06.1

18.1

10.5

32

54.7

IPI00

1872

89.2

Tran

smem

brane

prote

in 20

6Tm

em20

685

.14%

2516

84.7

50.7

16.9

62.1

46.4

611

108.6

IPI00

1373

36.1

Glyp

ican-1

Gpc1

67.50

%34

164

4.54

1.20

5.26

1.12

6.24

2020

4.9IP

I0012

3639

.1Ca

lretic

ulin

Calr

45.91

%13

931.4

70.9

21.2

50.9

81.7

15

44.0

1.57

0.96

1.65

0.91

1.51

1086

.81.5

01.9

51.7

50.9

64

29.1

IPI00

2726

90.2

Angio

tensin

-conv

erting

enzy

meAc

e50

.38%

5847

54.0

35.4

34.3

60.7

756

576.0

IPI00

4736

80.2

Tran

smem

brane

emp2

4 dom

ain-co

ntaini

ng pr

otein

9Tm

ed9

42.75

%12

444.0

20.6

94.0

11.0

35.2

68

60.0

IPI00

5151

73.1

H-2 c

lass I

histo

comp

atibil

ity an

tigen

, K-K

alph

aH2

-K1

63.61

%23

783.4

11.0

44.4

91.2

95.0

45

35.2

IPI00

1184

13.2

Throm

obos

pond

in-1

Thbs

137

.92%

4096

3.79

0.91

2.67

0.92

3.07

1075

.0IP

I0098

5828

.1H-

2 clas

s I hi

stoco

mpati

bility

antig

en, D

-P al

pha

H2-D

162

.50%

2914

02.8

74.2

12.3

20.9

63

53.1

IPI00

1247

00.1

Tran

sferrin

rece

ptor p

rotein

1Tfr

c58

.72%

5028

92.0

90.7

62.0

21.4

32.0

631

186.0

673

.7IP

I0097

2920

.140

S rib

osom

al pro

tein S

23Rp

s23

55.24

%13

106

1.12

1.15

0.80

0.99

1.50

1231

.71.1

31.2

31.4

90.7

20.3

21

2.32.3

71.3

91.4

80.8

60.8

632

99.9

3.50

2.78

2.28

0.87

1032

.8IP

I0023

0660

.540

S rib

osom

al pro

tein S

15a

Rps1

5a76

.92%

989

1.11

1.87

0.51

1.24

2.14

1452

.70.9

40.9

30.9

60.8

70.5

512

27.5

1.61

1.62

0.84

0.74

0.46

1442

.52.8

64.1

92.5

51.0

03

IPI00

1259

01.5

40S

ribos

omal

protei

n S13

Rps1

383

.44%

2085

1.01

0.93

1.18

1.38

0.97

634

.61.3

91.3

21.1

90.7

90.5

615

69.7

4.53

4.50

2.78

1.13

421

.3IP

I0098

6371

.160

S rib

osom

al pro

tein L

27a

Rpl27

a63

.80%

1349

1.22

1.41

0.56

0.89

1.84

936

.20.6

60.3

50.3

40.2

20.5

14

21.0

4.94

1.56

5.58

1.35

27.8

IPI00

9881

01.1

40S

ribos

omal

protei

n S2

Rps2

68.26

%31

260

1.07

1.44

0.55

0.87

1.78

1986

.31.0

41.0

10.8

91.1

30.7

712

50.4

1.54

1.33

1.19

0.75

0.70

4016

5.63.0

02.0

91.5

80.7

810

52.5

IPI00

5535

38.3

Histo

ne H

3.1Hi

st1h3

a73

.53%

1310

611.1

71.7

80.3

00.7

32.5

179

388.4

4.74

1.98

1.45

1.60

0.55

2733

7.32.8

31.7

11.4

30.4

20.3

415

279

5.30.6

51.2

81.2

11.0

418

96.9

IPI00

8765

49.2

Histo

ne H

2BHi

st1h2

b79

.85%

1613

651.6

41.7

70.3

40.7

22.9

481

327.9

3.59

2.59

1.16

1.65

0.38

5837

9.93.1

61.6

01.6

70.5

10.4

626

411

09.7

0.54

0.93

1.03

0.70

2210

8.6IP

I0078

5343

.2Hi

stone

H3.3

H3f3a

72.79

%12

1076

1.10

1.77

0.29

0.73

2.29

8243

7.64.3

71.9

61.7

02.1

90.3

638

399.9

2.74

1.75

1.41

0.42

0.34

148

762.9

0.65

1.28

1.21

1.04

1899

.4IP

I0028

2848

.120

kDa p

rotein

Hist1

h3e

69.06

%18

1489

1.17

1.83

0.30

0.73

2.52

119

634.5

3.60

1.92

1.56

2.04

0.35

4565

4.82.9

81.7

31.3

70.4

20.3

322

213

41.4

0.65

1.28

1.21

1.02

1910

5.6IP

I0040

7339

.7Hi

stone

H4

Hist1

h4a

70.87

%15

1317

1.04

1.79

0.38

0.63

2.03

123

447.6

4.40

1.59

1.41

1.51

0.38

9349

6.93.1

01.6

91.3

30.4

60.2

834

413

79.5

0.62

1.22

1.10

0.95

4421

3.8IP

I0023

0264

.5Hi

stone

H2A

.xH2

afx93

.71%

2474

61.1

01.5

60.3

30.7

32.2

048

157.1

2.54

1.95

1.76

2.04

0.55

3521

6.31.9

71.6

81.0

50.5

60.2

918

375

7.80.7

11.2

81.2

01.0

114

61.1

IPI00

1173

48.4

Tubu

lin al

pha-1

B ch

ainTu

ba1b

69.18

%30

720

1.40

1.06

1.02

1.23

1.86

7433

8.01.5

01.5

21.0

51.2

91.0

216

109.6

1.06

1.26

0.78

0.86

0.46

128

612.6

1.01

1.25

1.16

1.04

8036

7.2IP

I0011

0753

.1Tu

bulin

alph

a-1A

chain

Tuba

1b69

.40%

3072

11.3

81.0

51.0

21.2

21.8

368

319.5

1.31

1.38

1.13

1.38

0.89

2214

0.70.9

71.2

50.7

30.8

80.4

412

058

8.51.0

51.2

41.1

41.0

484

392.4

IPI00

1122

51.1

Tubu

lin be

ta-3 c

hain

Tuba

1a79

.78%

3363

61.4

81.0

90.8

61.1

91.9

349

227.9

0.95

1.14

0.92

1.03

0.92

2614

6.40.9

01.2

60.7

10.8

30.3

910

857

9.41.0

71.2

21.0

91.0

348

248.4

IPI00

1173

52.1

Tubu

lin be

ta-5 c

hain

Tubb

579

.95%

3797

01.3

71.0

30.8

71.2

41.9

191

394.2

0.90

1.14

0.89

1.03

0.91

3218

3.11.0

21.2

90.7

90.8

20.4

617

084

8.01.0

41.1

81.1

01.0

281

380.0

IPI00

1090

61.1

Tubu

lin be

ta-2B

chain

Tubb

2b79

.78%

3677

91.3

71.0

20.8

61.2

51.9

377

352.3

0.90

1.14

0.89

1.03

0.91

3217

9.60.9

61.2

70.7

50.8

30.4

413

069

5.81.0

61.2

31.0

71.0

156

279.4

IPI00

1694

63.1

Tubu

lin be

ta-2C

chain

Tubb

2c73

.71%

3472

81.3

91.0

80.8

61.1

91.8

467

310.2

0.92

1.20

0.85

1.09

0.83

2012

2.61.0

01.2

90.7

60.8

40.4

511

664

2.21.0

21.0

41.0

30.9

550

255.6

IPI00

1183

84.1

14-3-

3 prot

ein ep

silon

Ywha

e64

.31%

1933

30.7

91.3

50.5

00.8

51.3

755

212.7

1.63

4.20

0.94

1.19

0.73

3214

4.41.5

41.6

10.7

90.5

50.3

735

143.6

1.14

1.47

1.26

0.87

313

.6IP

I0022

7392

.514

-3-3 p

rotein

eta

Ywha

h47

.97%

1533

80.8

11.3

30.5

20.8

61.3

949

168.5

1.47

4.24

0.89

1.13

0.68

2710

9.61.5

41.6

10.8

00.5

50.3

741

179.6

1.14

1.47

1.26

0.87

313

.6IP

I0023

0707

.614

-3-3 p

rotein

gamm

aYw

hag

59.11

%20

410

0.79

1.33

0.52

0.92

1.35

5820

5.61.4

54.1

60.8

91.1

30.6

829

127.2

1.50

1.54

0.78

0.59

0.37

6531

5.71.1

41.4

71.3

81.2

25

13.6

IPI00

7545

45.3

14-3-

3 prot

ein th

eta-lik

eYw

haq

81.82

%29

406

0.81

1.33

0.53

0.89

1.41

5626

6.41.4

64.2

00.8

91.1

10.6

828

128.8

1.53

1.59

0.82

0.57

0.37

4320

5.41.0

41.3

61.2

40.8

84

40.7

IPI00

1164

98.1

14-3-

3 prot

ein ze

ta/de

ltaYw

haz

79.59

%26

627

0.87

1.29

0.52

0.88

1.47

114

532.6

1.36

2.16

0.75

1.11

0.68

6232

9.11.5

21.5

10.8

40.5

60.3

768

275.9

0.95

1.27

1.22

0.68

513

.6

Color

s sha

ding a

s for

Table

1. In

addit

ion, g

rey sh

ading

of di

fferen

t inten

sities

was

used

to vi

suall

y sep

arate

non-s

pecif

ic int

eracto

rs be

longin

g to d

istinc

t prot

ein fa

milie

s.

Page 106: The Prion Protein is Embedded in a Molecular Environment ... · The Prion Protein: What We Do and Don’t Know 1.1 Introduction The prion protein (PrP) is central to the pathogenesis

94

Supplementary Table S4.2. Global proteome analysis of NMuMG cells -/+ TGFΒ1 (datset I)

in PrP-deficient and wildtype cells (dataset II).

Supp

lem

enta

ry T

able

S4.

2. G

loba

l pro

teom

e an

alys

is o

f NM

uMG

cel

ls -/

+ TG

FB1

(dat

set I

) in

PrP-

defic

ient

and

wild

type

cel

ls (d

atas

et II

)

III

-TGF

B1+T

GFB1

-TGF

B1+T

GFB1

-TGF

B1Pr

P kd

1w

t1Pr

P kd

2w

t2Pr

P kd

3Ac

cess

ion

Mod

ified

des

crip

tion

C ove

rage

+TGF

B1+T

GFB1

+TGF

B1+T

GFB1

+TGF

B1Co

unt

wt3

wt3

wt3

wt3

wt3

Coun

t-/

+TGF

B1w

t IP

I002

3066

5.3

Neur

al ce

ll ad

hesio

n m

olec

ule

1 iso

form

373

.27%

0.36

70.

989

0.35

30.

943

0.37

828

0.76

20.

986

0.75

70.

944

0.75

157

0.36

60.

757

.102

416.0859.0

766.0369.0

726.0%65.57

1-ateb nirgetnI3.47423100IPI

022

0.97

41.

004

1.00

90.

993

270.

636

1.00

6.0

3617.0

708.0236.0

369.0895.0

%55.761-nidnopsob

morhT2.31481100IPI

989

0.95

11.

007

0.88

71.

000

100.

649

0.99

9.0

740.153

697.0831.1

208.0919.0

628.0%03.69

1-nitcelaG5.71592200IPI

963

1.13

31.

054

1.15

641

0.80

81.

112

69.0949.0

098.0%13.47

1 nietorp rotpecer nirrefsnarT1.00742100IPI

10.

972

0.96

837

0.95

61.

004

0.96

20.

937

0.89

536

0.94

00.

938

IPI0

0403

079.

4Le

ukoc

yte

surfa

ce a

ntige

n CD

4761

.06%

1.16

91.

103

1.10

11.

027

1.08

212

1.05

11.

023

0.96

70.

965

1.01

813

1.11

71.

012

IPI0

0311

682.

5So

dium

/pot

assiu

m-tr

ansp

ortin

g AT

Pase

subu

nit a

lpha

-61

.68%

1.31

41.

020

1.35

81.

043

1.35

089

0.92

61.

025

0.92

91.

014

0.93

976

1.34

10.

931

IPI0

0930

882.

14F

2 ce

ll-su

rface

anti

gen

heav

y ch

ain

isofo

rm a

76.4

6%1.

182

1.04

41.

209

0.95

61.

177

231.

027

1.06

00.

968

1.00

51.

063

221.

189

1.01

9IP

I001

2939

5.2

Larg

e ne

utra

l am

ino

acid

s tra

nspo

rter

smal

l sub

unit

142

.58%

1.51

41.

123

1.43

81.

071

1.42

511

1.12

11.

101

1.02

31.

200

1.16

59

1.45

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