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Page 1: Spectroscopic studies of dynorphin neuropeptides and the ...su.diva-portal.org/smash/get/diva2:197741/FULLTEXT01.pdf · Hirschberg, Tomas Bergman, Ülo Langel, Kurt F. Hauser, Aladdin

Spectroscopic studies of dynorphin neuropeptides and the amyloid β peptide

The consequences of biomembrane interactions

Loïc Hugonin

Department of Biochemistry and Biophysics

Stockholm University

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Doctoral thesis ©Loïc Hugonin, Stockholm 2007 ISBN 978-91-7155-536-6 Printed in Sweden by US-AB, Stockholm 2007 Distributor: Department of Biochemistry and Biophysics, Stockholm University Cover page: Schematic binding of dynorphin to the biomembrane and its effects viewed by different spectroscopic techniques

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A ma femme, mon père, ma mère, ma sœur et mon frère

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Abstract

Dynorphin A, dynorphin B and big dynorphin, collectively known as dynorphins, are endogenous opioid neuropeptides. Dynorphins are widely distributed in the central nervous system and are synthesized from the precursor protein prodynorphin in the striatum, amygdala, hippocampus and other brain structures and also in the spinal cord. They play an important role in a wide variety of physiological functions such as regulation of pain processing and memory acquisition. Such actions are generally mediated through the κ-receptors. Besides opioid receptor interactions, dynorphins have non-opioid physiological activities. The non-opioid activities of dynorphins result in excitotoxic effects in neuropathic pain, spinal cord and brain injury. The mechanisms behind these effects are still unknown. The receptors apparently involved in non-opioid effects are glutamate receptors (N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainite receptors).

In order to gain insight into the mechanisms of the non-opioid interactions of dynorphins with the cell, spectroscopic studies were performed. The analysis of the sequence of dynorphins revealed that they have a high content of basic and hydrophobic amino acid residues, thus dynorphins share certain common properties with cell penetrating peptides. Cell penetrating peptides are short water soluble peptides, which translocate across the plasma membrane with or without cargoes conjugated to the peptide. This translocation occurs without chiral receptors. We demonstrated that big dynorphin and dynorphin A, but not dynorphin B, penetrated into cells and were localized in the cytoplasm and associated with the endoplasmic reticulum. Fluorescence and circular dichroism (CD) experiments revealed that all dynorphins interact with a membrane model system (small unilamellar vesicles) with weak membrane-induced secondary structure. The calcein-entrapped and fura-entrapped large unilamellar vesicle (LUV) experiments show that big dynorphin and dynorphin A induce membrane perturbation, calcein leakage and cause permeability of the membrane to calcium. We have also investigated whether the dynorphins can translocate through the vesicle membranes and estimated the relative strength of interaction of the peptides with the vesicles by fluorescence resonance energy transfer. The results reveal that dynorphins do not translocate in the LUV membrane model system. There is a strong electrostatic contribution to the interaction of the peptides with the membrane bilayer.

In the second part of this thesis we investigated the amyloid β(1-40) peptide. This peptide is related to Alzheimer’s disease and its soluble oligomeric aggregates are reported to mediate toxic effects on neurons and to contribute to the pathology of the disease. In order to provide better insight into the aggregation processes we examined the membrane interaction of amyloid β(1-40) in a model system by CD and nuclear magnetic resonance (NMR) spectroscopy. The CD experiment reveals that gradual addition of small amounts of sodium dodecyl sulfate to an aqueous solution gives rise to a secondary structure conversion of amyloid β(1-40) peptide. The conversion can be described as a two state process, from random coil to β-sheet. At high detergent concentrations there is a transition from β-sheet to α-helix conformation. The NMR studies show that at low concentrations of lithium dodecyl sulfate almost all NMR signals of the peptide disappear. This observation indicates formation of high molecular mass complexes between peptide and detergent giving rise to the β-sheet structures, possibly mimicking the behavior of the peptide when aggregating at a cell membrane surface.

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Résumé

Dynorphine A, dynorphine B et “big” dynorphine, regroupées sous le nom de dynorphines, sont des neuropeptides opioïde endogènes. Les dynorphines sont localisées dans le système nerveux central et synthétisées, à partir de la protéine précurseur appelée prodynorphine, dans le striatum, le complexe amygdalien, l’hippocampe et d'autres aires cérébrales, ainsi que dans la moelle épinière. Ils jouent un rôle important dans de nombreuses fonctions physiologiques comme la régulation de la douleur et l'acquisition de mémoire. De tels mécanismes sont généralement mis en oeuvre par transduction des récepteurs-κ. En plus des interactions avec les récepteurs-κ opioïde, les dynorphines ont des activités physiologiques non-opioïdes. Ces évènements biologiques aboutissent à des effets excitotoxiques impliqués dans les douleurs neuropathiques, ainsi que les traumatismes occasionnés dans le cerveau et la moelle épinière. Bien que les mécanismes à l’origine de ces observations soient toujours inconnus, il semblerait que les récepteurs glutamate (des récepteurs N-méthyl-D-aspartate et alpha-amino-3-hydroxy-5-méthylisoazol-4-propionate/kainite) soient impliqués dans ces phénomènes non-opioïdes.

Les travaux basés sur des études spectroscopiques, ont été réalisés afin de mieux comprendre les interactions non-opioïdes entre les dynorphines et la cellule. L'analyse de la séquence des dynorphines a révélé un grand nombre d'acides aminés basiques et hydrophobes, montrant certaines propriétés communes avec les “cell penetrating peptides” (CPPs). CPPs sont de petits peptides hydrosolubles traversant la membrane cellulaire avec ou sans cargos conjugués au peptide, et sans médiation par un récepteur chiral. Nous avons démontré que “big” dynorphine et dynorphine A, mais pas la dynorphine B, pénétraient dans les cellules et s’associaient avec le réticulum endoplasmique dans le cytoplasme. D’une part, les expériences de fluorescence et de dichroïsme circulaire (CD) montrent que toutes les dynorphines interagissent avec le modèle membranaire (petites vésicules unilamellaires) avec une faible induction de structure secondaire. D’autre part, les expériences sur les larges vésicules unilamellaires (LUVs) calcéine-encapsulées et fura-encapsulées ont mis en évidence que “big” dynorphine et dynorphin A provoquaient une perturbation de la membrane, un efflux de la calcéine et la perméabilité de la membrane au calcium. Enfin, nous avons fait appel à la technique du transfert d’énergie de fluorescence pour montrer, d’une part, la forte contribution des interactions électrostatiques entre les peptides et la double couche membranaire et, d’autre part, que les dynorphines n’avaient pas vocation à traverser la membrane des LUVs.

Dans la deuxième partie de cette thèse, nous avons examiné le peptide β-amyloïde (1-40), impliqué dans la maladie d'Alzheimer. En formant des agrégats oligomériques solubles, il provoque une certaine toxicité neuronale et contribuer ainsi aux mécanismes physiopathologiques de la maladie. Afin de mieux appréhender le processus d'agrégation, nous avons examiné l’interaction du β-amyloïde (1-40) avec un modèle membranaire par CD et spectroscopie de résonance magnétique nucléaire (RMN). Le CD a révélé que l’addition graduelle de petites quantités de dodécyl sulfate de sodium à une solution aqueuse provoquait une conversion de la structure secondaire du peptide β-amyloïde d’un état désordonné vers un feuillet β. A concentration élevée du détergent il y a une transition du feuillet β à la conformation d’hélice α. Les études de RMN montrent qu'aux concentrations basses du dodécyl lithium de sulfate presque tous les signaux RMN du peptide disparaissent. Cette observation indique la formation de complexes d’une masse moléculaire élevée entre le peptide et le détergent provoquant la structuration en feuillets β et imitant probablement le comportement du peptide lors de son agrégation à la surface de la membrane cellulaire.

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List of publications

I. Zoya Marinova, Vladana Vukojević, Slavina Surcheva, Tatiana Yakovleva, Gvido Cebers,

Natalia Pasikova, Ivan Usynin, Loïc Hugonin, Weijie Fang, Mathias Hallberg, Daniel

Hirschberg, Tomas Bergman, Ülo Langel, Kurt F. Hauser, Aladdin Pramanik, Jane V.

Aldrich, Astrid Gräslund, Lars Terenius and Georgy Bakalkin. Translocation of dynorphin

neuropeptides across the plasma membrane. A putative mechanism of signal transmission.

J. Biol. Chem. 280 (2005), pp. 26360-26370

II. Loïc Hugonin, Vladana Vukojević, Georgy Bakalkin and Astrid Gräslund, membrane

leakage Induced by Dynorphins, FEBS Lett. 580 (2006), pp. 3201-3205

III. Loïc Hugonin, Vladana Vukojević, Georgy Bakalkin and Astrid Gräslund, Calcium influx

into phospholipid vesicles caused by dynorphin neuropeptides (2007) under revision for

Biochim. Biophys. Acta - Biomembranes

IV. Loïc Hugonin, Alex Perálvarez-Marín, Andreas Barth and Astrid Gräslund, Secondary

structure transitions and interfacial aggregation induced in dynorphin neuropeptides by a

membrane mimicking environment, (2007) submitted manuscript

V. Loïc Hugonin, Anna Wahlström, Jüri Jarvet and Astrid Gräslund, Secondary structure

conversions of Alzheimer’s Aβ(1-40) peptide induced by sodium dodecyl sulfate, (2007)

Manuscript

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Abbreviations

Aβ Amyloid β peptide

AD Alzheimer’s disease

AMPA alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate

APP Alzheimer’s precursor protein

CD Circular dichroism

CHO Chinese-hamster ovary

CMC Critical micellar concentration

CPPs Cell Penetrating Peptides

Dansyl-DHPE N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-

glycero-3-phosphoethanolamine triethylammonium salt

DABF Dynorphin A-binding factor

EL Extracellular loop regions

FCS Fluorescence correlation spectroscopy

FRET Fluorescence resonance energy transfer

FT-IR Fourier transform infrared

GAG Glycosaminoglycan

HIV-1 Human Immunodeficiency Virus type 1

HSPG Heparan sulfate proteoglycans

HSQC Heteronuclear single quantum correlation

Leu-enkephalin-Arg Leucine-enkephalin-Arginine

LiDS lithium dodecyl sulfate

L/P Lipid to peptide molar ratio

LUVs Large unilamellar vesicles

NMDA N-methyl-D-aspartate

NMR Nuclear magnetic resonance

ORL1 Opioid receptor-like receptor

pAntp Penetratin

PC2 Proprotein convertase 2

POPC 1-palmitoyl-2-oleyl-phosphatidylcholine

POPG 1-palmitoyl-2-oleyl-phosphatidylglycerol

Rh-Dyn A Rhodamine labeled dynorphin A

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RNase A Ribonuclease A

SDS Sodium docecyl sulfate

SUVs Small unilamellar vesicles

TROSY Transverse relaxation optimized spectroscopy

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Contents Background....................................................................................................................................................1

1. Neuropeptides ............................................................................................................................................2

2. Dynorphins.................................................................................................................................................3

a. Opioid activities of dynorphins ...............................................................................................................5

b. Non-opioid activities of dynorphins........................................................................................................7

3. Cell penetrating peptides ........................................................................................................................10

a. Introduction ...........................................................................................................................................10

b. Classes of cell penetrating peptides.......................................................................................................11

Penetratin ..............................................................................................................................................11

HIV-1 Tat ..............................................................................................................................................12

Transportan...........................................................................................................................................13

c. Mechanisms ...........................................................................................................................................14

4. Alzheimer’s disease .................................................................................................................................19

Aims ..............................................................................................................................................................21

5. Methods ....................................................................................................................................................22

a. Cell cultures ...........................................................................................................................................22

b. Lipid membranes ...................................................................................................................................22

Membranes ............................................................................................................................................22

Biomembrane mimicking solvents .........................................................................................................23

c. Fluorescence spectroscopy ....................................................................................................................25

Introduction...........................................................................................................................................25

Tryptophan fluorescence .......................................................................................................................27

Calcein leakage .....................................................................................................................................27

Calcium influx .......................................................................................................................................28

Fluorescence resonance energy transfer ..............................................................................................29

d. Circular dichroism spectroscopy ...........................................................................................................32

e. Other methods........................................................................................................................................35

Fluorescence correlation spectroscopy.................................................................................................35

Fourier transform infrared spectroscopy..............................................................................................36

Nuclear magnetic resonance spectroscopy ...........................................................................................36

6. Results and discussion.............................................................................................................................38

a. Translocation of dynorphins across the plasma membrane and the membrane model systems ............38

Penetration into cells ............................................................................................................................38

Peptide translocation in a membrane model system .............................................................................40

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b. Interaction and structure induction of dynorphins in the presence of membrane model systems .........41

Membrane interaction ...........................................................................................................................41

Secondary structure induction...............................................................................................................42

c. Membrane perturbation of dynorphins using LUV model systems, including calcium permeability ...43

Calcein leakage from phospholipid vesicles .........................................................................................44

Calcium influx into phospholipid vesicles .............................................................................................45

d. Structure induction of Aβ(1-40) in the presence of SDS micelles as a membrane model system.........46

Conclusion ....................................................................................................................................................48

Acknowledgement........................................................................................................................................50

References ....................................................................................................................................................52

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Background

Sumerians (∼4500 BC) and Egyptians (∼1600 BC) knew the opium poppy to have

analgesic, anti-diarrheic and cough suppressant effects. The opium poppy was also widely used

during the eighteenth and nineteenth centuries because of its euphoric effect. In 1805 Friedrich

Wilhelm Sertürner, a young pharmacist’s assistant, isolated the principal active component of the

opium poppy, morphine, which was immediately used in medicine as an analgesic. The modes of

action of morphine and its derivatives were systematically studied in the middle of the twentieth

century, with the results suggesting the presence of several opioid receptor types in the central

nervous system. At the beginning of the 1970s, the existence of specific receptors of morphine

and its derivatives, was demonstrated using biochemical binding assays with radioactive ligands

(Pert et al., 1973; Simon et al., 1973; Terenius, 1973). Initially, the opioid receptors were

classified according to the pharmacological effects of the various prototype agonists in dogs: µ

(for morphine), κ (for ketocyclazocine) and σ (for N-allylnormetazocine) (Martin et al., 1976).

The existence of specific agonists and antagonists for the receptors µ, κ and σ, as well as their

cloning, allowed researchers to characterize them at the pharmacological, biochemical and

molecular levels. The three subtypes of opioid receptors, µ, κ and σ, have similar primary

structures. They have an extracellular N-terminal region, seven transmembrane domains and an

intracellular C-terminal tail structure. Pharmacological testing indicates that the σ-receptors are

activated by drugs unrelated to opioids, and the opiate antagonists do not have an effect on them.

Based on these investigations the σ-receptor is no longer regarded as an opioid receptor (Iwamoto,

1981; Su, 1981).

The discovery of opioid receptors that can bind morphine and its analogues indicated the

existence of endogenous ligands capable of interacting with these receptors. The presence of

endogenous opiates as neurotransmitters was proposed, because the human body does not contain

any morphine at birth. The endogenous ligands of the opioid receptors were discovered in 1975,

with enkephalins being the first endogenous opioid peptides isolated and sequenced (Hughes et

al., 1975). Some years later, an extraordinarily potent opioid peptide was isolated. This peptide

has been named "dynorphin" (dyn- from Greek dynamis = power) (Goldstein et al., 1979) and was

originally proposed as a ligand of the κ-opioid receptor (Chavkin et al., 1982).

It may appear simple to view each of the major opioid peptides as selective for only one type

of major receptor. However, each of these ligands shows a significant affinity for more than one

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receptor type. The behavior of the dynorphins is no exception, and, even though dynorphin

peptides exhibit some preference to bind to the κ receptor, they have an affinity for all the major

opioid receptor types (κ, µ, δ and ORL1) (Smith and Lee, 1988; Zhang et al., 1998).

1. Neuropeptides

Neurons use chemical signals, called neurotransmitters, to communicate information.

Neurotransmitters are released from an axon terminal of the synapse, diffuse across the narrow

synaptic cleft, bind to a specific receptor on the surface of the dendrite (postsynaptic cell), and

either inhibit or excite the target cell. The neurotransmitters are divided into several categories,

e.g. amino acids and their metabolites (such as glutamate and glycine), biogenic monoamines

(such as serotonin, histamine and norepinephrine), gaseous molecules (such as nitric oxide) and

neuropeptides.

Neuropeptides are peptides with between 3 and 50 amino acid residues that are extensively

expressed in neurons of the central and peripheral nervous system, and they are also present in

various types of glial cells. About 300 different neuropeptides are known, and they are up to 50

times larger in molecular mass than classical neurotransmitters. The major difference between

neuropeptides and classical neurotransmitters is their mode of synthesis and their replacement

after release. Neuropeptides are synthesized in a complex process in the cell bodies, while classic

neurotransmitters can be synthesized locally at the nerve ending. Neuropeptides are often

involved in slow transmission (modulator action), with their actions generally longer than those of

the classical neurotransmitters. They also have an indirect effect on ionic channels via G proteins.

Neuropeptides are degraded by peptidases in the extracellular space, and several of these enzymes

have been successfully identified (Toques and Noble, 1995). Replacement of degraded peptides

after release occurs via axonal transport from the cell bodies to the nerve ending. In contrast,

classical neurotransmitters often have a membrane reuptake mechanism, using specific transporter

molecules, which allows reutilization of the transmitters. One of the important families of

neuropeptides is the family of opioid peptides, which can be produced endogenously (Table 1) or

can be absorbed from food (like exorphins and casorphins).

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Table 1: Major classes of endogenous opioid peptides

Precursor Endogenous peptide Preferred receptor

Proopiomelanocortin β-Endorphin µ and δ Proenkephalin [Met]enkephalin

[Leu]enkephalin Metorphamide

δ and µ

Prodynorphin Big Dynorphin Dynorphin A Dynorphin B α-neoendorphin β-neoendorphin

[Leu]enkephalin

κ

δ and µ

2. Dynorphins

Big dynorphin, dynorphin A and dynorphin B are endogenous opioid peptides derived

from prodynorphin (or proenkephalin B – 256 animo acids) (Table 2). Big dynorphin consists of

dynorphin A as its N-terminus and dynorphin B as its C-terminus, although the two constituent

dynorphins cannot be directly derived from it (Day et al., 1998). Prodynorphin is widespread in

the mammalian nervous system and the prodynorphin gene is located in human chromosome 20

(Litt et al., 1988; Merchenthaler et al., 1997). Proprotein convertase 2 (PC2), also called

prohormone convertase, is the major enzyme involved in the reaction of prodynorphin to give

mature dynorphin peptides (Day et al., 1998) (Fig. 1). Prodynorphin also contains several leucine-

enkephalin sequences, and some studies have proposed that Leu-enkephalin can be derived from

prodynorphin, rather than from proenkephalin in areas of the brain rich in dynorphin peptides

(Zamir et al., 1984; Zamir and Quirion, 1985; Taquet et al., 1985). In contrast to dynorphin A and

dynorphin B, Leu-enkephalin can be directly derived from big dynorphin by the PC2 enzyme

(Day et al., 1998).

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Table 2: Amino acid sequences of dynorphin neuropeptides

Peptide Sequence

Big Dynorphin YGGFLRRIRPKLKWDNQKRYGGFLRRDFKVVT

Dynorphin A YGGFLRRIRPKLKWDNQ

Dynorphin A(1-13) YGGFLRRIRPKLK

Dynorphin B YGGFLRRDFKVVT

Big Dynorphin central fragment RRIRPKLKWDNQKRYGGFLRR

NH2 COOHProdynorphin

α-NE Big Dyn

Dyn A Dyn B

Leu-enkephalin-Arg

Leu-enkephalin

Dyno

rphi

n Pro

cess

ing

by PC

2 KR

KRKR KRRR RR RR

R R

Figure 1: Summary of the major posttranslational products of prodynorphin. PC2 is the proprotein

convertase 2 enzyme.

Neuropeptides are known to be synthesized in the cell body in the following steps: 1)

transcription of the pre-propeptide gene and creation of pre-propeptide RNA; 2) translation into

pre-propeptide, which enters the endoplasmic reticulum ; 3) formation of the propeptide, which is

the direct precursor of neuropeptides and migrate to the Golgi apparatus; and 4) conversion of

propeptide into neuropeptides by peptidase cleavage in vesicles released from the Golgi

apparatus, during the time when the vesicles are transported from neuronal soma to axon

terminals.

Normally, all the propeptide should be totally convert into neuropeptides by the time the

vesicles reach the axon terminal, but prodynorphin has been detected in the axon terminal in the

brains of rats. In this case, the processing of prodynorphin into dynorphin is stimulated by the

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depolarization of the axon, which is followed by the secretion of dynorphin. The regulation of

prodynorphin storage and its processing at synapses by neuronal activity may provide a new

model for the local mechanism of regulation of synaptic transmission (Yakovleva et al., 2006).

Dynorphins are present at relatively high concentrations in the dorsal spinal cord, the

autonomic nervous system, adrenal medulla and various brain areas, particularly the hippocampus

and the striato-nigral pathway (Khachaturian et al., 1982; Fischli et al., 1982; Fallon et al., 1986;

Xie et al., 1987; Mansour et al., 1994; Pierce et al., 1999). Dynorphin A is most prevalent in the

spinal cord, while dynorphin B is the major dynorphin neuropeptide in the hippocampus, cortex

and striatum (Healy and Meador-Woordruff, 1994).

Big dynorphin and dynorphin A are rich in Arg and Lys residues and are the most basic of the

neuropeptides known. They have the same N-terminal hexapeptide sequence (called Leu-

enkephalin-Arg6 = Tyr Gly Gly Phe Leu Arg) as dynorphin B (Kakidani et al., 1982), with the N-

terminal Tyr crucial for preserving their opioid activity (Walter et al., 1982). Dynorphin A is

highly conserved and rapidly metabolized by peptidase (Herman et al., 1980; Leslie and

Goldstein, 1982), and is identical in many species, such as humans, mice, bovines and porcine

species, and amphibians (Goldstein, 1987; Evans et al., 1988; Kakidani et al., 1982; Danielson et

al., 2002). Big dynorphin and dynorphin A show a similar affinity for the human κ-opioid

receptor, which is significantly greater than the affinity of dynorphin B. Big dynorphin activates G

proteins through the κ-opioid receptor with much greater potency, efficacy and selectivity than

other dynorphins (Merg et al., 2006).

Dynorphin peptides are specific in their opioid activity via an opioid receptor. However, at

high dynorphin concentrations they start to exhibit non-opioid activities that are toxic for the cell.

a. Opioid activities of dynorphins

Like other opioids, the dynorphin neuropeptides play an important role in a wide variety

of physiological functions, including pain regulation, motor activity, cardiovascular regulation,

respiration, temperature regulation, feeding behavior, hormone balance, and the response to shock

or stress (De Vries and Shippenberg, 2002; Kreek et al., 2002; Costigan and Woolf, 2002) (Table

3). Dynorphins have the highest affinity for κ-opioid receptors thar have three extracellular loop

regions (EL1-EL3) and belong to the superfamily of G-protein-coupled receptors. Chimera and

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molecular studies indicated that the negatively charged EL2 loop binds dynorphin neuropeptides

by electrostatic interaction, due to the basic nature of the peptides (Wang et al., 1994; Xue et al.,

1994; Paterlini et al., 1997). Activation of κ-opioid receptors leads to the inhibition of the

electrical activity of the neuron through the N-type channel, by increasing K+ conductance

(opening of K+ channels) and decreasing Ca2+ conductance (closing of Ca2+ channels) (Macdonald

and Werz, 1986; Huang, 1995). Activation of these receptors can result in many effects, including

dysphoria, antinociception, modulation of memory formation, neuroendocrine response and a

pschotometric effect (Costigan and Woolf, 2002; De Vries and Shippenberg, 2002). Epilepsy

connected experiments (Solbrig and Koob, 2004; Loacker et al., 2007) also showed that

dynorphin A and its analogues are anticonvulsant-like and tissue-protective. Furthermore,

activation of the κ-opioid receptors by dynorphin has shown neuroprotection activity in a variety

of models (Genovese et al.1994; Sheng et al., 1997), although the mechanism for this is not

completely understood.

Dynorphin A does not have analgesic activity in the brain (Friedman et al., 1981; Walker et

al., 1982), but analgesis activity at very high concentrations has been reported (Petrie et al., 1982;

Nakazawa et al., 1985). Dynorphins have been reported to be involved in the modulation of

memory and learning under stress, and also in tissue injury and pain (Sandin et al., 1998; Ukai et

al., 1998). They are upregulated in rodents with age and this upregulation contributes to age-

associated cognitive impairment (Jiang et al., 1989; Nguyen et al., 2005)

As mentioned previously, the dynorphins do not behave as analgesics in the brain, although

early studies demonstrated their analgesic activity after intrathecal administration in the spinal

cord. Dynorphin A (1-13) is 6-10 times more potent than morphine after intrathecal injection (Han

and Xie, 1984; Herman and Goldstein, 1985; Nakazawa et al.; 1985), 700 times more potent than

Leu-enkephalin, and 50 times more potent than β-endorphin (Goldstein et al.; 1979). The

analgesic action of dynorphin is strong and longasting, with dynorphin A (1-13) shown to produce

analgesia in human for an average of 7 hours (Wen et al., 1985, 1987). More recent analysis

however, showed that the strong and long effects are produced due to the ability of dynorphin A

to induce paralysis and motor impairment (see section 2.b). Unfortunately, dynorphins do not

have robust antinociception.

Dynorphins are also suspected to have a significant role in regulating body fat and in

attenuation weight loss during negative balance, based on experiments on dynorphin knockout

mice, which showed reduced fat mass and increased weight loss during a fasting period compared

to control mice (Sainsbury et al., 1998).

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Table 3: Opioid activities of dynorphin A

Effect Results Reference Cardiovascular Effects Lower blood pressure

Heart rate

Laurent and Schmitt, 1983 Feuerstein and Faden, 1984 Gautret B and Schmitt, 1985

Temperature Control

Sligth hyperthermic effect Cavicchini et al., 1988

Respiration enhanced morphine’s depression

Woo et al., 1983

Hormonal Effects induce hormone secretion in some isolated tissue systems

Guaza et al., 1986

Feeding Behavior Stimulated food intake Yim and Lowy, 1984 Morley and Levine, 1985 Sainsbury et al., 1998

Paradoxically, dynorphins or potentially other endogenous opioid peptides have been

observed that they can contribute to secondary central nervous system injury via their opioid

activity (Faden et al., 1987; McIntosh et al., 1987).

b. Non-opioid activities of dynorphins

In the early studies, researchers were particularly interested in dynorphins because of

their role in pain regulation. In the mid 1980s, dynorphins were suspected to act as profound and

long lasting analgesics in the spinal cord (Han and Xie, 1984; Herman and Goldstein, 1985), since

earlier studies had showed that they have no analgesic effect in the brain at normal physiological

concentrations (Friedman et al., 1981; Walker et al., 1982). However, studies on rodents showed

that high concentrations of dynorphin A often resulted in hind limb paralysis, and even low doses

could result in transient paralysis (Herman and Goldstein, 1985). Hence, the previous conclusions

about the strong and longlasting analgesic effects of dynorphin A were not interpreted correctly

since the tail or limb movements were the dependent measure in the essays. Fortunately, so far

there have been no adverse effects reported in human studies (Wen et al., 1985, 1987), although,

because of the findings for rodents, it is still feared that high concentrations might induce

paralysis in patients. At paralytic doses, these peptides are neurotoxic, cause death of sensory

neurons, motor neurons, and interneurons in the spinal cord (Long et al., 1988). Paralysis has also

resulted when using the non-opioid dynorphin-derivative dynorphin A (2-17), which does not

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contain the tyrosine residue (Y1, see Table 2) necessary for interaction with opioid receptors. This

suggests that the paralytic effect is non-opioid (Faden and Jacobs, 1984). These non-opioid

dynorphin activities are critical for the development of chronic neuropathic pain. Prodynorphin

knockout mice do not maintain a chronic pain state induced by spinal nerve ligation (Wang et al.,

2001). Effects caused by dynorphin (Table 4) can be prevented by antagonists (e.g. MK-801) and

modulators of the N-methyl-D-aspartate (NMDA) receptors (Long and Skolnick, 1994; Woolf and

Thompson, 1991). The NMDA receptor is an ionotropic receptor for glutamate, and forms a

heterodimer between the NR1 (glycine binding domain) and the NR2 (glutamine binding domain)

subunits (Stephenson, 2006). In the central nervous system, NMDA receptors are thought to play

important roles in synaptic transmission, neuronal development, and certain types of learning and

memory (Hollmann and Heinemann, 1994 and McBain and Mayer, 1994). On the other hand,

neurotoxic activities of glutamate on postsynaptic NMDA receptors may contribute to the

pathophysiological processes involved in strokes, epileptic seizures, and several

neurodegenerative diseases (Choi and Viseskul, 1988).

Table 4: Some non-opioid effects of dynorphin A (Adapted from Wollemann and Benyhe, 2004): Effect Target Receptor Reference Hyperalgesia Inflammation Locomotory effects Injury, and neurological dysfunction Central motor Electric current potentiation Increase in EAA extracellular level Antianalgesia ACTH release Increased heart rate and arterial pressure

spinal cord rat hindpaw, spinal cord spinal cord hippocampus PAG neurons Hippocampus CA3 region spinal cord pituitary cell line

NMDA NMDA NMDA NMDA NMDA Cholecystokinin non-opioid non-opioid

Trujillo and Akil, 1991 Dubner and Ruda, 1992 Walker et al., 1982 Caudle et al., 1998 Bakshi and Faden, 1990 Shukla and Lemaire, 1994 Lai et al., 1998 Faden, 1992 Rady et al., 1999 Yarygin et al., 1998 Rochford et al., 1991

Abbreviations: NMDA N-methyl-D-aspartate; CNS central nervous system; EAA excitatory amino acids; ACTH

adrenocorticotrophic hormone

The NMDA receptors were proposed as mediators for the non-opioid activities of dynorphin

A. It has been shown that dynorphin interacts directly with NMDA receptors with a high affinity

(Massardier and Hunt, 1989; Chen et al., 1995A; Tang et al., 1999). Dynorphin A seems to bind

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to the NR1 subunit (Woods et al., 2006), although it can also have indirect interaction with this

receptor; dynorphin A can induce the release of excitatory amino acids (such as glutamate and

aspartate) which will stimulate NMDA receptors (Faden, 1992). Direct interaction studies indicate

that dynorphin A associates more strongly with NMDA receptors that are in a relatively closed

state than those in the open state (Tang et al., 1999). This interaction shortens the mean open time,

decreases the probability of opening of NMDA channels, but has no effect on the single channel

conductance (Chen et al., 1995B). In rat brain membranes, dynorphin and its related fragments

attenuate the binding of radioligands such as [3H]glutamate (agonist) (Massardier and Hunt, 1989)

and [3H]MK-801 (antagonist) (Shukla et al., 1992) by being in direct competition with them. This

binding profile suggests that the effects of dynorphin are both activating and inhibiting on the

NMDA receptors. The inhibition of the NMDA receptors by dynorphins is dependent on

extracellular pH in the range of 6.7-8.3 (Kanemitsu et al., 2003). However, the precise mechanism

for the inhibition of the NMDA receptor function by dynorphin has not been established.

Furthermore, depending on the neuronal population under studied, dynorphins can also activate

NMDA receptors (Jarvis et al., 1997; Zhang et al., 1997), and the concentration of glycine seems

to determine the action of dynorphin A on the NMDA receptor. More specifically, in a medium of

high glycine concentration, e.g. 10 µM, dynorphin A inhibits the activities of the NMDA receptor

gate (Zhang et al., 1997). The justification of the opposing effects of dynorphin A are unclear at

present. Big dynorphin seems to produce memory facilitation, locomotor activation and anxiety-

like behavior mediated through NMDA receptors, whereas dynorphin A and dynorphin B do not

have these effects (Kuzmin et al, 2006).

Several non-opioid activities of dynorphins cannot be assigned to NMDA-dependent

mechanisms. Dynorphin A can also modulate alpha-amino-3-hydroxy-5-methylisoxazole-4-

propionate (AMPA)/kainite receptor responses in neurons, which will exert neurotoxicity effects

on the neurons (Kolaj et al., 1995; Goody et al., 2003; Singh et al., 2003). Cell imaging

experiments have shown that dynorphin A can induce a time- and dose-dependent increase in

intra-neuronal calcium concentration through a non-opioid and non-NMDA mechanism (Tang et

al., 2000), with this effect attributed to the activation of the bradykinin receptors (Lai et al., 2006).

Normally, the opioid activities of dynorphins decrease the intra-neuronal calcium concentration

(Hu et al., 1998).

Being arginine rich and among the most basic naturally occurring peptides, dynorphins share

certain common properties with cell penetrating peptides (CPPs). Like CPPs, dynorphins may also

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be translocated across the plasma membrane, which may be relevant for interneuronal

communication in the central nervous system.

3. Cell penetrating peptides a. Introduction

Several peptides have been demonstrated to translocate across the plasma membrane of

eukaryotic cells by a seemingly energy-independent pathway, i.e. they internalize even when cells

are incubated at 4°C. Green et al. (1988), Frankel et al. (1988) and Joliot et al. (1991) were the

first groups to demonstrate the “spontaneous” cell membrane translocation of two proteins (the

HIV-1 Tat protein and the 60 amino acid homeodomain of the Antennapedia protein of

Drosophila). These proteins were found to contain short sequences responsible for their

translocation across the cell membranes. The first of the cell-penetrating peptides (CPPs)

identified was the penetratin peptide (pAntp), a peptide composed of 16 amino acids from the

third helix of the Antennapedia homeodomain (Derossi et al., 1994). Other natural and synthetic

peptides with similar properties have since then been identified.

CPPs are defined as a class of natural or synthetic, short (<30 amino acids), highly basic and

water-soluble peptides that are capable of translocation across various cell membranes while

carrying a “cargo” (e.g. oligonucleotides or proteins). The mechanism for this is dependent on the

peptide, and in addition to endocytosis there are probably to be other receptor– and transporter –

independent pathways. Overall these peptides are characterized by high efficiency and low lytic

activity and are generally non-cell-type specific (Derossi et al., 1998; Lindgren et al., 2000).

These translocating peptides have been applied as vectors for the delivery of hydrophilic

biomolecules and drugs into cytoplasmic and nuclear compartments of cells, both in vivo and in

vitro (Derossi et al., 1998; Lindgren et al., 2000), and retain they their translocating ability when

covalently linked to a cargo. They are useful vectors for pharmacologically interesting substances,

such as antisense oligonucleotides, proteins and peptides, that have many times their molecular

mass, and also for larger cargoes like plasmids and gold particles. These studies have opened up

new opportunities for the delivery of hydrophilic biomolecules and drugs into cells without

destroying the integrety of the cell membrane and with seemingly low toxic effect.

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b. Classes of Cell penetrating peptides

These interesting peptides can be divided into three different main classes: penetratins,

Tat-derived peptides, and transportans (Table 5).

Table 5. Amino acid sequences and the origin of three well-known cell-penetrating peptides.

Peptide Origin Sequence

Penetratin (pAntp) Antennapedia HD RQI KIWFQ50NRRMK WKK

Tat (48-60) fragment HIV-1 Tat protein GRK50KRRQRRRPPQ60

Transportan Synthetic chimeric GWTLNSAGYL10LGKINLKALAA20LAKKIL

Penetratin

Joliot et al (1991) showed that the Antennapedia homeoprotein of Drosophila internalizes

into living cells. The Antennapedia homeoprotein belongs to a class of transactivating factors that

binds to DNA through a specific sequence of 60 amino acids, the homeodomain. The

translocation ability can be attributed to the sequence of 16 amino acids within the third helix of

the Antennapedia homeodomain (residues 43-58) (Derossi et al., 1994). The peptide from this

sequence is called penetratin peptide (pAntp) (Table 5). It was the first reported example of a

homeodomain-derived translocating peptide and is one of the most widely studied of all CPPs. In

model membrane systems, pAntp can undergo an α to β secondary structure transition when there

is an increase in the concentration of the peptide or an increase in the negative charge of the

membrane surface (Magzoub et al., 2002). Derossi et al. (1996) showed that a helical structure

alteration of the peptide by substitution of some amino acids (Gln50 or Ile45, Gln50 and Lys55) by

Pro had no influence on the internalization of pAntp. The only difference between pAntp and the

proline substituted analogues is their cellular localization –pAntp is in the nucleus while proline

substituted analogues are in the cytoplasm. Therefore, the helical structure is not necessary for the

translocation mechanism of pAntp. On the other hand, N- and C terminal residues of the peptide

do appear to be important for cellular internalization. Most of the amino acids that constitute

terminal residues are basic amino acids (Lys and Arg). As a consequence, Ala-substitution of any

charged residue results in a large decrease in the uptake of the peptide (Fischer et al., 2000), in

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apparent contrast to the experiment of Derossi et al. (1996) regarding Lys55 (see above). The two

Trp residues also seem important in uptake of pAntp, and the Trp48 residue is more important than

Trp56, possibly related to the fact that Trp48 is localized deeper within the membrane (Christiaens

et al., 2002). Substitution of either of these residues by Ala or Phe has been shown to reduce the

translocation of the peptide (Fischer et al., 2000; Drin et al., 2001; Christiaeans et al., 2004) The

interfacial interactions of Trp with the membrane may significantly contribute to membrane

organization, which will allow the peptide to translocate (Derossi et al, 1996; Dom et al., 2003).

However, the discovery of the artifact problem due to the cell fixation process (see section 3.c) by

Lundberg and Johansson (2002) questions the role of Trp in CPPs.

HIV-1 Tat

The HIV-1 Tat peptide is derived from the Tat protein, an 86 amino acid transcription-

activating protein involved in the replication of Human Immunodeficiency Virus type 1 (HIV-1).

In 1988, two different groups (Green and Loewenstein, 1988; Frankel and Pabo, 1988) found that

the HIV-1 protein is taken up by cells. Translocation of the Tat protein is fast, and 50 % and 80%

of the protein was found to be bound to the plasma membrane after 1 min and 15 min,

respectively, at 37 °C. After translocation, the cellular localization of the Tat protein is in the

nucleus and the cytosol (Frankel and Pabo, 1988), with incubation at low temperature having no

effect on the rate of binding (Mann and Frankel, 1991). After chemical cross-linking with other

proteins (e.g. RNase A and β-galactosidase), two segments of the Tat protein, Tat (1-70) and Tat

(37-72), were localized in the nucleus and cytosol (Fawell et al., 1994). Tat (37-72) includes both

the putative α-helical domain (residues 34-47) and a basic region (residues 49-57). Vivès et al.

(1997) concluded that the shorter segment Tat (48-60), which contains the whole basic region of

the protein, was more efficiently transduced than the other active peptide sequences of the Tat

protein. The (48-60) segment is rich in the basic amino acids (Arg and Lys) and the Arg residues

have been found to play a crucial role in internalization of the Tat peptide (Werder et al., 2000). It

does not possess the putative α-helical domain, and as such, the α-helical region seems to be

unnecessary for its translocation. Initially, researchers thought the translocation mechanism of Tat

to be via an energy-independent pathway, as is the case for pAntp. However, the discovery of the

artifact problem due to the cell fixation process once again showed these conclusions to be

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misleading. Live cell experiment have since then shown that the uptake mechanism of the Tat

peptide is via the endocytotic pathway (Richard et al., 2003).

Transportan

Transportan is a synthetic chimeric peptide that is a combination of the 12 N-terminal

amino acids of the neuropeptide galanine and the full length 14 amino acid wasp venom peptide

mastoparan, linked by an extra Lys (K13) (Langel et al., 1996). This peptide, like the other CPPs,

is capable of coupling different cargoes and translocating. Its uptake kinetics is more rapid than

that of both pAntp and the Tat peptide (Hällbrink et al., 2001). However, at high concentrations it

causes leakage in the cell, reveals membrane perturbation in model systems and has GTPase

activity that may cause unwanted side effects when used in biological systems (Hällbrink et al.,

2001; Magzoub et al., 2003). In an attempt to prevent these side effects, an analogue to

transportan was developed by deleting the first six N-terminal amino acids

(AGYLLGKINLKALAALAKKIL) and the new peptide was named Tp10 (Soomets et al., 2000).

Unfortunately, further investigation revealed that also Tp10 induces membrane perturbation,

causing leakage in model systems (Magzoub et al., 2003; Bárány-Wallje et al., 2007). The

toxicity of transportan is probably related to the mastoparan part of the peptide. Mastoparan, like

the peptide melittin, originates from venom and forms pores in the membrane (Matsuzaki et al.,

1995 and 1996). The amphipatic α-helix within C-terminus of transportan, the mastoparan part,

has been shown to be very important for peptide translocation (Soomets et al., 2000; Lindgren et

al., 2000). Transportan and Tp10 do not contain arginine residues and are not highly charged in

comparison to pAntp and the Tat peptide. Based on these considerations, it can be assumed that

transportan and Tp10 have at least partially different mechanisms of internalization into the cell

from pAntp and the Tat peptide. A significant decrease in membrane leakage has been found

when Tp10 is linked with a large cargo (Bárány-Wallje et al., 2007). Therefore, it has been

proposed that the non-endocytotic pathway of Tp10, and probably also that of transportan, can be

inhibited by the presence of large hydrophilic cargoes.

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c. Mechanisms

Hypothetically, if certain amino acid sequences are responsible for the cell membrane

translocation, they have been developed by nature to facilitate transport of certain proteins. One

should keep in mind that this process may not have been developed only in the “good” sense,

since such translocation sequences of proteins may also be involved in infectious diseases (e.g. the

prion protein) (Lundberg et al., 2002).

Even today, the translocation mechanisms of CPPs are under debate, but it is clear that the

peptide-membrane interactions must be of fundamental importance for the translocation process.

Certain CPPs show an energy-independent translocation, in fact non-reduced uptake, either at 4ºC

(Derossi et al., 1994) or in the presence of endocytosis inhibitors (Vivès et al., 1997). Many

peptides show these characteristics in experiments with fixed cells. In 2002 it was shown that in

the cell handling procedure and the positively charged proteins may appear to translocate across

the membrane because of the presence of the artifacts (Lundberg and Johansson, 2002). The

artifacts are the result of the cell fixation procedure which causes the disruption of the cell

membrane and thus certain peptides could have been mistaken for CPPs. Further reports, e.g.

Koppelhus et al (2002) and Richard et al. (2003), demonstrated that the uptake of the Tat

fragment and penetratin is mainly endocytotic and is cargo dependent. There are most likely

various competing mechanisms contributing to the cellular uptake of the peptides, and, due to the

physico-chemical diversity of CPPs, different mechanisms of internalization for different CPPs

are likely.

Almost all CPPs have a high content of the basic amino acids, resulting in a positive net

charge. This charge is important for the interaction between the peptides and the membranes, but

it is not sufficient for translocation (Futaki et al., 2001). The secondary structure of the peptides

has been studied under a number of different conditions. In contact with model phospholipid

membranes, CPPs generally have a α-helical secondary structure induced by the membrane

interaction, although penetratin can also adopt a β-sheet structure depending on the charge of the

lipid bilayer and total lipid-to-peptide (L/P) molar ratio. The proportion of β-sheets is higher when

penetratin is in contact with vesicles containing at least 30% negatively charged headgroups at

low L/P ratio, or when the charge density of vesicles is increased. Furthermore, if the charge

density is fixed, reduction of the L/P ratio results in an α→β structure conversion (Magzoub et

al., 2002).

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Currently, there are several proposals for the translocation mechanism of CPPs. Since 2002 a

large number of studies have investigated the uptake mechanism by live cell experiments, with the

most favored mechanism being endocytotic uptake (Drin et al., 2003; Richard et al., 2003; Vivès

et al., 2003; Potocky et al., 2003; Fischer et al., 2004; Nakase et al, 2004), which requires ATP

and is thus energy-dependent. There are two different types of endocytosis (Fig. 2). The best

known is “phagocytosis” (literally, cell-eating) and is used by specialized mammalian cells,

macrophages, for the ingestion of large particles, such as dead cells, bacteria and viruses. The

second endocytotic type, which occurs in all cells, is “pinocytosis” (literally, cell-drinking). This

process concerns the uptake of solutes and single molecules, such as proteins from outside the

cell, through the formation of vesicles called endosomes. There are two different subtypes of

pinocytosis – micropinocytosis and macropinocytosis (Conner and Schmid, 2003).

Macropinocytosis is defined as the series of events initiated by extensive plasma membrane

reorganization or ruffling, in order to form an external macropinocytic structure that is later

enclosed and internalized. Macropinocytosis is induced in many cell types, which do not undergo

phagocytosis upon stimulation by growth factors or other signals (Dowrick et al., 1993; Hewlett et

al., 1994).

Micropinocytosis includes caveolae-mediated endocytosis, clathrin-mediated endocytosis and

caveolin- & clathrin-independent endocytosis. Caveolae-mediated endocytosis occurs in

membrane domains rich in cholesterol and sphingolipid (Pike et al., 2002). Caveolae contain a

dimeric protein known as caveolin that binds with great affinity to cholesterol (1:1 ratio) (Murata

et al., 1995). Cholesterol is a key component of the caveolae structure and helps to regulate

caveolae functions. Caveolae and lipid rafts have similar lipid and protein components, but

caveolae do not contain glycosyl–phosphatidylinositol-anchored proteins and the lipid rafts do not

contain caveolin (Brown and Waneck, 1992 and Smart et al., 1999). The scientific community is

still debating whether caveolae should be considered as a type of lipid raft, or caveolae and rafts

are two microdomains acting as completely separate entities.

Clathrin-independent endocytosis normally occurs at special sites called coated pits, covering

about 0.5–2% of the cell surface (Brown and Petersen, 1999), which concentrate surface proteins

(such as receptors and dynamin) before internalization. The mechanism of caveolin- & clathrin-

independent endocytosis is still poorly understood (Conner and Schmid, 2003).

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Macropinocytosis(>1 µm)

Phagocytosis

Plasma membrane

Cytoplasm

Extracelluar fluid

Clathrin-mediatedEndocytosis (85-110 nm)

Particle

Receptor

Dynamin

Clathrin coat

Caveolin

Caveolae-mediatedEndocytosis (50-100 nm)

Pinocytosis

Plasma membrane

Plasma membrane

Figure 2. Schematic overview of the major classes of endocytotic pathways.

Several studies now indicate that macropinocytosis can function as one of the pathways for

the cellular uptake of some CPPs (Nakase et al., 2004 and 2007; Wadia et al., 2004; Kaplan et al.,

2005, Magzoub et al., 2006). Nakase et al. (2004) observed the uptake of pAntp to be rather

insensitive to EIPA (inhibitor of macropinocytosis), suggesting that pAntp has other

internalization mechanisms. The presence of inhibitors of caveolin- & clathrin-mediated

endocytosis and phagocytosis have been shown not to have any significant effect on the cellular

uptake of a Tat peptide conjugate (Wadia et al., 2004). Richard at al (2004) confirmed

transduction to be caveolin-mediated endocytosis independent, but showed that free Tat peptide is

clathrin-mediated endocytosis dependent. However, other studies with different cell types have

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suggested that uptake has a caveolae-dependent pathway (Ferrari et al., 2003; Fittipaldi et al.,

2003). Therefore the cellular uptake mechanisms of Tat are probably dependent on several

factors, e.g. the presence and nature of the cargo or the cell type.

Membrane-associated proteoglycans, such as heparan sulfate proteoglycans (HSPG), seem to

be important for cellular uptake of arginine-rich CPPs (Suzuki et al., 2002; Console et al., 2003;

Richard et al., 2005; Nakase et al, 2007). Proteoglycans belong to a special class of glycoproteins

and are the combination of one protein and one or more glycosaminoglycan (GAG) chain(s).

Proteoglycans are the major component of the mammalian extracellular matrix and are involved

in binding cations (such as calcium and sodium) and water due to their high negative charge

caused by the GAG chains. Proteoglycans also contribute to movement regulation of the

molecules through the matrix. There is a strong and rapid electrostatic interaction between the Tat

peptides and HSPG, and this interaction is more probable than the binding of the peptides directly

with the lipid bilayer. Moreover, this HPSG interaction can be viewed as the primary step of

peptide translocation (Zieger and Seelig, 2004). According to Fuchs and Raines (2004), the Tat

translocation pathway follows the steps:

1) The peptide binds with HSPG

2) The complex undergoes uptake by endocytosis

3) The complex dissociates in the endocytic vesicles due to hydrolytic degradation of heparan

sulfate by heparanase (Yanagishita and Hascall, 1984)

4) The peptide escapes from the endocytic vesicles.

HSPG has been shown to be crucial in the induction of macropinocytosis (Nakase et al,

2007). Experiments with one type of proteoglycan-deficient Chinese-hamster ovary (CHO) cells

were performed in order to examine the importance of proteoglycans in the cellular uptake of the

Tat peptides. The observations did not show a very large decrease in the uptake of the peptides

(Nakase et al, 2007), and based on these results it was suggested that proteoglycans are not the

only receptor for the cellular uptake of arginine-rich peptides. This conclusion is in agreement

with the proposal that the uptake pathways are dependent on several factors, which have been

mentioned previously.

The endocytosis pathways are important, but it should not be forgotten that they may

operate in parallel with direct energy-independent membrane-based mechanisms (Padari et al.,

2005; Deshayes et al., 2005). Some of the proposed models for the non-endocytotic mechanisms

are: the inverted micelle model, the carpet model, the pore formation model and the membrane-

thinning model (Fig. 3).

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Alain Prochiantz and coworkers proposed the hypothesis of the inverted micelle model, Fig.

3B (Derossi et al., 1996). This mechanism could occur due to the interaction of the hydrophobic

amino acids with the membrane. The positive charge of the peptide would be attracted by the

negative charge of the membrane, followed by membrane shuttling aided by of the interaction of

the hydrophobic amino acids with the membrane. This mechanism would be valid only for

peptides containing a certain fraction of hydrophobic amino acids (e.g. penetratin), and has not

been considered for the Tat peptide.

Figure 3: Energy-independent internalization mechanisms proposed for CPPs. Models with no proposal toxic effect: a) direct penetration and b) ‘inverted micelle’ model (e.g. structure-mediated). Models based on the membrane inter actions similar to those of certain antimicrobial peptides, toxins and venoms: c) ‘carpet’ model, d) ‘toroidal pores’, and e) ‘barrel-stave pores’ (adapted from Magzoub and Gräslund, 2004)

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Another suggestion for a non-endocytotic mechanism is the carpet model, Fig. 3C (Pouny et

al., 1992). In this model the peptides bind to the phospholipid membrane surface until a threshold

concentration is reached, and the peptides permeate the membrane in a detergent-like manner,

with transient holes formed as peptides cover the cell surface. The carpet mechanism requires

neither a specific peptide structure, nor the formation of structured channels, in contrast to the

pore formation mechanism.

The third hypothesis concerns pore formation models, Fig. 3 D and E (Matsuzaki et al., 1996;

Lundberg et al., 2002). Pore formation is caused by associated bundles, formed by amphipathic α-

helical peptides. The outwardly facing hydrophobic residues interact with the lipid membrane, and

the inwardly facing hydrophilic surfaces form the pore. Pores may be stable or transient.

The last hypothesis describes the membrane-thinning model (Ludtke et al., 1995 and Lee et

al., 2005) and suggests that carpeting of the membrane by the peptides is followed by electrostatic

perturbations in the outer leaflet. The thinning of the membrane is then the result of lateral

rearrangement of the lipids.

In accordance with the endocytosis mechanisms for the cellular uptake of CPP, the peptides,

with or without cargo, will be found in the large vesicles – endosomes or macropinosomes – after

internalization. The next important question that has to be answered in order to understand the

mechanisms is: How do the peptides escape from the large vesicles to be released in the cytosol?

The details of this mechanism are unclear. One possible explanation could be found in the

endosome acidification caused by enzymatic activity inside the vesicles, resulting in pH decreases

from 7.4 to 5.5 (Potocky et al., 2003; Fischer et al, 2004). This concept is supported by model

system studies, where CPPs enclosed within large unilamellar vesicles are observed to escape

from the vesicles once a transmembrane pH gradient is created whereupon the resulting inner

vesicle environment became acidic (Magzoub et al., 2005A; Björklund et al., 2006).

4. Alzheimer’s disease

In 1901, a German psychiatrist, Dr. Aloïs Alzheimer, observed that a patient suffered from

a mental disorder that could not be explained by the knowledge of medicine at the time. Five

years later, after the patient’s death, Alzheimer microscopically examined her brain and

discovered the presence of senile (neuritic) plaques (fibrils) and neurofibrillar degeneration at the

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level of the cerebral cortex (Alzheimer, 1907). In the 1980s, Glenner and Wong (1984) reported

that the extracellular senile plaques mainly consisted of the amyloid β peptide (Aβ), while

intracellular neurofibrillary tangles mainly contained the tau protein. Even though tau proteins

alone cause a type of dementia other than Alzheimer’s disease (AD), the Aβ alone form senile

plaques and may induce tau deposition which subsequently results in AD (Hardy and Selkoe,

2002). Most commonly Aβ is composed of 40-42 residues cleaved from the Alzheimer’s

precursor protein (APP) (Hardy and Selkoe, 2002). Usually, the APP is cleaved by two proteases,

α- and γ-secretase, and the released fragments form neither toxic oligomers nor fibrils (Hardy and

Selkoe, 2002). The pathological cleavage pathway of APP to give Aβ peptides still involves γ-

secretase, but the second protease is β-secretase rather than α-secretase (Table 6).

Table 6: Amino acid sequence of amyloid β peptide

Peptide Sequence

Aβ (1-42) DAEFR5HDSGY10EVHHQ15KLVFF20AEDVG25SNKGA30IIGLM35VGGVV40IA

Aβ (1-40) DAEFR5HDSGY10EVHHQ15KLVFF20AEDVG25SNKGA30IIGLM35VGGVV

In solution and at sufficiently low concentration Aβ appears as a monomer with unstructured

conformation. At higher concentrations, aggregation occurs and results in the formation of a β-

sheet structure. Soluble oligomeric aggregates of Aβ, which assemble into amyloid material, are

reported to mediate toxic effects on neurons and synapses (Kuo et al. 1998; Hardy and Selkoe

2002). The aggregation of Aβ seems to be related to the fact that the peptide is built of a

hydrophilic N-terminal domain and a more hydrophobic C-terminal domain, with a central

hydrophobic cluster (residues 17-21). The Aβ (1-40) and Aβ (1-42) fragments have different

critical concentrations to initiate spontaneous aggregation. The critical concentration is slightly

lower for Aβ (1-42), and therefore the monomeric form of this peptide is less stable (Harper and

Lansbury, 1997). The concentration of Aβ in the cerebrospinal fluid is well below the critical

concentration, so Aβ needs to be enriched in a specific region of the brain in order to reach the

critical concentration to aggregate. Membrane-peptide interactions can be one of the processes

that contribute to increased Aβ concentration (Terzi et al., 1997). The estimated number of AD

patients in the world at present is approximately 20-25 million.

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Aims

The main objectives of the work presented in this thesis were as follows:

1. To determine whether dynorphin peptides have the capacity to penetrate across the plasma

membrane; based on the sequence similarity to CPPs in their high content of basic and

hydrophobic amino acid residues.

2. To study the membrane interactions of dynorphin neuropeptides in different model

systems, in order to obtain insight into the translocation mechanism(s).

3. To define if the penetration or the membrane interaction of dynorphin may give rise to a

non-opioid activity to the peptide, in the context of intracellular biological activities as

well as calcium signaling.

4. To examine the membrane interaction of Aβ (1-40) in a model system, in order to provide

better insight into the aggregation processes.

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5. Methods a. Cell cultures

Different types of cells were used to study the ability of dynorphins to translocate across

the plasma membrane into different cells – HeLa, Cos-1 and PC12. The HeLa cells and the COS-1

cells belong to immortal cell lines (cells with the ability to proliferate indefinitely either through

random mutation or deliberate modification) derived from a cervical cancer tissue obtained from

Henrietta Lack in 1951 and African green monkey kidney, respectively. The opioid receptors are

not expressed at detectable levels in the HeLa cells (Kim et al., 2004). The PC12 cells, established

from a rat pheochromocytoma, have many properties in common with primary sympathetic

neurons and chromaffin cell cultures. This cell line is primarily used as a neuron model.

b. Lipid membranes Membranes

The cell membrane is what defines the cell and keeps its components separate from

outside cells or organisms. The membrane is not only responsible for creating a “wall”, it must

also act as a threshold through which selected molecules enter and exit the cell when necessary.

The cell membrane is composed of a phospholipid bilayer in which membrane proteins are

embedded. It is the structure of the self-assembled lipid bilayer, which prevents free passage for

most molecules through the membrane and the lipid bilayer is the basic component of any

membrane in living cells.

The function of the lipid bilayer as a barrier can be understood from its molecular structure.

Each phospholipid molecule contains a hydrophilic part, the so-called polar head group region,

and a hydrophobic part, also known as the nonpolar tail region. The fatty acid tail is composed of

a string of carbons and hydrogens, attached to glycerol. The phosphate group also attached to the

glycerol forms the polar head group region and the phosphate group can contain choline, glycerol,

ethanolamine or etc. Such molecules, containing both polar and nonpolar regions, are also called

amphipathic molecules. The hydrophilic region is attracted by the aqueous environment while the

hydrophobic region is repelled from such environment. The phospholipids self-organize

themselves in the bilayer to hide their hydrophobic tail regions and to expose the hydrophilic

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regions to water. This organization is spontaneous and does not require energy. The bilayer,

functions as the “wall” between the inner and out outer cell environment.

Biological membranes typically include a mixture of lipids. Sphingolipids in the plasma

membrane outer leaflet tend to separate from glycerophospholipids, and co-localize with

cholesterol in microdomains called lipid rafts (Simons and Ikonen, 1995). The close packing of

sphingolipids in association with cholesterol has been attributed to the lack of double bonds in

sphingolipid hydrocarbon chains. Glycerophospholipids often include at least one kinked fatty

acid, with one or more double bonds.

Biomembrane mimicking solvents

Phospholipid vesicles are an artificial membrane system and have been intensively used as

a tool to determine the properties of biological membranes. The use of the vesicles allows easier

manipulation of various parameters of the membrane systems and thus, the membrane transport

systems can be investigated without potentially interfering reactions that occur in the cell

membrane.

Various unilamellar phospholipid vesicles are also useful model systems. There are three

types of vesicles commonly used for biophysical studies: A) Small unilamellar vesicles (SUVs),

produced by ultrasound (sonication). The resulting vesicles are less than 100 nm in diameter. The

vesicle has a strong curvature, causing nonuniform tension on the inner and outer leaflets of the

bilayer. B) Large unilamellar vesicles (LUVs), produced by repeated extrusion through a

polycarbonate microfilter. The diameter of LUVs varies from 100 to 200 nm (Fig. 4, left). C)

Giant unilamellar vesicles (GUVs), produced by an electro-formation method. The diameter of

these vesicles is in the range 10 to 100 µM.

The larger vesicles are better mimics of the plasma membrane belonging to a true biological

cell, but the light scattering from large particles makes them problematic to be used for optical

spectroscopic studies.

LUVs were prepared by the following steps: 1) Drying a solution of the desired composition

of lipids in an organic solvent by lyophilization or evaporation. 2) Hydrating the lipid film using a

suitable buffer, to give a large multilamellar suspension. 3) To increase the efficiency of

entrapment of water-soluble compounds and thus to decrease lamellarity, the hydrated lipid

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suspension is subjected to 4 freeze/thaw cycles. 4) LUVs with a narrow size distribution were

obtained by repeated extrusions (20 times) of the sample through polycarbonate filters with 100

nm pore size.

Figure 4: Schematic illustration of a large unilamellar vesicle (LUV) (left-hand side) and SDS detergent micelle

(right-hand side)

Two types of phospholipid were used to prepare the vesicles for this study: lipids with a

negative charge on the head group, POPG (1-palmitoyl-2-oleyl-phosphatidylglycerol), and lipids

with a neutral (or zwitterionic) head group, POPC (1-palmitoyl-2-oleyl-phosphatidylcholine),

illustrated in Figure 5.

Figure 5: Illustration of the structure of POPG (1-palmitoyl-2-oleyl-phosphatidylglycerol), and lipids with a neutral or zwitterionic head group, POPC (1-palmitoyl-2-oleyl-phosphatidylcholine) with their different regions.

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Small spherical micelles can also serve as good mimics of a membrane for certain

spectroscopic studies (Öhman et al.,1995). Micelles are produced by various amphiphiles. The

most common type is formed by sodium docecyl sulfate (SDS), Fig 4, right. SDS is an anionic

surfactant forming micelles (approximately 35-40 Å in diameter) above the critical micellar

concentration (CMC) which is about 8 mM in water.

c. Fluorescence Spectroscopy Introduction

Fluorescence is the emission of light from a molecule that has been excited by the

absorption of light. Fluorescence arises when molecules (fluorophores) in the electronically

excited state (S1 or S2), due to absorption of light of specific wavelength, spontaneously return to

the ground state (S0) by emission of a photon. The emission has to take place between two singlet

states for fluorescence to occur. If there is a transition from a triplet excited state to a singlet

ground state, the deexcitation process called phosphorescence takes place instead. The underlying

photophysical processes can be illustrated by a Jablonski diagram (Fig. 6).

Figure 6: Jablonski scheme of excitation and deexcitation. Bold horizontal lines represent the energy level of the vibrational ground state of electronic states and thin horizontal lines represent vibrationally excited states. Absorption (Abs), fluorescence (F), internal conversion (IC), intersystem crossing (ISC), phosphorescence (Ph), vibrational relaxation (VR).

ISC

VR

IC IC

Abs

Abs

F

VR

VR

VR

VR

VR

VR

Ph

S0

S1

S2

T1

T2

VR

IC

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The excited state can lose its energy through many other processes besides direct emission of

light. The nonradiative processes that compete with fluorescence are the following:

• Internal conversion is a transition between two energy states not characterized by emission

of a photon because the energy is instead transferred to an inner orbital electron, which is

ejected from the atom (with the rate constant kic).

• Intersystem crossing is the transition from a singlet to a triplet state (T1) and vice versa

(characterized by rate constant kisc).

• Quenching processes of various types (characterized by rate constant kq[Q]).

All these processes compete directly to depopulate the excited singlet state. The overall rate

constant (k) for the depopulation of the excited state is given by the equation:

[ ] ∑+=+++= ifqiscICf kkQkkkkk (1)

where kf is the fluorescence rate and Σki is the sum of the rate constants for all the other

processes. An important quantity is the quantum yield of fluorescence. It is the number of emitted

photons divided by the number of absorbed photons which is equal to the relative probability of

fluorescence with respect to the probability of all other deexcitations.

kkf=Φ (2)

The fluorescence intensity can be derived from the quantum yield (Φ).

ΦΨ= .I.F Aλ (3)

In the equation, Ψ is the instrumental correction factor and IA is the initial population of the

excited state.

Fluorescence spectroscopy is a highly sensitive optical spectroscopy.

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Tryptophan fluorescence

Proteins and peptides may contain three types of aromatic amino acid residues

(tryptophan, tyrosine, phenylalanine) that may contribute to their intrinsic fluorescence.

Tryptophan (Trp) has much stronger fluorescence than the other two aromatic amino acids,

although the quantum yield is almost the same for Trp and tyrosine (Tyr) – ΦTrp =0.13±0.01; ΦTyr

=0.14±0.01 (Lakowicz, 1999). The higher brightness of Trp is resulting from a higher extinction

coefficient (ε280Trp= 5690 M-1.cm-1 – ε280

Tyr= 1280 M-1.cm-1) (Gill and von Hippel, 1989), which is

defined as the quantity of light absorbed by the Trp, whereas the quantum yield represents the

amount of absorbed energy which is released as energy (fluorescence light). So Trp is the best

naturally occurring fluorophore suitable for the study of proteins and peptides. Trp emission is

very sensitive to the polarity of the solvent. Trp fluorescence gives important information on the

structure and function of proteins and peptides, such as in protein folding or in membrane

interactions. Because of the shift in the wavelength of the Trp fluorescence emission maximum,

the polarity of the peptide’s environment can be determined. There is a shift from about 356 nm in

a hydrophilic environment to about 320 nm in a hydrophobic environment.

When there is no Trp in the protein or peptide under study, Tyr can be used as an intrinsic

fluorophore. Tyr like Trp has strong absorption bands around 280 nm. When excited by light at

this wavelength, Tyr has a characteristic emission profile, but is a weaker emitter than Trp, as

mentioned previously. Phe is typically a too weak intrinsic fluorophore to be suitable for

biophysical studies of proteins and peptides

Calcein leakage

An efficient method to investigate membrane perturbation effects of the peptides is by

calcein leakage from LUVs entrapping high concentrations of the fluorophore calcein. The

advantages of calcein (a fluorescein derivative) are its high solubility, self-quenching at high

concentration and its negative charge at neutral pH (negative charge minimizes its interaction with

the membrane).

In the presented studies LUVs with entrapped calcein were prepared by hydrating a lipid film

of the desired composition with 70 mM calcein present in the buffer (final pH was adjusted to 7.2

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by addition of NaOH from a 10 M stock solution). The vesicles were prepared as described in

section 5.b (Biomembrane mimicking solvents). Calcein that was not entrapped was separated

from the LUVs on a Sephadex-G25 column. The fluorescence emitted by calcein at 70 mM was

low (self-quenching), but increased considerably upon dilution. The release of calcein from the

LUVs was consequently monitored by an increase in the fluorescence intensity. The fluorescence

intensity was measured at 520 nm (excitation at 490 nm).

The maximum fluorescence intensity (100 % leakage) was determined by lysing the vesicles

with a 10 % (w/v) solution of the detergent Triton X-100. The percent leakage value was

calculated according to the following equation:

%FF

FF100leakagepercent0

0

max⎟⎠⎞

⎜⎝⎛

−−

= (4)

where F0 is the fluorescence intensity of the intact vesicle, F and Fmax represent the intensity

before and after the addition of the detergent.

Calcium influx

Fura-2 is a ratiometric fluorescence dye derived from EGTA and BAPTA that binds to

free calcium. It is called ratiometric because a ratio between two fluorescence intensities is used to

measure the calcium concentration. It was invented to study free intracellular calcium

(Grynkiewicz et al., 1985) and to replace an existing common fluorescence dye (quin2). The

previously used dye had exactness problems because its fluorescence emission was too close to

the autofluorescence from the cell. It increased its fluorescence intensity without substantial shift

in either excitation or emission wavelength when the indicator bound to calcium. Fura-2 has high

bright fluorescence.

In our experiments, LUVs with entrapped fura-2 (fura-LUVs) were prepared by hydrating a

lipid film of the desired composition with 100 µM fura-2 present in the buffer (final pH was

adjusted to 7.4 by addition of KOH from a 5 M stock solution). The vesicles were prepared as

described in section 5.b (Biomembrane mimicking solvents). Fura-2 that was not entrapped was

separated twice from the LUVs on a Sephadex G-25 column. The rate of calcium influx was

monitored by measuring the ratio of fluorescence emission of fura-2 at 510 nm, using the two

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excitation wavelengths 340 and 380 nm, F340/F380, as a function of time. The use of the ratio

gives an advantage of omitting a large number of confounding factors, such as ambient light, dye

concentration and autofluorescence background.

The calcium concentration inside the vesicles, [Ca2+]i was calculated using the equation given

in (Grynkiewicz et al.,1986):

[ ] ⎟⎟⎠

⎞⎜⎜⎝

⎛⎟⎟⎠

⎞⎜⎜⎝

⎛−

−=+

2b

2f

max

mindi

2SS

RRRR

KCa (5)

R is F340/F380, Rmin is F340/F380 at zero calcium and Rmax is F340/F380 at calcium saturation.

Sf2 is the fluorescence intensity at the excitation wavelength 380 nm for free dye; and Sb2 is the

fluorescence intensity at calcium saturation.

It is important to perform a number of control experiments in the study of calcium influx into

phospholipid vesicles in order to verify that the results are not influenced by fura-2 leakage out of

the vesicles or due to spontaneous influx of calcium. In the first control experiment calcium was

added to a medium with fura-LUVs. If there is no change observed as a function of time of the

F340/F380 ratio, the spontaneous penetration of calcium into the vesicles is not significant.

However, an immediate increase of the F340/F380 ratio was detected once the calcium was added

to the medium. The increase should be a result of the small amounts of fura-2 remaining outside

of the vesicles after purification. In the second control experiment 4 mM EDTA was added at the

end of the experiments where the vesicles had been incubated with peptide and calcium. If the

addition of EDTA does not have any effect on the F340/F380 ratio, it shows that the fura-2-Ca2+

complexes were present within the phospholipid vesicles and fura-2 does not leak outside of the

LUVs. Addition of EDTA may have a small effect on the F340/F380 ratio, again due to the small

amounts of fura-2 remaining outside of the vesicles after purification. It was also verified that if

calcium was added to a medium with lysed vesicles where fura-2 was no longer entrapped, the

addition of the same EDTA concentration changed the fluorescence ratio F340/F380 to that of

fura-2 alone without bound calcium.

Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) (also known as Förster resonance energy

transfer or singlet-singlet energy transfer) is the non-radiative energy transfer from a donor

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molecule to an acceptor molecule. It is a result of dipole-dipole interactions. The emission and

reabsorbtion of a photon are not involved (Förster, 1948), because each dye molecule acts like a

classical dipole antenna, emitting and absorbing energy. A fluorescent donor is excited at its

specific fluorescence excitation wavelength and its excited state energy is transferred to an

acceptor. The emission spectrum of the donor needs to overlap with the absorption spectrum of

the acceptor. The efficiency of energy transfer (E) depends also on the distance between the

donor-acceptor pair (between 10-75 Å) and FRET can therefore be used to measure the distances

between molecules. The measured efficiency must be related to the distance between the two

fluorophores, to obtain useful structure information from energy transfer. The efficiency of energy

transfer (E) can be obtained by measuring the fluorescence intensities of the donor with acceptor,

FT and without acceptor, FD:

D

TFF1E −= (6)

The relation between the transfer efficiency and the donor-acceptor distance R is given by the

equation:

)RR(

RE 66

0

60

+= (7)

where R0 is called Förster distance and it is the distance at which energy transfer is 50% efficient.

In our FRET experiment we studied the quenching of Trp fluorescence due to resonance

energy transfer in LUVs symmetrically labeled with 10 mole% dansyl-DHPE, to detect peptide

translocation through the membrane of the model system. Dansyl labeled vesicles (dansyl-LUVs)

were prepared by hydrating the lipid film composed of POPC, POPG, and dansyl-DHPE

(70:20:10 molar ratio). Dried lipids were dispersed in a buffer solution containing 50 mM

phosphate buffer at pH 7.4. The vesicles were prepared as described in section 5.b (Biomembrane

mimicking solvents). The experiment setup followed the protocol described by Matsuzaki

(Matsuzaki et al., 1995, 1996 and 1997).

In the experiment the Dansyl-LUVs were mixed with peptide (Fig. 7A). The peptides binding

to the vesicle reduced the Trp fluorescence intensity due to quenching by FRET to dansyl (Fig.

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7B). After incubation, a large excess of a second population of dansyl-free LUVs was added (Fig.

7C). Increased intensity indicates the relief from FRET caused by the redistribution between the

two vesicle populations of the peptide molecules which had been bound to the outer surface of the

first population of vesicles. To determine the minimum of energy transfer, both populations of

vesicles were added simultaneously to the peptide solution (F0). The peptide remains on the outer

leaflet of the first population of vesicles and does not translocate through the phospholipid bilayer

(Fig. 7D), if there was no change in the final fluorescence intensity observed in two types of

experiments. Once the peptide translocates the redistribution between the two vesicle populations

will be not total (Fig. 7E) and will result in a decrease of fluorescence intensity compared to F0.

W

W

Incubation

TranslocationNo TranslocationW

W

dansyl-labeled phospholipid

A B

C

Peptide remains on the outer leaflet of dansyl-LUVs

D E

Figure 7: Schematic illustration of the FRET experiment

In a different approach, FRET was also used to determine the relative strength of interaction

of the peptides with a membrane model system. Initially the potassium concentration outside the

vesicles was kept low. The FRET experiment was performed as previously described. After the

addition of dansyl-free LUVs, potassium chloride was added to the sample. Different kinetics of

the redistribution between the peptide and the two vesicle populations could be observed.

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d. Circular Dichroism Spectroscopy

Circular dichroism (CD) is defined as the differential absorption by a chiral molecule of

left and right hand circularly polarized light (Fig. 6). In circularly polarized light the electric and

magnetic field vectors are not oscillating in amplitude but rotate around the direction of

propagation (Fig. 7). Circularly polarized light can be thought to be composed of two waves

which are plane polarized perpendicular to each other and have a 90° phase difference.

1

5

y y

x

Before sampley yAfter sample

1 23

4

1 23

45

5

2

3

4

θ1

5

2

34

12

3

45

1234

5

RH RHLH LH

y y

x x

A C

B D

Figure 8: (A) and (B). Combination of two circularly polarized waves to a plane polarized wave seen along the direction of propagation. The two circularly polarized waves have the same amplitude. The electric field vector of the plane polarized wave oscillates between positions 1 and 5. Positions 2, 3 and 4 give arbitrary intermediate values. (C) and (D): Different absorption of right hand circularly polarized and left-hand circularly polarized light produce elliptically polarized light from plane polarized light. Elliptically polarized light is a combination of two circularly polarized waves with different amplitudes. (Adapted from CR Cantor, PR Schimmel, Biophysical Chemistry, Fig. 8.1)

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Figure 9: Circularly polarized light is composed of two plane polarized waves. Only the electric field components are shown. The vector is the electric field vector that results from the addition of the two components. (Adapted from Halliday, Resnick, Krane, Physics, Fig. 44-17)

Both right- and left-handed circularly polarized light obey Beer-Lambert’s law, and CD is

defined as the difference in extinction coefficient:

[ ] c.l.c.l.)()()(A)(A)(A RLRL ελελελλλ Δ=−=−=Δ (8)

where A is the absorption, )(λε the molar absorptivity (dm3.mol-1.cm-1), εΔ the extinction

coefficient M-1.cm-1, c the concentration of the sample (mol.dm-3), and l the optical path length

(cm).

Although ΔA (or Δε) is measured, the parameter [θ ]λ, the molar ellipticity, is often used. It

relates the measurement to optical rotatory dispersion (θ is an angle of twist of plane polarized

light). These parameters can be related by the expression:

[ ] εθ λ Δ= 3300 degree.cm2.dmol-1 (9)

Alternatively, for CD measurements on proteins, the mean residue molar ellipticity is often

reported. The mean residue molar ellipticity is defined as:

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[ ]Rcl

obsres

...10θ

θ λ = (10)

where θobs is the observed ellipticity in degrees and R is the number of residues.

The peptide bond is one of the major chromophores in proteins (or peptides) and it is formed

by joining two amino acids. The peptide bond absorbs light between 190 nm and 240 nm. Thus

the phenomenon of circular dichroism is very sensitive to the secondary structures of polypeptides

and proteins, such as β-sheet and α-helix (Fig. 8).

250240230220210200190

120

100

80

60

40

20

0

-20

-40

-60

Wavelength / nm

[ θ] /

10-3

deg

cm

2 dm

ol-1

antiparalel beta

31-helix

α-helix

parallel beta

310-helix

random coil

Left-handed 31-helix

Figure 10: Circular dichroism spectra of different secondary structures (Jüri Jarvet, personal communication).

The secondary structures of proteins or peptides can be quantitatively evaluated from their

CD spectra using different methods, e.g. VARSELEC program. This program uses a procedure

based on the statistical method of "variable selection". The basis set of the analysis is composed of

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the CD spectra of 33 proteins for which the secondary structures were determined by X-ray

crystallography (Manavalan and Johnson, 1987).

Another simpler analysis method assumes that only random coil and α-helix secondary

structures are present (which is often the case for short peptides). The equation is as follows:

100.AR

Rhelix% ⎟⎠⎞

⎜⎝⎛

−Θ−

=− λα (11)

where A stands for the mean residue molar ellipticity for an α-helix model at wavelength λ, R is

the mean residue molar ellipticity for a random coil model at a wavelength λ, and Θλ is the mean

residue molar ellipticity of the sample at a wavelength λ. The calculation is typically performed

using the ellipticity at 222 nm where the α-helix ellipticity contribution has its most negative

value (-38000 deg.cm2.dmol-1) and the contribution of random coil is small (3900

deg.cm2.dmol-1).

The determinations of percentages secondary structures components are not absolute

measures, but they are particularly useful in studies of conformational changes in peptides due to

varying environment.

e. Other methods Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is an ultrasensitive spectroscopic technique

using fluorescence labeled molecules that allows single-molecule detection in a confocal volume.

The observation volume from which fluorescence is detected is typically about 2×10-16 liters.

From the temporal autocorrelation of fluorescence, from the spontaneous fluctuations in

fluorescence intensity generated by confocal illumination, it is possible to evaluate the dynamics

of the labeled molecule in real-time resolution, such as translational diffusion constants,

hydrodynamic radii, average concentrations or kinetic chemical reaction rates (Vukojević et al.,

2005).

In this thesis, FCS was used to investigate the kinetics of translocation and to analyze the

microenvironment of the peptides in vivo within a single living cell and in real time.

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Fourier Transform Infrared Spectroscopy

Like circular dichroism, Fourier transform infrared (FT-IR) spectroscopy can be used for

secondary structure analyses of proteins and peptides. The infrared spectra of peptides or

polypeptides show different vibrational modes of the peptide bond represented by a number of

bands, known as amide bands. The most used amide band for secondary structure analyses is the

amide I band. The amide I band results from the C=O stretching vibration of the amide group

coupled to the bending of the N-H bond and the stretching of the C-N bond. This IR absorption

band is sensitive to the secondary structure, because it depends on hydrogen bonding and coupling

between the transition dipoles of adjacent peptide bonds. The amide I absorption of the

polypeptide backbone is between 1610 and 1700 cm-1 (Table 7). Water (H2O) has an intense IR

band at 1643.5 cm-1 due to bending of the H-O, which overlaps with the amide I absorption. A

deuterated water solvent (heavy water, D2O) is therefore generally used for the secondary

structure analyses by FT-IR (Barth and Zscherp, 2002).

Table 7: Amide I band positions and corresponding secondary structure based on experimental

data and assignments from various reports collected and evaluated by Goormaghtigh et al. (1994)

Band position [cm-1] Secondary structure Average Extremes

α-helix β-sheet β-sheet Turns

Disordered

1654 1633 1684 1672 1654

1648–1657 1623–1641 1674–1695 1662–1686 1642–1657

Nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectroscopy (NMR) uses the fact that nuclei with non-zero

spin quantum number (such as 1H, 15N or 13C with spin one half) have a magnetic moment due to

their spin and placed in a strong magnetic field their two spin states are not equal (Hore, 1995).

Depending on the strength of the interaction between the field and the nucleus, these two spin

state are separated by a certain energy. This energy is detected by applying electromagnetic

radiation frequency pulses, followed by Fourier transformation for a more readily comprehensible

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frequency domain spectrum. Nuclei with spin quantum number equal to zero (such as 12C) do not

give NMR signals.

The electronic environment of the molecule (such as in a peptide) interferes with nuclear

magnetic energy levels. Thus a nucleus has non-identical resonance frequencies depending on the

local electron distribution in the molecule. The difference between the resonance frequencies of a

given nucleus and its standard reference nucleus is called chemical-shift. Therefore different

nuclei in a peptide have different chemical shifts and they can be affected by the secondary

structure of the peptide. In two-dimensional NMR, multiple pulses are used to excite the molecule

and to give more than one time-domain, useful for large molecules.

In the present thesis two-dimensional NMR spectroscopy was carried out on the 15N-labeled

the Aβ(1-40) peptide. Heteronuclear single quantum correlation (HSQC) and Transverse

relaxation optimized spectroscopy (TROSY) spectra showed cross peaks from the 15N-H groups

of the peptide, which could be regarded as a “fingerprint” of the peptide backbone.

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6. Results and discussion

a. Translocation of dynorphins across the plasma membrane and the membrane model systems (paper I and III)

The basic nature and/or hydrophobicity are general properties of CPPs. Dynorphin

neuropeptides being arginine rich and among the most basic naturally occurring peptides, share

certain common properties with CPPs. Therefore, dynorphins may also be translocated across the

plasma membrane into the cell and by this penetration process dynorphins may signal information

to the cell interior. We studied the ability of dynorphin peptides to translocate into cell or

membrane model systems in order to test this hypothesis. The kinetics of translocation and the

intracellular distribution of dynorphins were also investigated.

Several dynorphin neuropeptides were investigated in these studies: big dynorphin, dynorphin

A, dynorphin B and big dynorphin central fragment (Table 2). Big dynorphin central fragment is a

synthetic peptide and as indicated by its name, it constitutes the central part of big dynorphin,

which contains the most basic part of the sequence. This peptide was only used in the studies of

the translocation across the plasma membrane.

Penetration into cells (Paper I)

Various experiments with HeLa cells were performed to establish whether the dynorphin

neuropeptides translocate into cells. HeLa cells were incubated with biotinylated big dynorphin,

dynorphin A, dynorphin B or big dynorphin central fragment, followed by the fixation of the cells

and probed with streptavidin–Alexa Fluor 633 conjugate. The confocal microscopy experiments

in this cell preparation indicated cellular uptake of big dynorphin at 1 µM concentration with a

localization in the cytoplasm, and with negligible signal on the plasma membrane and in the cell

nucleus. Only very weak signals were detected for the other dynorphins (Fig. 1 in paper I). These

weak signals were considered as artifactual, thus these three peptides do not translocate into the

cell at the given concentration, using this particular technique. Hence, only big dynorphin was

seen to translocate.

A second experiment used HeLa cells incubated with dynorphins peptides, fixed and labeled

with antibodies against dynorphin A or dynorphin B. At the end these antibodies were visualized

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by anti-antibodies conjugated to a fluorescence dye. The experiment did not include big

dynorphin central fragment because there was no antibody available. The result concerning big

dynorphin is identical with the previous experiment. The translocation of dynorphin A was

detected at 10 µM concentration with the same distribution in the cell as big dynorphin (Fig. 2 in

paper I). In contrast, dynorphin B did not translocate into the cell, not even at 10 µM

concentration. The experiment with COS-1 and PC12 gave results equivalent to those with HeLa

cells. This set of experiments indicates higher translocation potential for big dynorphin than for

dynorphin A, whereas dynorphin B does not penetrate into the cells. The ranking of the ability of

the dynorphins to translocate across the plasma membrane correlates well with the potency of

these peptides to induce non-opioid nociceptive effects (Tan-No et al., 2002). It is possible to

speculate that peptide translocation is relevant for physiological processes such as the

development of chronic pain or for toxic effects of the peptides.

At first the HeLa live cells experiments were used as control experiments, in order to avoid

the problem in the potentially artefactual detection of the cellular uptake of peptide, caused by the

fixed cell preparation. This problem was found during some observations with other peptides (see

section 3.c). The results with confocal microscopy showed that rhodamine-labeled dynorphin A

(Rh-Dyn A) was located inside the HeLa live cells. Almost all peptides were in the cytoplasm and

some bright fluorescent domains were visible in the cell nucleus and in the plasma membrane

(Fig. 3, A and C in paper I). In contrast, rhodamine-labeled dynorphin B did not show any

penetration into the cell (Fig. 3, B and D in paper I). Therefore, we could conclude that the cell

fixation procedure did not cause any obvious artifacts to the study of dynorphin translocation into

cells.

The study of Rh-Dyn A with fluorescence correlation spectroscopy (FCS) was applied to

examine the translocation at low peptide concentration and the kinetics of translocation. The FCS

experiment confirmed the penetration of peptides into the cell at all concentrations tested (10 to

200 nM) and Rh-Dyn A could form complexes with intracellular components. The kinetics of Rh-

Dyn A showed a hyperbolic profile with a rapid initial uptake phase lasting for about 1 to 5

minutes, followed by a steady-state phase (Fig. 4C in paper I).

The distribution pattern of big dynorphin and dynorphin A in the cells, visualized in the

previous experiments, suggests that both peptides are associated to subcellular membrane

structures. Colocalization experiments with markers of the endoplasmic reticulum (ERp29), Golgi

complex (GM 130), and clathrin-mediated endocytosis (transferrin) were made to determine

which subcellular membrane structures are involved. The results shown the colocalization of big

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dynorphin and ERp29 and no colocalization was observed with the other two markers (Fig. 8 in

paper I). Since colocalisation of big dynorphin and transferrin was not detected, this indicates that

clathrin-mediated endocytosis was not involved in the cellular uptake of dynorphins. However,

our preliminary data suggests that the cellular uptake of dynorphins can be mediated by caveolar

endocytosis.

A recent article by Woods et al. (2006) considers noncovalent interaction of dynorphin A

with the conserved acidic epitope of the NR1 subunit (594-599, EEEEED) of NMDA-receptors,

and this sequence appears to be in the intracellular part of the subunit. The addition of synthetic

peptides containing this epitope prevents the potentiation of NMDA-mediated currents and

reduces cell death in spinal cord cultures elicited by dynorphin. Based on these results it can be

assumed that translocation of dynorphin A across the plasma membrane may be a condition for

interactions of dynorphin-NMDA receptor inside the cells. G proteins may be another potential

target for dynorphin neuropeptides after cellular uptake. A previous study on substance P

neuropeptide demonstrated translocation across the membrane of mast cells, followed by a direct

interaction of peptides with G protein α-subunits and induction of mast cell degranulation (Lorenz

et al., 1998). G proteins may be activated by other cationic peptides, like dynorphins, besides

substance P. This assumption is supported by the fact that dynorphin A stimulates histamine

release accompanied by degranulation of mast cells via a non-opioid activity (Shanahan et al.,

1984; Sugiyama and Furuta, 1984). Recently a new protein target for translocated dynorphins was

discovered inside the neurons (spinal cord and brain) (Marinova et al., 2004). This protein called

dynorphin A-binding factor (DABF) binds to dynorphin A and big dynorphin with high affinity

(virtually irreversible). DABF seems to function as an oligopeptidase by converting dynorphin A

into Leu-enkephalin to regulate its levels in the cells.

Peptide translocation in a membrane model system (Paper III)

We have examined the translocation ability of dynorphin peptides through a protein-free

phospholipid membrane, using a suitable model system for assessing the translocation of

amphipathic peptides in a Trp-dansyl FRET experiment, which was described by Matsuzaki et al.

(1995 and 1996). Dynorphin B was not included in the investigation since it does not contain any

Trp. POPC/POPG (70/30 molar ratio) and POPC/POPG/dansyl-DHPE (70/20/10) LUVs were

used as model systems. It was seen that the big dynorphin and dynorphin A interacted with the

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phospholipid vesicles, because the Trp emission intensity decreased during the addition of labeled

vesicles to both peptide solutions under investigation (Fig. 4 in paper III). Nevertheless, the data

from FRET experiment revealed that neither of the dynorphin peptides translocated across the

membrane in the model system. Persson et al. (2004) demonstrated that the translocation of pAntp

analogues is vesicle size dependent, and these peptides are able to translocate across the

membrane of GUVs but not of LUVs. Comparing our results with this observation, dynorphins

may have similar properties or they may simply translocate only by endocytotic pathways.

However, preliminary data suggested that big dynorphin could penetrate into HeLa cells through

non-endocytotic pathways (energy independent mechanisms) (Paper I).

b. Interaction and structure induction of dynorphins in the presence of membrane model systems (paper I, III and IV)

In the previous section, we showed that big dynorphin and dynorphin A translocate into

cells like CPPs. One of the first events in the cellular uptake of CPPs is their interaction with the

plasma membrane. Different spectroscopic techniques (CD, fluorescence and FT-IR) were used to

explore in more detail the membrane-peptide interactions by using membrane model systems

(with partly or totally negatively charged head groups).

Membrane interaction

Since all peptides have intrinsic fluorophores, Trp (dynorphin A, big dynorphin and

central fragment of big dynorphin) or only Tyr (dynorphin B), fluorescence spectroscopy was

used to probe the interaction between the peptides and negatively charged phospholipid SUVs

(composition POPC/POPG – 70/30). In the presence of the negatively charged SUVs a blue shift

of the maximum intensity of the Trp emission was observed for the three peptides compared to the

values observed in the buffer (between 9 and 12 nm depending on the peptide) (Fig. 9 in paper I).

Dynorphin B showed increase of the Tyr emission intensity upon interaction with the vesicles.

The results demonstrate that all dynorphin peptides interact with the phospholipid membrane in

these vesicles. Membrane interactions of both dynorphin A and dynorphin B were also observed

in FCS experiments using live cells (Paper I) and for big dynorphin and dynorphin A in the first

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set of FRET experiments with LUVs (Paper III). Dynorphin B and big dynorphin central fragment

do not penetrate into cells, even if both peptides do interact with cells and the model phospholipid

membranes. In summary, we conclude that the binding of dynorphins to a membrane is not

sufficient for it to enter into the cell.

The FRET experiments were performed to visualize the electrostatic contributions and

strength of the membrane binding of the peptides, using a variant of the original protocol from

Matsuzaki et al. (1995). The K+ concentration was kept at 100 mM inside the vesicles and it was

significantly decreased in the surrounding environment to 1 mM. Under this condition the results

for big dynorphin and dynorphin A indicate different kinetics in the redistribution of the peptides

between the two populations of vesicles (with and without FRET partner, see section 5.c

Fluorescence Resonance Energy Transfer, Fig. 7). The kinetics of big dynorphin are slower than

that of dynorphin A (Fig. 5 in paper III). An increase of the K+ concentration in the medium after

the addition of the second population of vesicles resulted in an increase of the rate of

redistribution but their relative order was unchanged (Fig. 5 in paper III). Therefore, the

membrane binding of the peptides has a strong electrostatic component and dynorphin A is a

weaker binder compared to big dynorphin, which can explain the ranking of translocation of these

peptides.

Secondary structure induction

The secondary structure induced in the big dynorphin, dynorphin A, dynorphin B and big

dynorphin central fragment by the interaction with the phospholipid SUVs (POPC/POPG – 70/30)

and SDS micelles was studied by CD and FT-IR spectroscopy. The dynorphin peptides showed to

be unstructured to a high extent in aqueous solution (Fig. 10 in paper I). In the presence of

negatively charged phospholipid SUVs at high lipid/peptide (P/L) molar ratio (~30 for dynorphin

B and ~100 for the other), all four peptides showed only surprisingly weak induction of secondary

structures (Fig 10 in paper I). The estimation from VARSELEC showed some α-helix induction

(~10% in aqueous solution increased to ~30 % in the presence of the vesicles). Normally CPPs,

antimicrobial or lytic peptides have a strong induction of secondary structure when they interact

with a membrane (Magzoub et al., 2003 and 2005B).

The study of the three main dynorphin peptides with SDS micelles by CD revealed a gradual

loss of the CD signal of the peptides by increasing the SDS concentration (L/P molar ratio

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between ~4 to ~25). The CD signal practically disappeared at L/P molar ratio 7.5 due to high light

scattering of the aggregate (Fig. 1 in paper IV). The aggregate formed was around 2 µm in size.

The FT-IR technique was chosen to determine and to quantify the secondary structure of the

peptide in this particle, which is impossible to determine by CD. The results indicated that the

peptides adopted mainly β-sheet structure (40%). The aggregated state and β-sheet transition may

reflect what could occur when a high concentration of peptides appear in the synapse at a specific

lipidic domain and may be responsible for the non-opioid activities of dynorphin neuropeptides.

For a high L/P molar ratio (375/1) CD experiments also indicated a weak induction of

secondary structure. The estimation from a quantitative estimation using the program JFIT

revealed an α-helix induction around 50%, confirmed by FT-IR experiment with the same L/P

molar ratio. A hypothesis about the behavior of dynorphins in the synapse can be based on these

experiments. The dynorphins may partially undergo random coil to α-helix transition when a

small peptide concentration interacts with the membrane and will start to have opioid activities. It

has been suggested that interaction with the membrane of the target cell may induce a

conformation and orientation of neuropeptide that will be recognized by the receptor (Moroder et

al., 1993). This process may be followed by an α to β secondary structure transition when there is

an increase of the concentration of the peptide and dynorphins would start their non-opioid

activities. Dynorphin A was shown to have non-opioid activities at high concentrations (Kajander

et al., 1990). Previous studies with pAntp and the Aβ (1-40) peptide illustrated a similar behavior

with a secondary structure transition with LUVs when the L/P decreases (Terzi et al., 1997;

Magzoub et al. 2002).

c. Membrane perturbation of dynorphins using LUV model systems, including calcium permeability (paper II and IV)

Previously we had established that big dynorphin and dynorphin A, in contrast to

dynorphin B and the central fragment of big dynorphin, translocate across the plasma membrane.

However, all dynorphin peptides interact with the membrane. We were interested in knowing

whether the interactions were connected to membrane perturbations or pore formation, and

whether calcium is able to enter into the vesicle during these perturbations. Studies on membrane

leakage were performed to probe the above mentioned phenomena. The membrane perturbations

by the dynorphin peptides were compared to melittin in calcein leakage experiments and pAntp in

calcium influx experiments.

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Calcein leakage from phospholipid vesicles

The membrane perturbation effect of the peptides was determined by studying calcein

leakage from phospholipid LUVs. Addition of big dynorphin and dynorphin A to the

POPG/POPC (30/70) LUVs provoked a substantial degree of leakage. An interesting observation

is the apparent saturation of calcein leakage with 5 µM of peptides and saturation of leakage

around 20% for dynorphin A and 60% for big dynorphin after 10 min (Fig. 1 in paper II). Melittin

caused maximal leakage (100%) under the same conditions. Dynorphin A induced calcein leakage

also with zwitterionic head group vesicles (Fig. 2 in paper II). Both zwitterionic and 30%

negatively charged LUVs gave the same degree of leakage at high peptide concentration (>20

µM), while at lower peptide concentration the degree of leakage was comparatively stronger with

charged vesicles. With 50% negatively charged vesicles, the overall leakage is higher at all

peptide concentrations (Fig. 2 paper II). The higher the charge of the vesicles, the higher the

degree of induced leakage. This contrasts to the effects of mellitin, for which negatively charged

phospholipids included into zwitterionic LUVs decrease leakage of calcein (Benachir and Lafleur,

1995). Dynorphin B did not induce any leakage despite its interaction with the phospholipid

membranes, possibly due to its smaller positive charge or/and its short length. An NMR study

showed that dynorphin A inserts its N-terminus into the bilayer of the membrane, while dynorphin

B was found to reside at the surface of the bilayer (Lind et al., 2006). This observation can also

explain why dynorphin B does not cause leakage.

Big dynorphin and dynorphin A cause perturbations and leakage in a membrane model

system. This observation provides a strong link between the observed translocation across cell

membranes and the induction of leakage in the LUV model systems. It suggests that these potent

translocating peptides (dynorphin A and big dynorphin) may cause direct membrane damage upon

entry into the cells. In turn this can perhaps explain their reported toxicity (Tan-No et al., 2001),

which may at least in part be brought about by direct membrane interactions.

The vesicle leakage is saturated in the presence of high peptide concentrations, which may be

due to the formation of aggregates by the peptide with the membrane, similar to what was

reported in the SDS micelle studies for low L/P ratios (see section 6.b). The saturation of vesicle

leakage as a function of time at constant peptide concentration has been explained as transient

pore formation with a nucleation step during an initial bilayer perturbation period. This is

followed by a transient restabilization of the peptide/lipid bilayer structure, which should give rise

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to the observed decrease of the spontaneous leakage (Schwarz and Arbuzova, 1995; Arbuzova and

Schwarz, 1999). An alternative explanation for both types of saturation is that the peptide induces

a stress in the membrane. To reduce the stress, molecular rearrangements in the membrane are

induced, so that further addition or activity of the peptide does not give any further perturbation of

the membrane (Andersson et al.,2007). However, to my best knowledge, the molecular details of

this process have never been adequately explained for any of the investigated peptides.

The interactions between the membrane and peptides cannot be attributed only to electrostatic

interactions, because dynorphin A causes leakage from LUVs with zwitterionic headgroups.

Nevertheless, a higher membrane charge density generally results in a higher degree of leakage

and so the electrostatic interactions are of importance for the the binding.

Calcium influx into phospholipid vesicles

The questions concerning the ability of calcium to enter into the cell during the membrane

perturbation by dynorphins was investigated by using the calcium indicator fura-2 entrapped in

phospholipid LUVs with partially negatively charged headgroups (POPC/POPG – 70/30). The

concentrations of peptides (0.25 to 1 µM) and calcium (500 µM) employed are reasonably close

to physiologically relevant conditions. Dynorphin A(1-13) was also investigated in this study.

This peptide has the same number of residues as dynorphin B and a higher net charge (+5) at pH 7

than dynorphin A.

Big dynorphin and dynorphin A generated an increase in intravesicular calcium

concentrations in LUVs, and like in the previous experiments big dynorphin is more potent.

Dynorphin A(1-13) and pAntp had only very small effects and dynorphin B was found to be

inactive in causing calcium influx into the vesicles. The results show that both charge and length

seem to play a role for dynorphins to allow calcium to permeate the phospholipid bilayer of the

vesicles. Dynorphin A(1-13) has a weaker effect than dynorphin A, even though it has higher

positive charge (+5) but shorter length. Dynorphin B has the lower positive charge (+3) among the

peptides and was found to be inactive. pAntp on the other hand is hardly active at all despite a

high charge (+7) and practically the same length than dynorphin A. We conclude that there is no

simple relation between the calcium influx potency and the sequence properties for the

investigated peptides. Instead there may be differences in structure and membrane interaction that

determine the potencies of the different peptides.

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The measured calcium influx into the vesicles due to big dynorphin and dynorphin A gives

rise to sub-µM concentration inside the vesicles. This approximately corresponds to the

intracellular calcium concentrations inside cells after stimuli (Heidelberger et al., 1994). The

comparison suggests that the dynorphin effects on calcium permeability may be biologically

relevant to neuron cell excitability and to maintain neuropathic pain.

d. Structure induction of Aβ(1-40) in the presence of SDS micelles as a membrane model system (paper V)

The amyloid β-peptide (Aβ) is the major component of the amyloid plaques found in

Alzheimer’s disease. Soluble oligomeric aggregates are reported to mediate toxic effects on

neurons and to contribute to the pathology of the disease. The peptide must be in high

concentrations to form the aggregates and membrane-peptide interactions is one of the suggested

mechanisms to increase Aβ concentration in a specific region in the brain (Terzi et al., 1997). To

mimic the behavior of Aβ(1-40) during its aggregation on a cell membrane surface, the secondary

structure conversion of the peptide with SDS or lithium dodecyl sulfate (LiDS) micelles was

studied by CD and NMR. The same peptide sample was used for all measurements with each

spectroscopic method and the detergents were titrated to the sample. The peptide concentration

was approximately the same in the CD and NMR experiments.

The CD experiments showed that titration of SDS into the aqueous solution of Aβ(1-40)

increases the β-sheet contribution, at SDS concentrations well below the CMC of SDS (Fig.1 in

paper V), in agreement with recently published observations (Rangachari et al., 2006). Here we

observed an approximately isodichroic point at 211 nm, indicating that the change in peptide

conformation is a simple two-state equilibrium between random coil and β-sheet conformation.

Close to the CMC of SDS, the peptide begins to show a transition from β-sheet to α-helix

conformation. At the very high SDS concentration, when there is more than one SDS micelle per

peptide, the peptide secondary structure consists of two α-helices and the C-terminal helix will be

inserted into the micelle (Jarvet et al., 2007). In the CD spectrum, this is seen as a strong α-helical

character of the peptide. Around the detergent/peptide molar ratio (D/P) 10, when the Aβ(1-40)

starts to have a high degree of β-sheet conformation, the NMR signal of the peptide in a HSQC

experiment almost disappears and has vanished at D/P 25. Therefore, the peptide together with

detergent forms large complexes, which are not easily studied by NMR. From the CD experiment

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we know that the peptide has a major β-sheet conformation in the complex. The NMR signal

reappears after a further large increase of the LiDS concentration, but then the resonances are

shifted. The Aβ(1-40) peptide has converted its structure, from β-sheet to α-helix.

In order to further study the state induced at D/P 25, the NMR-TROSY experiment was used.

TROSY makes it possible to study large proteins or complexes since it reduces the transverse

relaxation rates (Pervushin et al., 1997). In the TROSY spectrum at the same conditions as for the

HSQC spectrum where all signals had disappeared, two C-terminal residues are observable (V39

and V40) (Fig. 2 in paper V). This could be due to the fact that the charged carboxy terminus of

Aβ(1-40) is still flexible to some extent.

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Conclusions

The primary objective of the work described in this thesis was to obtain an understanding

of the consequences of biomembrane interaction of dynorphin neuropeptides and amyloid β

peptide which may have an impact on the physiology and pathology involving these peptides.

We discovered a novel property of the opioid peptides big dynorphin and dynorphin A to

translocate across the plasma membrane of cells. The cellular uptake is rapid within 1-5 min and

the peptides are localized to the cytoplasm where they interact with the endoplasmic reticulum.

The translocation ability of dynorphins seems to be correlated with their ability to induce some

non-opioid activities. Their interactions with the membrane cause perturbation and permeability

of the membrane to Ca2+. These three observations suggest that their translocation and interactions

with the membrane are relevant for physiological processes such as development and the

maintenance of chronic pain. The direct membrane interaction of the peptides may be described

by the carpet model and this may be related to a potential non-endocytotic pathway of entry into

cells. However, the membrane translocation may be affected by more than one mechanism,

depending on conditions. One can speculate that these special properties of dynorphins including

cell membrane translocation may represent an ancient cell signaling mechanism that does not

require specialized receptors.

Dynorphin A and big dynorphin share some properties of CPPs, but they should rather be

classified only as CPPs-like. It would be interesting to study a dynorphin-cargo conjugate with a

large cargo. In any case dynorphins do not seem to be good potential drug delivery agents because

they are involved in a variety of physiological processes (toxic or non-toxic). They can be very

toxic to the cell at high concentrations.

Many questions remain to be answered and experimental methodologies must be developed

further in order to understand and know in molecular detail the cellular uptake mechanisms of

dynorphins. First, it would be interesting to determine whether dynorphins penetrate into the cell

by caveolae endocytosis or macropinocytosis and if non-endocytosis pathways are active in the

cellular uptake of the peptides in vivo. Second, if there are endocytotic memchanisms in

operation, how the translocated dynorphins are able to escape from the endosome. Another

interesting physiological question is why most of translocated peptides interact with the

endoplasmic reticulum and if this has particular repercussions on the cell (non-toxic or toxic).

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For Aβ(1-40) the membrane interactions were mimicked in an SDS-detergent environment.

The extreme structural variability of the peptide was shown in its transition from random coil to β-

structure and further to α-helix as more SDS was added to the sample. In the aggregated β-

structure rich state, preliminary NMR experiments suggested that the residues in the C-terminus

were more mobile than the rest of the peptide. Further NMR and perhaps dynamic light scattering

experiments under varying conditions are required to explain this unexpected observation.

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Acknowledgments

Thanks to all the people who supported me directly or indirectly during my Ph.D studies.

Especially I would like to thank to:

Professor Astrid Gräslund, my supervisor, for always offering me help and guidance and for your enthusiasm for science. Thank you also for the great help during the preparation of this thesis and for being the greatest human being I could have as supervisor.

Vladana Vukojević and Georgy Bakalkin, for allowing me to work on dynorphin and for your help. Thank you for this great collaboration.

Lena Mäler, Göran Erikson and Andreas Barth, for your great enthusiasm for science and for being so nice and helpful.

I am grateful for expert assistance provided by Torbjörn and Haidi Astlind, Britt-Marie Olsson, Bengt Eurenius and Bogos Adali, without whom the department would not work so well.

Alex for bringing the Latin culture I was missing, to the department and for being a good collaborator. Thanks also for the great scientific and football discussions. Good luck for the preparation of your wedding!

Henrik for our conversations about Manga and Japan. Only pity is your passion for vegetables! I hope one day you will come back from the dark side and you will eat again some meat! Good luck for finishing your thesis.

Jesper for his enthusiasm, for the discussions about football and about life in general. But be careful! You work too much and you will be sick one day! Take care and good luck for your thesis.

August and Jens, for the good moments in L.A., for the great scientific discussions and for making me discover Hammarby.

Jüri and Anna for having a good collaboration on the Aβ paper.

Bruce for letting me to be a friend with a real American super-hero.

Thanks to all roommates who spent the time in our office - for the good time I spent with them: Elka, Maz and Sebastien.

Thanks to all other nice people at the department which I was able to meet:

Amin, Ana-Julia, Elsa, Evangelos, Andrea, Fatemeh, Håkan, Joachim, Jon, Joshua, Julia, Kamila, Kristina, Maria, Marta, Martin, Mirjam, Nina, Peter, Shashi, Sofia,Tara and Tariq.

Thanks also to the people who played football with me Thursday morning: Daniel, David (both), Mats, Pavel, Peter, Tomas and Vasco.

Ann-Britt, Hillevi and Jaja for the good discussions and your very good mood during the lunches at Inorganic chemistry department.

Tim, for the time you spent to read my thesis and improve my English. It is a pity France beat New-Zealand for nothing.

Reneta, to be a very good flat mate and for all the lunches you cooked for me, but it is a pity there was not the meat all the time.

Jan for being very helpful every time.

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Merci à tous les professeurs du lycée Gregor Mendel de Vincennes que j’ai pu avoir au lycée et en BTS et qui m’ont permis d’apprendre à aimer les sciences. Tout particulièrement à Mme Etancelin, Mme Berthe, Mr Ziarczyk, Mme Netter, Mr Tétreau, Mr Juliot, Mme Guermont, Mme Cardona, Mme Delacroix, Mme Vaxelaire, Mme Chrisostome et Mr Nobilio.

Arnaud Berland, mon ancien entraîneur d’athlétisme, pour m’avoir appris à me surpasser. Et sans qui je ne serais sûrement pas où j’en suis.

Patrick, Mounia et Amin de m’avoir tant aidé quand je suis arrivé en Suède.

Mon meilleur ami Nico et son frère Alex avec qui j’ai passé et je vais encore passer de bons moments. Grâce à vous je sais que je ne suis pas le seul fou à supporter le PSG, quand on y pense, on est quand même des “bon vieux gros sado-masos”.

Mon second meilleur ami David qui m’a apporté beaucoup. J’espère que tu deviendras un photographe connu. Merci aussi pour tous ces bons moments passés ensemble.

Clélie pour avoir arrêté de te cacher sous le lit quand je venais voir Nico, mais surtout pour être quasiment toujours de bonne humeur.

Guillaume, Jimmy et Marco pour tous les bons moments passés ensemble en Ardèche et sur Paris, la prochaine fois je gagne la “boule d’or” et le “strike in the night”. Et d’avoir toujours été là quand j’ai eu besoin de votre soutien.

Igor pour être resté le seul vrai sportif du groupe et pour tous tes très longs emails sur le compte-rendu de ta vie qui m’ont bien occupé pendant mes pauses.

Caroline pour ta gentillesse et ton enthousiasme permanent.

Bertrand et Amélie pour m’avoir donné un peu de vie sociale en dehors du labo. Merci aussi pour les lasagnes et les crêpes parties. J’espère que tu continueras à aller à la gym après que je sois parti de Suède. Dommage que vous ne soyez par arrivés plus tôt à Stockholm.

Finalement je dédie cette thèse aux personnes que j’aime le plus sur Terre :

A mon père qui m’a toujours soutenu et approuvé dans mes décisions et des sacrifices qu’il a dû faire pour cela.

A ma mère qui m’a aussi toujours soutenu et approuvé dans mes décisions et pour être son “ fils adoré ”.

A Sophie et Sylvain pour avoir la chance d’avoir une sœur et un frère comme eux. Et vous me faites bien rire quand vous êtes jaloux que je sois le “fils adoré”, cela ne veut pas dire que je sois le préféré.

Et surtout à ma femme Zuzana, mon rayon de soleil dans ce pays obscur, à qui je dois en grande partie l’accomplissement de ce travail par ton soutien de tout le temps et de tout l’amour que tu me donnes. Tous les mots du monde ne peuvent décrire comment je suis heureux avec toi.

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