Spectroscopic studies of dynorphin neuropeptides and the...
Transcript of Spectroscopic studies of dynorphin neuropeptides and the...
Spectroscopic studies of dynorphin neuropeptides and the amyloid β peptide
The consequences of biomembrane interactions
Loïc Hugonin
Department of Biochemistry and Biophysics
Stockholm University
II
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
A ma femme, mon père, ma mère, ma sœur et mon frère
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.
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.
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
X
XI
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
XII
RNase A Ribonuclease A
SDS Sodium docecyl sulfate
SUVs Small unilamellar vesicles
TROSY Transverse relaxation optimized spectroscopy
XIII
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
XIV
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
1
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
2
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).
3
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).
4
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
5
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
6
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).
7
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
8
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
9
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
10
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.
11
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
12
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
13
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.
14
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).
15
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).
16
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
17
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).
18
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)
19
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
20
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.
21
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.
22
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
23
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
24
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.
25
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
26
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.
27
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
28
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
29
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
30
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.
31
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.
32
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)
33
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:
34
[ ]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
35
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.
36
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
37
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.
38
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
39
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
40
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
41
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
42
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
43
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.
44
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
45
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.
46
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
47
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.
48
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).
49
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.
50
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.
51
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.
52
References
Alzheimer A, Uber eine eigenartige Erkankung der Hirnrinde, Allg. Z. Psychiatr. 64 (1907), pp. 146-148
Andersson A, Danielsson J, Gräslund A and Mäler L, Kinetic models for peptide-induced leakage from vesicles and cells, Eur. Biophys. J. 36 (2007), pp. 621-635
Arbuzova A and Schwarz G, Pore-forming action of mastoparan peptides on liposomes: a quantitative analysis, Biochim. Biophys. Acta. 1420 (1999), pp. 139-152
Bakshi R and Faden AI, Competitive and non-competitive NMDA antagonists limit dynorphin A-induced rat hindlimb paralysis, Brain Res. 507 (1990), pp. 1-5
Bárány-Wallje E, Gaur J, Lundberg P, Langel U and Gräslund A, Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo, FEBS Lett. 581 (2007), pp. 2389-2393
Barth A and Zscherp C, What vibrations tell us about proteins, Q. Rev. Biophys. 35 (2002), pp. 369-430
Benachir T and Lafleur M, Study of vesicle leakage induced by melittin, Biochim. Biophys. Acta. 1235 (1995), pp. 452-460
Brown CM and Petersen NO, Free clathrin triskelions are required for the stability of clathrin-associated adaptor protein (AP-2) coated pit nucleation sites, Biochem. Cell Biol. 77 (1999), pp. 439–448
Brown D and Waneck GL, Glycosyl–phosphatidylinositol-anchored membrane proteins, J. Am. Soc. Nephrol. 3 (1992), pp. 895–906
Caudle RM and Dubner R, Ifenprodil blocks the excitatory effects of the opioid peptide dynorphin 1-17 on NMDA receptor-mediated currents in the CA3 region of the guinea pig hippocampus, Neuropeptides 32 (1998), pp. 87-95
Cavicchini E, Candeletti S and Ferri S, Effects of dynorphins on body temperature of rat, Pharmacol. Res. Commun. 20 (1988), pp. 603-604
Chavkin C, James IF and Goldstein A. Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science 215 (1982), pp. 413-415.
Chen L, Gu Y and Huang LY, The opioid peptide dynorphin directly blocks NMDA receptor channels in the rat. J. Physiol. 482 (1995A), pp. 575-581
Chen L, Gu Y and Huang LY, The mechanism of action for the block of NMDA receptor channels by the opioid peptide dynorphin, J. Neurosci. 15 (1995B), pp. 4602-4611
Choi DW and Viseskul V, Opioids and non-opioid enantiomers selectively attenuate N-methyl-D-aspartate neurotoxicity on cortical neurons, Eur. J. Pharmacol. 155 (1988), pp. 27–35
Christiaens B, Symoens S, Verheyden S, Engelborghs Y, Joliot A, Prochiantz A, Vandekerckhove J, Rosseneu M and Vanloo B, Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes, Eur. J. Biochem. 269 (2002), pp. 2918-2926
Christiaens B, Grooten J, Reusens M, Joliot A, Goethals M, Vandekerckhove J, Prochiantz A and Rosseneu M, Membrane interaction and cellular internalization of penetratin peptides, Eur. J. Biochem. 271 (2004), pp.1187-1197
Console S, Marty C, García-Echeverría C, Schwendener R and Ballmer-Hofer K, Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans, J. Biol. Chem. 278 (2003), pp. 35109-35114
Costigan M and Woolf CJ, No DREAM, No pain. Closing the spinal gate, Cell 108 (2002), pp. 297-300.
Danielson P, Walker D, Alrubaian J and Dores RM, Identification of a fourth opioid core sequence in a prodynorphin cDNA cloned from the brain of the amphibian, Bufo marinus: deciphering the evolution of prodynorphin and proenkephalin, Neuroendocrinology 76 (2002), pp. 55-62
Day R, Lazure C, Basak A, Boudreault A, Limperis P, Dong W and Lindberg I, Prodynorphyn processing by proprotein convertase 2, J. Biol. Chem. 273 (1998), pp. 829-836
53
De Vries TJ and Shippenberg TS, Neural systems underlying opiate addiction, J. Neurosci. 22 (2002), pp. 3321-3325
Derossi D, Joliot AH, Chassaing G, and Prochiantz A, The third helix of the Antennapedia homeodomain translocates through biological membranes, J. Biol. Chem. 269 (1994), pp. 10444–10450
Derossi D, Calvet S, Trembleau A, Brunissen A, Chassaing G and Prochiantz A, Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent, J. Biol. Chem. 271 (1996), pp. 18188-18193
Derossi D, Chassaing G and Prochiantz A, Trojan peptides: the penetratin system for intracellular delivery, Trends. Cell. Biol. 8 (1998), pp. 84-87
Deshayes S, Morris MC, Divita G and Heitz F, Cell-penetrating peptides: tools for intracellular delivery of therapeutics, Cell Mol. Life Sci. 62 (2005), pp. 1839-49
Dom G, Shaw-Jackson C, Matis C, Bouffioux O, Picard JJ, Prochiantz A, Mingeot-Leclercq MP, Brasseur R and Rezsohazy R, Cellular uptake of Antennapedia Penetratin peptides is a two-step process in which phase transfer precedes a tryptophan-dependent translocation, Nucleic Acids Res. 31 (2003), pp. 556-561
Dowrick P, Kenworthy P, McCann B and Warn R, Circular ruffle formation and closure lead to macropinocytosis in hepatocyte growth factor/scatter factor-treated cells, Eur. J. Cell. Biol. 61 (1993), pp. 44-53
Drin G, Mazel M, Clair P, Mathieu D, Kaczorek M and Temsamani J, Physico-chemical requirements for cellular uptake of pAntp peptide. Role of lipid-binding affinity, Eur. J. Biochem. 268 (2001), pp.1304-1314
Drin G, Cottin S, Blanc E, Rees AR and Temsamani J, Studies on the internalization mechanism of cationic cell-penetrating peptides, J. Biol. Chem. 278 (2003); pp. 31192-31201
Dubner R and Ruda MA, Activity-dependent neuronal plasticity following tissue injury and inflammation, Trends. Neurosci. 15 (1992), pp. 96-103
Evans CJ, Hammond DL and Frederickson RCA, The opioid peptides. In: The Opiate Receptors. Humana, Ed: Pasternak, GW, Clifton, New Jersey (1988), pp. 23-71
Faden AI and Jacobs TP, Dynorphin-related peptides cause motor dysfunction in the rat through a non-opiate action, Br. J. Pharmacol. 81 (1984), pp. 271-276
Faden AI, Takemori AE and Portoghese PS, Kappa-selective opiate antagonist nor-binaltorphimine improves outcome after traumatic spinal cord injury in rats, Cent. Nerv. Syst. Trauma. 4 (1987), pp. 227-237
Faden AI, Dynorphin increases extracellular levels of excitatory amino acids in the brain through a non-opioid mechanism, J. Neurosci. 12 (1992), pp. 425-429
Fallon JH, and Leslie FM, Distribution of dynorphin and enkephalin peptides in the rat brain, J. Comp. Neurol. 249 (1986), pp. 293-336.
Fawell S, Seery J, Daikh Y, Moore C, Chen L, Pepinsky B and Barsoum J, Tat-mediated delivery of heterologous proteins into cells, Proc. Natl. Acad. Sci. U. S. A. 91 (1994), pp. 664–668
Ferrari A, Pellegrini V, Arcangeli C, Fittipaldi A, Giacca M and Beltram F, Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time, Mol. Ther. 8 (2003), pp. 284-294
Feuerstein G and Faden AI, Cardiovascular effects of dynorphin A-(1-8), dynorphin A-(1-13) and dynorphin A-(1-17) microinjected into the preoptic medialis nucleus of the rat, Neuropeptides 5 (1984), pp. 295-298
Fischer PM, Zhelev NZ, Wang S, Melville JE, Fåhraeus R and Lane DP, Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin, J. Pept. Res. 55 (2000), pp. 163-172
Fischer R, Köhler K, Fotin-Mleczek M and Brock R, A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides, J. Biol. Chem. 279 (2004), pp. 12625-12635
Fittipaldi A, Ferrari A, Zoppé M, Arcangeli C, Pellegrini V, Beltram F and Giacca M, Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins, J. Biol. Chem. 278 (2003), pp. 34141-34149
Frankel AD and Pabo CO, Cellular uptake of the tat protein from human immunodeficiency virus, Cell 55 (1988), pp. 1189-1993
54
Friedman HJ, Jen MF, Chang JK, Lee NM and Loh HH, Dynorphin: a possible modulatory peptide on morphine or beta-endorphin analgesia in mouse, Eur. J. Pharmacol. 69 (1981), pp.357-360
Fischli W, Goldstein A, Hunkapiller MW and Hood LE, Isolation and amino acid sequence analysis of a 4000-dalton dynorphin from porcine pituitary. Proc. Natl. Acad. Sci. USA 79 (1982), pp. 5435–5437.
Fuchs SM and Raines RT, Pathway for polyarginine entry into mammalian cells, Biochemistry 43 (2004), pp. 2438-2444
Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K and Sugiura Y, Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001), pp. 5836-5840
Gautret B and Schmitt H, Central and peripheral sites for cardiovascular actions of dynorphin-(1-13) in rats, Eur. J. Pharmacol. 111 (1985), pp. 263-266
Gill SC and von Hippel PH, Calculation of protein extinction coefficients from amino acid sequence data, Anal. Biochem. 182 (1989), pp. 319-326
Glenner GG and Wong CW, Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys. Res. Commun. 120 (1984), pp. 885-890
Goldstein A, Tachibana S, Lowney LI, Hunkapiller M and Hood L, Dynorphin-(1-13), an extraordinarily potent opioid peptide, Proc Natl. Acad. Sci. USA. 76 (1979), pp. 6666-6670
Goldstein A, Binding selectivity profiles for ligands of multiple receptor types: focus on opioid receptors, Trends Pharmacol. Sci. 8 (1987), pp. 456-459
Goody RJ, Martin KM, Goebel SM and Hauser KF, Dynorphin A toxicity in striatal neurons via an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor mechanism, Neuroscience 116 (2003), pp. 807-816
Goormaghtigh E, Cabiaux Vand Ruysschaert JM, Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. III. Secondary structures, Subcell. Biochem. 23 (1994), pp. 405–450.
Green M and Loewenstein PM, Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein, Cell 55 (1988), pp. 1188-1189
Grundke-Iqbal I, Iqbal K, Quinlan M, Tung YC, Zaidi MS and Wisniewski HM, Microtubule-associated protein tau. A component of Alzheimer paired helical filaments, J. Biol. Chem. 261 (1986), pp. 6084-6089
Grynkiewicz G, Poenie M and Tsien RY, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 (1985), pp. 3440-3450
Guaza C, Zubiaur M and Borrell J, Corticosteroidogenesis modulation by beta-endorphin and dynorphin1-17 in isolated rat adrenocortical cells, Peptides 7 (1986), pp. 237-240
Hällbrink M, Florén A, Elmquist A, Pooga M, Bartfai T and Langel Ü, Cargo delivery kinetics of cell-penetrating peptides, Biochim. Biophys. Acta 1515 (2001), pp. 101-109.
Han JS and Xie CW, Dynorphin: potent analgesic effect in spinal cord of the rat, Sci. Sin. 27 (1984), pp. 169-177
Hardy J and Selkoe DJ, The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics, Science 297 (2002), pp. 353-356
Harper JD and Lansbury PT Jr, Models of amyloid seeding in Alzheimer's disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins, Annu. Rev. Biochem. 66 (1997), pp. 385-407
Healy DJ and Meador-Woodruff JH, Prodynorphin-derived peptide expression in primate cortex and striatum, Neuropeptides 27 (1994), pp. 277–284
Heidelberger R, Heinemann C, Neher E and Matthews G, Calcium dependence of the rate of exocytosis in a synaptic terminal, Nature 371 (1994), pp. 513-515
55
Herman BH, Leslie F and Goldstein A, Behavioral effect and in vivo degradation of intraventricularly administered dynorphin-(1-13) and D-Ala2-dynorphin-(1-11) in rats, Life Sci. 27, (1980), pp. 883-892
Herman BH and Goldstein A, Antinociception and paralysis induced by intrathecal dynorphin A, J. Pharmacol. Exp. Ther. 232 (1985), pp. 27-32
Hewlett LJ, Prescott AR and Watts C, The coated pit and macropinocytic pathways serve distinct endosome populations, J. Cell. Biol. 124 (1994), pp. 689-703
Hollmann M and Heinemann S, Cloned glutamate receptors, Annu. Rev. Neurosci. 17 (1994), pp. 31–108
Hore PJ, Nuclear magnetic resonance, Oxford University Press, Oxford Chemistry Primers 32, (1995)
Hu WH, Zhang CH, Yang HF, Zheng YH, Liu N, Sun XJ, Jenc J, Jen MF. Mechanism of the dynorphin-induced dualistic effect on free intracellular Ca2+ concentration in cultured rat spinal neurons, Eur. J. Pharmacol. 342 (1998), pp. 325–332
Huang LYM, Cellular mechanisms of excitatory and inhibitory actions of opioids., in Pharmacology of Opioid Peptides (Tseng LF ed) (1995), pp. 131-149, Harwood Academic Press, Singapore
Hughes J, Smith T, Morgan B and Fothergill L, Purification and properties of enkephalin - the possible endogenous ligand for the morphine receptor, Life Sci. 16 (1976), pp. 1753-1758
Iwamoto ET, Locomotor activity and antinociception after putative mu, kappa and sigma opioid receptor agonists in the rat: influence of dopaminergic agonists and antagonists, J. Pharmacol. Exp. Ther. 217 (1981), pp. 451–460
Jarvet J, Danielsson J, Damberg P, Oleszczuk M and Gräslund A, Positioning of the Alzheimer Aβ(1-40) peptide in SDS micelles using NMR and paramagnetic probes. J. Biomol. NMR 39 (2007), pp. 63-72
Jarvis CR, Xiong ZG, Plant JR, Churchill D, Lu WY, MacVicar BA and MacDonald JF, Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons, J. Neurophysiol. 78 (1997), pp. 2363-2371
Jhamandas K, Sutak M and Lemaire S, Comparative spinal analgesic action of dynorphin1-8, dynorphin1-13, and a kappa-receptor agonist U50,488, Can. J. Physiol. Pharmacol. 64 (1986), pp. 263-268.
Joliot A, Pernelle C, Deagostini-Bazin H and Prochiantz A, Antennapedia homeobox peptide regulates neural morphogenesis, Proc. Natl. Acad. Sci. USA. 88 (1991), pp. 1864-1868
Kajander KC, Sahara Y, Iadarola MJ and Bennett GJ, Dynorphin increases in the dorsal spinal cord in rats with a painful peripheral neuropathy, Peptides 11 (1990), pp. 719–728
Kakidani H, Furutani Y, Takahashi H, Noda M, Morimoto Y, Hirose T, Asai M, Inayama S, Nakanishi S and Numa S, Cloning and sequence analysis of cDNA for porcine beta-neo-endorphin/dynorphin precursor, Nature 298 (1982), pp. 245–249
Kanemitsu Y, Hosoi M, Zhu PJ, Weight FF, Peoples RW, McLaughlin JS and Zhang L, Dynorphin A inhibits NMDA receptors through a pH-dependent mechanism, Mol. Cell. Neurosci. 24 (2003), pp. 525-537
Kaplan IM, Wadia JS and Dowdy SF, Cationic TAT peptide transduction domain enters cells by macropinocytosis, J. Control. Release. 102 (2005), pp. 247-253
Khachaturian, H, Watson, SJ, Lewis, M E, Coy, D, Goldstein, A, and Akil, H, Dynorphin immunocytochemistry in the rat central nervous system. Peptides 3 (1982), pp. 941-954.
Kolaj M, Cerne R and Randic M, The opioid peptide dynorphin modulates AMPA and kainate responses in acutely isolated neurons from the dorsal horn, Brain Res. 671 (1995), pp. 227-244
Koppelhus U, Awasthi SK, Zachar V, Holst HU, Ebbesen P and Nielsen PE, Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates, Antisense, Nucl. Acid. Drug. Dev. 12 (2002), pp. 51–63
Kreek MJ, LaForge KS and Butelman E, Pharmacotherapy of addictions. Nat. Rev. Drug Discov. 1 (2002), pp. 710-726.
56
Kuo YM, Webster S, Emmerling MR, De Lima N and Roher AE, Irreversible dimerization/tetramerization and post-translational modifications inhibit proteolytic degradation of Aβ peptides of Alzheimer's disease, Biochim. Biophys. Acta 1406 (1998), pp. 291-298
Kuzmin A, Madjid N, Terenius L, Ogren SO and Bakalkin G, Big Dynorphin, a Prodynorphin-Derived Peptide Produces NMDA Receptor-Mediated Effects on Memory, Anxiolytic-Like and Locomotor Behavior in Mice, Neuropsychopharmacology 31 (2006), pp. 1928-1937
Lai SL, Gu Y and Huang LY, Dynorphin uses a non-opioid mechanism to potentiate N-methyl-D-aspartate currents in single rat periaqueductal gray neurons, Neurosci. Lett. 247 (1998), pp. 115-118
Lai J, Luo, M.C, Chen Q, Ma S, Gardell, L.R, Ossipov, M.H and Porreca, F, Dynorphin A activates bradykinin receptors to maintain neuropathic pain. Nat Neurosci. 9 (2006), pp. 1534-1540
Lakowicz JR, Principles of Fluorescence Spectroscopy, Kluwer Academic/Plenum Publishers, 2nd Ed., New York, London, Moscow, Dordrecht (1999), pp. 53
Langel Ü, Pooga M, Kairane C, Zilmer M and Bartfai T, A galanin-mastoparan chimeric peptide activates the Na+,K+-ATPase and reverses its inhibition by ouabain, Regul. Pept. 62 (1996), pp. 47–52
Laurent S and Schmitt H, Central cardiovascular effects of kappa agonists dynorphin-(1-13) and ethylketocyclazocine in the anaesthetized rat, Eur. J. Pharmacol. 96 (1983), pp. 165-169
Lee MT, Hung WC, Chen FY and Huang HW, Many-body effect of antimicrobial peptides: on the correlation between lipid's spontaneous curvature and pore formation, Biophys. J. 89 (2005), pp. 4006-4016
Leslie FM and Goldstein A, Degradation of dynorphin-(1-13) by membrane-bound rat brain enzymes, Neuropeptides 2 (1982), pp. 185-196
Lind J, Gräslund A and Mäler L, Membrane interactions of dynorphins. Biochemistry 45 (2006), pp. 15931-15940
Lindgren M, Hallbrink M, Prochiantz A and Langel Ü, Cell-penetrating peptides, Trends. Pharmacol. Sci. 21 (2000), 99-103
Litt M, Buroker NE, Kondoleon S, Douglass J, Liston D, Sheehy R and Magenis RE, Chromosomal localization of the human proenkephalin and prodynorphin genes, Am. J. Hum. Genet. 42 (1988), 327-334
Long JB, Petras JM, Mobley WC and Holaday JW, Neurological dysfunction after intrathecal injection of dynorphin A (1-13) in the rat. II. Nonopioid mechanisms mediate loss of motor, sensory and autonomic function, J. Pharmacol. Exp. Ther. 246 (1988), pp. 1167-1174
Long JB and Skolnick P, 1-Aminocyclopropanecarboxylic acid protects against dynorphin A-induced spinal injury, Eur. J. Pharmacol. 261 (1994), pp. 295-301
Lorenz D, Wiesner B, Zipper J, Winkler A, Krause E, Beyermann M, Lindau M and Bienert M, Mechanism of peptide-induced mast cell degranulation. Translocation and patch-clamp studies, J. Gen. Physiol. 112 (1998), pp. 577-591
Ludtke S, He K, and Huang H, Membrane thinning caused by magainin 2, Biochemistry 34 (1995), pp. 16764-16769
Lundberg P, Magzoub M, Lindberg M, Hallbrink M, Jarvet J, Eriksson LE, Langel Ü and Gräslund A, Cell membrane translocation of the N-terminal (1-28) part of the prion protein, Biochem. Biophys. Res. Commun. 299 (2002), pp. 85-90
Lundberg M and Johansson M, Positively charged DNA-binding proteins cause apparent cell membrane translocation, Biochem. Biophys. Res. Commun. 291 (2002), pp. 367-371
Macdonald RL and Werz MA, Dynorphin A decreases voltage-dependent calcium conductance of mouse dorsal root ganglion neurones, J. Physiol. 377 (1986), pp. 237-249
Magzoub M, Eriksson LE and Gräslund A, Conformational states of the cell-penetrating peptide penetratin when interacting with phospholipid vesicles: effects of surface charge and peptide concentration, Biochim. Biophys. Acta. 1563 (2002), pp. 53-63
57
Magzoub M, Eriksson LE and Gräslund A, Comparison of the interaction, positioning, structure induction and membrane perturbation of cell-penetrating peptides and non-translocating variants with phospholipid vesicles, Biophys. Chem. 103 (2003), pp. 271-288
Magzoub M and Gräslund A, Cell-penetrating peptides: small from inception to application, Q. Rev. Biophys. 37 (2004), pp. 147-195
Magzoub M, Pramanik A and Gräslund A, Modeling the endosomal escape of cell-penetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers, Biochemistry 44 (2005A), pp. 14890-14897
Magzoub M, Oglecka K, Pramanik A, Göran Eriksson LE and Gräslund A, Membrane perturbation effects of peptides derived from the N-termini of unprocessed prion proteins, Biochim. Biophys. Acta. 1716 (2005B), pp. 126-136
Magzoub M, Sandgren S, Lundberg P, Oglecka K, Lilja J, Wittrup A, Göran Eriksson LE, Langel U, Belting M and Gräslund A, N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis, Biochem. Biophys. Res. Commun. 348 (2006), pp. 379-385
Manavalan P and Johnson WC Jr, Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra, Anal. Biochem. 167 (1987), pp. 76-85
Mann D and Frankel A, Endocytosis and targeting of exogenous HIV-1 Tat protein, EMBO J. 10 (1991), pp. 1733–1739
Mansour A, Fox CA, Burke S, Meng F, Thompson RC, Akil H and Watson SJ, Mu, delta, and κ-opioid receptor mRNA expression in the rat CNS: an in situ hybridization study. J. Comp. Neurol. 350 (1994), pp. 412-438.
Martin WR, Eades CG, Thompson JA, Huppler RE and Gilbert PE, The effects of morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal dog, J. Pharmacol. Exp. Ther. 197 (1976), pp.517-32
Massardier D and Hunt PF, A direct non-opiate interaction of dynorphin-(1-13) with the N-methyl-D-aspartate (NMDA) receptor, Eur. J. Pharmacol. 170 (1989), pp. 125-126
Matsuzaki K., Murase O, Fujii, N and Miyajima K. Translocation of a channel-forming antimicrobial peptide, magainin 2, across lipid bilayers by forming a pore. Biochemistry 34 (1995), pp. 6521-6526.
Matsuzaki K, Yoneyama S, Murase O, and Miyajima K, Transbilayer transport of ions and lipids coupled with mastoparan X translocation, Biochemistry 35 (1996); pp. 8450-8456
Matsuzaki K, Yoneyama S, and Miyajima K. Pore formation and translocation of melittin. Biophys. J. 73 (1997), 831-838.
McBain CJ and Mayer ML, N-methyl-D-aspartic acid receptor structure and function, Physiol. Rev. 74 (1994), pp. 723–760
McIntosh TK, Hayes RL, DeWitt DS, Agura V and Faden AI, Endogenous opioids may mediate secondary damage after experimental brain injury, Am. J. Physiol. 253 (1987), pp. E565-E574
Merchenthaler I, Maderdrut JL, Cianchetta P, Shughrue P and Bronstein D, In situ hybridization histochemical localization of prodynorphin messenger RNA in the central nervous system of the rat, J. Comp. Neurol. 384 (1997), pp. 211–232
Merg F., Filliol D, Usynin I, Bazov I, Bark N, Hurd YL, Yakovleva T, Kieffer BL and Bakalkin G, Big dynorphin as a putative endogenous ligand for the κ-opioid receptor, J. Neurochem. 97 (2006), pp. 292-301
Morley JE and Levine AS, Dynorphin, an endogenous stimulator of feeding, Prog. Clin. Biol. Res. 192 (1985), pp. 293-300
Moroder L, Romano R, Guba W, Mierke DF, Kessler H, Delporte C, Winand J and Christophe J, New evidence for a membrane-bound pathway in hormone receptor binding, Biochemistry. 32 (1993), pp. 13551-13559
Murata M, Peränen J, Schreiner R, Wieland F, Kurzchalia TV and Simons K, VIP21/caveolin is a cholesterol-binding protein, Proc Natl Acad Sci USA 92 (1995), pp. 10339–10343
58
Nakase I, Niwa M, Takeuchi T, Sonomura K, Kawabata N, Koike Y, Takehashi M, Tanaka S, Ueda K, Simpson JC, Jones AT, Sugiura Y and Futaki S, Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement, Mol. Ther. 10 (2004), pp. 1011-1022
Nakase I, Tadokoro A, Kawabata N, Takeuchi T, Katoh H, Hiramoto K, Negishi M, Nomizu M, Sugiura Y and Futaki S, nteraction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis, Biochemistry 46 (2007), pp. 492-501
Nakazawa T, Ikeda M, Kaneko T and Yamatsu K, Analgesic effects of dynorphin-A and morphine in mice, Peptides. 6 (1985), pp. 75-78
Öhman A, Lycksell PO, Andell S, Langel U, Bartfai T and Gräslund A, Solvent stabilized solution structures of galanin and galanin analogs, studied by circular dichroism spectroscopy, Biochim. Biophys. Acta. 1236 (1995), pp. 259-265
Padari K, Säälik P, M. Hansen, Koppel K, Raid R, Langel Ü, and Pooga M, Cell transduction pathways of transportans, Bioconjugate Chem. 16 (2005), pp. 1399-1410
Paterlini G, Portoghese PS and Ferguson DM, Molecular simulation of dynorphin A-(1-10) binding to extracellular loop 2 of the kappa-opioid receptor. A model for receptor activation, J. Med. Chem. 40 (1997), pp. 3254-3262
Pierce JP, Kurucz OS and Milner TA, Morphometry of a peptidergic transmitter system: dynorphin B-like immunoreactivity in the rat hippocampal mossy fiber pathway before and after seizures. Hippocampus 9 (1999), pp. 255-276.
Pike LJ, Han X, Chung KN and Gross RW, Lipid rafts are enriched in enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis, Biochemistry 41 (2002), pp. 2075–2088
Persson D, Thorén PE, Esbjörner EK, Goksör M, Lincoln P and Nordén B, Vesicle size-dependent translocation of penetratin analogs across lipid membranes, Biochim. Biophys. Acta. 1665 (2004), pp. 142-155
Pert CB, Pasternak G and Snyder SH, Opiate agonists and antagonists discriminated by receptor binding in brain, Science 182 (1973), pp. 1359-1361
Pervushin K, Riek R, Wider G and Wüthrich K, Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution, Proc. Natl. Acad. Sci. U S A 94 (1997), pp. 12366-12371
Petrie EC, Tiffany ST, Baker TB and Dahl JL, Dynorphin (1-13): analgesia, hypothermia, cross-tolerance with morphine and beta-endorphin, Peptides. 3 (1982), pp. 41-47
Potocky TB, Menon AK and Gellman SH, Cytoplasmic and nuclear delivery of a TAT-derived peptide and a beta-peptide after endocytic uptake into HeLa cells, J. Biol. Chem. 278 (2003), pp. 50188-50194
Pouny Y, Rapaport D, Mor A, Nicolas P and Shai Y, Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes, Biochemistry 31 (1992), pp. 12416–12423
Rady JJ, Holmes BB and Fujimoto JM, Antianalgesic action of dynorphin A mediated by spinal cholecystokinin, Proc. Soc. Exp. Biol. Med. 220 (1999), pp. 178-183
Rangachari V, Reed DK, Moore BD and Rosenberry TL, Secondary structure and interfacial aggregation of amyloid-beta(1-40) on sodium dodecyl sulfate micelles, Biochemistry 45 (2006), pp. 8639-8648
Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ, Chernomordik LV and Lebleu B, Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake, J. Biol. Chem. 278 (2003), pp. 585-590
Richard JP, Melikov K, Brooks H, Prevot P, Lebleu B and Chernomordik LV, Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors, J. Biol. Chem. 280 (2005), pp. 15300-15006
Rochford J, Godin C and Henry JL, Intrathecal administration of dynorphin A and its fragments increase heart rate and arterial pressure in the urethane anesthetized rat: mediation by a nonopioid mechanism, Brain Res. 565 (1991), pp. 67-77
59
Roques BP and Noble F, Dual inhibitors of enkephalin-degrading enzymes (neutral endopeptidase 24.11 and aminopeptidase N) as potential new medications in the management of pain and opioid addiction, NIDA. Research. Monographs. 147 (1995), pp. 104–145
Sainsbury A, Lin S, McNamara K, Slack K, Enriquez R, Lee NJ, Boey D, Smythe GA, Schwarzer C, Baldock P, Karl T, Lin EJ, Couzens M and Herzog H, Dynorphin knockout reduces fat mass and increases weight loss during fasting in mice, Mol. Endocrinol. 21 (2007), pp. 1722-1735
Sandin J, Nylander I, Georgieva J, Schött PA, Ogren SO and Terenius L, Hippocampal dynorphin B injections impair spatial learning in rats: a kappa-opioid receptor-mediated effect, Neuroscience. 85 (1998), pp. 375-82
Schwarz G and Arbuzova A, Pore kinetics reflected in the dequenching of a lipid vesicle entrapped fluorescent dye, Biochim. Biophys. Acta. 1239 (1995), pp. 51-57
Shanahan F, Lee TD, Bienenstock J and Befus AD, The influence of endorphins on peritoneal and mucosal mast cell secretion, J. Allergy Clin. Immunol. 74 (1984), pp. 499-504
Shukla VK, Bansinath M, Dumont M and Lemaire S, Selective involvement of kappa opioid and phencyclidine receptors in the analgesic and motor effects of dynorphin-A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro, Brain Res. 591 (1992), pp. 176-180
Shukla VK and Lemaire S, Non-opioid effects of dynorphins: possible role of the NMDA receptor, Trends. Pharmacol. Sci. 15 (1994), pp. 420-424
Simon EJ, Hiller JM and Edelman I, Stereospecific binding of the potent narcotic analgesic (3H) Etorphine to rat-brain homogenate, Proc. Natl. Acad. Sci. USA. 70 (1973), pp. 1947-1949
Simoms K and Ikonen E, Functional rafts in cell membranes, Nature 387 (1997), pp. 569-572
Singh IN, Goody RJ, Goebel SM, Martin KM, Knapp PE, Marinova Z, Hirschberg D, Yakovleva T, Bergman T, Bakalkin G and Hauser KF, Dynorphin A (1-17) induces apoptosis in striatal neurons in vitro through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome c release and caspase-3 activation, Neuroscience 122 (2003), pp.1013-1023
Smart EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T and Lisanti MP, Caveolins, liquid-ordered domains, and signal transduction, Mol. Cell. Biol. 19 (1999), pp. 7289–7304
Smith AP and Lee M, Pharmacology of dynorphin, Annu. Rev. Pharmacol. Toxicol. 28 (1988), pp. 123–140
Soomets U, Lindgren M, Gallet X, Hällbrink M, Elmquist A, Balaspiri L, Zorko M, Pooga M, Brasseur R and Langel Ü, Deletion analogues of transportan, Biochim. Biophys. Acta 1467 (2000), pp. 165–176
Stevens CW and Yaksh TL, Dynorphin A and related peptides administered intrathecally in the rat: a search for putative kappa opiate receptor activity, J. Pharmacol. Exp. Ther. 238 (1986), pp. 833-838
Su TP, Psychotomimetic opioid binding: Specific binding of [3H]-SKF-10047 to etorphine-inaccessible sites in guinea-pig brain, Eur. J. Pharmacol. 75 (1981), pp. 81–82
Sugiyama K and Furuta H, Histamine release induced by dynorphin-(1-13) from rat mast cells, Jpn. J. Pharmacol. 35 (1984), pp. 247-252
Suzuki T, Futaki S, Niwa M, Tanaka S, Ueda K and Sugiura Y, Possible existence of common internalization mechanisms among arginine-rich peptides, J. Biol. Chem. 277 (2002), pp. 2437-2443
Tan-No K, Cebers G, Yakovleva T, Hoon Goh B, Gileva I, Reznikov K, Aguilar-Santelises M, Hauser KF, Terenius L and Bakalkin G, Cytotoxic effects of dynorphins through nonopioid intracellular mechanisms, Exp. Cell. Res. 269 (2001), pp. 54-63
Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L and Kisara K, Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-D-aspartate receptor mechanism, Brain Res. 952 (2002), pp. 7-14
Tang Q, Gandhoke R, Burritt A, Hruby VJ, Porreca F and Lai J, High-affinity interaction of (des-Tyrosyl)dynorphin A(2-17) with NMDA receptors, J. Pharmacol. Exp. Ther. 291 (1999), pp. 760-765
60
Tang Q, Lynch RM, Porreca F and Lai J, Dynorphin A elicits an increase in intracellular calcium in cultured neurons via a non-opioid, non-NMDA mechanism, J. Neurophysiol 83 (2000), pp. 2610-2615
Taquet H, Javoy-Agid F, Giraud P, Legrand JC, Agid Y and Cesselin F, Dynorphin levels in parkinsonian patients: Leu5-enkephalin production from either proenkephalin A or prodynorphin in human brain, Brain Res. 341 (1985), pp. 390-392
Terenius L, Stereospecific interaction between narcotic analgesics and a synaptic plasm a membrane fraction of rat cerebral cortex, Acta Pharmacol. Toxicol. 32 (1973), pp. 317-320
Terzi E, Hölzemann G and Seelig J, Interaction of Alzheimer beta-amyloid peptide(1-40) with lipid membranes, Biochemistry 36 (1997), pp. 14845-14852
Trujillo KA and Akil H, Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801, Science 251 (1991), pp. 85-87
Ukai M, Itoh J, Kobayashi T, Shinkai N and Kameyama T, Effects of the kappa-opioid dynorphin A(1-13) on learning and memory in mice, Behav Brain Res. 83 (1997), pp. 169-172
Vivès E, Brodin P and Lebleu B, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem. 272 (1997), pp. 16010–16017
Vivès E, Richard JP, Rispal C and Lebleu B, TAT peptide internalization: seeking the mechanism of entry, Curr. Prot. Pep. Sci. 4 (2003), pp.125–132
Vukojević V, Pramanik A, Yakovleva T, Rigler R, Terenius L and Bakalkin G, Study of molecular events in cells by fluorescence correlation spectroscopy, Cell. Mol. Life Sci. 62 (2005), pp. 535-550
Wadia JS, Stan RV and Dowdy SF, Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis, Nat. Med. 10 (2004), pp.310-315
Walker JM, Moises HC, Coy DH, Young EA, Watson SJ and Akil H. Dynorphin (1-17): lack of analgesia but evidence for non-opiate electrophysiological and motor effects. Life Sci. 31(1982), pp. 1821-1824
Walker JM, Moises HC, Coy DH, Baldrighi G and Akil H, Nonopiate effects of dynorphin and des-tyr-dynorphin, Science 218 (1982), pp. 1136–1139
Wang JB, Johnson PS, Wu JM, Wang WF and Uhl GR, Human kappa opiate receptor second extracellular loop elevates dynorphin's affinity for human mu/kappa chimeras, J. Biol. Chem. 269 (1994), pp. 25966-25969
Wang Z, Gardell LR, Ossipov MH, Vanderah TW, Brennan MB, Hochgeschwender U, Hruby VJ, Malan TP Jr, Lai J and Porreca F, Pronociceptive actions of dynorphin maintain chronic neuropathic pain, J. Neurosci. 21 (2001), pp. 1779-1786
Wen HL, Mehal ZD, Ong BH, Ho WK and Wen DY, Intrathecal administration of beta-endorphin and dynorphin-(1-13) for the treatment of intractable pain, Life Sci. 37 (1985), pp. 1213-1220
Wen HL, Mehal ZD, Ong BH and Ho WK, Treatment of pain in cancer patients by intrathecal administration of dynorphin, Peptides 8 (1987), pp. 191-193
Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L and Rothbard JB, The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters, Proc Natl. Acad. Sci. U S A. 97 (2000), pp. 13003-13008
Wollemann M and Benyhe S, Non-opioid actions of opioid peptides, Life Sci. 75 (2004), pp. 257-270
Woo SK, Tulunay FC, Loh HH and Lee NM, Effect of dynorphin-(1-13) and related peptides on respiratory rate and morphine-induced respiratory rate depression, Eur. J. Pharmacol. 96 (1983), pp. 117-122
Woods AS, Kaminski R, Oz M, Wang Y, Hauser K, Goody R, Wang HY, Jackson SN, Zeitz P, Zeitz KP, Zolkowska D, Schepers R, Nold M, Danielson J, Gräslund A, Vukojevic V, Bakalkin G, Basbaum A and Shippenberg T, Decoy peptides that bind dynorphin noncovalently prevent NMDA receptor-mediated neurotoxicity, J. Proteome Res. 5 (2006), pp. 1017-1023
61
Woolf CJ and Thompson SW, The induction and maintenance of central sensitization is dependent on N-methyl-D-aspartic acid receptor activation; implications for the treatment of post-injury pain hypersensitivity states, Pain 44 (1991), pp. 293-299
Xie GX and Goldstein A, Characterization of big dynorphins from rat brain and spinal cord, J. Neurosci. 7 (1987), pp. 2049-2055
Xue JC, Chen C, Zhu J, Kunapuli S, DeRiel JK, Yu L and Liu-Chen LY, Differential binding domains of peptide and non-peptide ligands in the cloned rat kappa opioid receptor, J. Biol. Chem. 269 (1994), pp. 30195-30199
Yakovleva T, Bazov I, Cebers G, Marinova Z, Hara Y, Ahmed A, Vlaskovska M, Johansson B, Hochgeschwender U, Singh IN, Bruce-Keller AJ, Hurd YL, Kaneko T, Terenius L, Ekström TJ, Hauser KF, Pickel VM and Bakalkin G, Prodynorphin storage and processing in axon terminals and dendrites, FASEB J. 20 (2006), pp. 2124-2126.
Yanagishitia M, and Hascall VC, Metabolism of proteoglycans in rat ovarian granulosa cell culture, J. Biol. Chem. 259 (1984), pp. 10207-10283
Yarygin KN, Zhang XH and Lee NM, Non-opioid dynorphin binding site on secretory vesicles of a pituitary-derived cell line, Brain Res. 791 (1998), pp. 99-107
Yim GK and Lowy MT, Opioids, feeding, and anorexias, Fed. Proc. 43 (1984), pp. 2893-2897
Zamir N, Weber E, Palkovits M and Brownstein M, Differential processing of prodynorphin and proenkephalin in specific regions of the rat brain, Proc. Natl. Acad. Sci. USA 81 (1984), pp. 6886-6889
Zamir N and Quirion R, Dynorphinergic pathways of leu-enkephalin production in the rat brain, Neuropeptides 5 (1985), pp. 441-444
Zhang, L, Peoples RW, Oz M, Harvey-White J, Weight FF and Brauneis U, Potentiation of NMDA receptormediated responses by dynorphin at low extracellular glycine concentrations, J. Neurophysiol. 78 (1997), pp. 582-590
Zhang S, Tong Y, Tian M, Dehaven RN, Cortesburgos L, Mansson E, Simonin F, Kieffer B and Yu L, Dynorphin A as a potential endogenous ligand for four members of the opioid receptor gene family, J. Pharmacol. Exp. Ther. 286 (1998), pp. 136-141.
Ziegler A and Seelig J, Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters, Biophys. J. 86 (2004), pp. 254-263