Membrane Transport Group, Department of Chemistry, The Faculties, The Australian National University, Canberra City, Australian Capital Territory, 0200 Australia
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ABSTRACT |
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Cytotoxic peptides are relatively small cationic
molecules such as those found 1) in venoms, e.g., melittin in
bee, scorpion toxins in scorpion, pilosulin 1 in jumper ant, and
lycotoxin I and II in wolf spider; 2) in skin secretions (e.g.,
magainin I and II from Xenopus laevis, dermaseptin from frog,
antimicrobials from carp) and cells of the immune system (e.g., insect,
scorpion, and mammalian defensins and cryptdins); 3) as
autocytotoxicity peptides, e.g., amylin cytotoxic to pancreatic
-cells, prion peptide fragment 106-126
[PrP-(106-126)], and amyloid
-protein (A
P)
cytotoxic to neurons; and 4) as designed synthetic peptides based on the sequences and properties of naturally occurring cytotoxic peptides. The small cytotoxic peptides are composed of
-sheets, e.g., mammalian defensins, A
P, amylin, and PrP-(106-126),
whereas the larger cytotoxic peptides have several domains composed of both
-helices and
-sheets stabilized by cysteine bonds, e.g., scorpion toxins, scorpion, and insect defensins. Electrophysiological and molecular biology techniques indicate that these structures modify
cell membranes via 1) interaction with intrinsic ion transport proteins and/or 2) formation of ion channels. These two
nonexclusive mechanisms of action lead to changes in second messenger
systems that further augment the abnormal electrical activity and
distortion of the signal transduction causing cell death.
toxins; cytotoxic peptides; amyloids; ion channels; calcium homeostasis; cell death
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INTRODUCTION |
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CYTOTOXIC PEPTIDES ARE SMALL PROTEINS that interact with lipid bilayers, resulting in an alteration of the cell's membrane permeability that leads to cell death. The peptides are thought to contribute to this process through the formation of new ion channels in the cell membrane and/or by changing the activity of existing channels. Many cytotoxic peptides have been shown to act against bacterial cells, e.g., magainin I and II (24, 31), Cyprinus carpio peptides (55), a 9-kDa polypeptide originally extracted from porcine lymphocytes (NK-lysin) (1-5), melittin (reviewed in Ref. 13), lycotoxins (87), insect defensin (20), and scorpion defensin (21). Dermaseptin acts against fungal cells and pilosulin 1 attacks mammalian cells (67, 86). Mammalian defensins are active against all enveloped viruses (43).
The characterization of cytotoxic channel-forming peptides, which modify the cellular signal transduction, forms the basis of understanding how they function in the living organism. This is of great significance to the fundamental science needed for the design of new pharmaceutical agents and for insight into cytotoxic diseases such as Creutzfeldt-Jacob disease or "mad cow" disease. Aside from the benefits flowing from improvement to health and pharmaceutical development, the investigations of cytotoxic peptides could have a marked impact on the meat industries. As shown in the United Kingdom, the meat industry needs to understand and combat cytotoxic disease, which can have devastating economic consequences, while efficiently minimizing the more common pathologies that increase production costs and reduce market acceptance.
Because the molecular mechanisms of interactions between proteins
and membranes are an important frontier of cell biology, the
investigations of these peptides could make a significant contribution
by clarifying the means of interaction between small peptides and cell
membranes and by indicating how this process can be modulated. The new
information about the membrane-binding properties of peptides, which
underlie intracellular signaling, is of fundamental benefit to this
increasingly vital field of cytotoxic peptide research. The
understanding of the properties of cytotoxic peptides could allow us to
1) formulate models of bilayer insertion and conditions of
lipid composition, voltage, pH and Ca2+, which may be
required for peptide interaction and channel formation; 2)
model how highly charged, water-soluble peptides come together and
interact with the membrane to form channels; 3) determine the
roles of peptide domains in the biophysical and pharmacological properties of the channel functions by locating configurations, regions, and polar and nonpolar amino acids that are involved in
determining the conductance levels, ionic permeability and selectivity,
voltage dependency, gating, and kinetics of the channel; 4)
clarify the domain structure of cytotoxic pore-forming peptides, e.g.,
prion and amyloid -protein (A
P), an apparently disparate group of
cytotoxins that may possess some common structural features; and
5) provide models of how these peptides can modify cellular physiological functions and cause pathologies.
Cytotoxic peptides have been isolated from a variety of natural
sources, including venoms and antimicrobial secretions, and are
involved in the mammalian immune response, being present in a range of
cells of the immune system. In addition, there is some evidence that
molecules of similar activity may be involved in "autocytotoxic"
conditions, where the body produces peptides that act against its own
cells, causing disease. Possible examples include the role of amylin in
type II diabetes and apoptosis (11, 65), the prion peptide in the mad
cow disease (58), and AP in Alzheimer's disease (6-9, 66, 73).
It is thought that these cytotoxic-formed ion channels cause these
diseases by modifying a second messenger system, e.g., Ca2+
homeostasis (see Fig. 7B).
The structure, biological activity, and electrical activity of some cytotoxic peptides found in toxins and antimicrobial secretions have been investigated extensively. This information can be used to characterize molecules with cytotoxic activity and may aid in the identification of other, naturally occurring, cytotoxic peptides. Interest in the identification and characterization of such molecules stems from their potential as pharmacological agents, either as antimicrobial or tumoricidal compounds. Knowledge of the mechanisms of their action also provides insight into conditions caused by similar molecules and possible treatments for such conditions. This review examines a selection of not-well-characterized cytotoxic molecules found in venoms, antimicrobial secretions, and the mammalian immune system. We have surveyed and summarized our current understanding of the properties of these cytotoxic-peptide-formed ion channels and provided a framework for future research. These peptides are grouped according to their having several common features. They are small molecules, often cationic and amphipathic in nature. Their amino acid sequences vary, but certain motifs and secondary structures are common. Few of the ion channels formed by cytotoxic peptides have been characterized extensively, but those that have show very diverse characteristics and include anion-selective, cation-selective, and nonselective channels. Some cytotoxic peptides have been well characterized and reviewed previously (see Ref. 13). The natriuretic peptides, some of which are present in venoms and toxins, have been reviewed elsewhere (46-49).
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TYPES OF CYTOTOXIC PEPTIDES |
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The cytotoxic peptides discussed here can be divided into three main
groups based on the origin of the molecule. Perhaps the most
well-studied group is that of the cytotoxic peptides found in various
venoms. Melittin, found in bee venom, is particularly well
characterized, providing a model for other cytotoxic molecules (see
review in Ref. 13). Cytotoxic peptides are also found in scorpion venom
(long- and short-chain scorpion toxins), jumper ant (Myrmecia
pilosula) venom (pilosulin 1), and the venom of the wolf spider
(Lycosa carolinensis; lycotoxin I and II). The second group of
cytotoxic peptides is found in antimicrobial secretions and cells
involved in immunity. Magainin I and II secreted from Xenopus
laevis skin are the most extensively characterized (also reviewed
in Ref. 13). Dermaseptin is another peptide isolated from frog skin.
Antimicrobials have been identified in skin secretions of carp
[C. carpio (55)] and in cells of the immune system
(e.g., insect defensins, scorpion defensins, mammalian defensins, and cryptdins). The third group of cytotoxic peptides is involved in
pathological conditions and is believed to be produced by the organism
itself (autocytotoxicity). Amylin, which is cytotoxic to pancreatic
-cells and implicated in the pathogenesis of type II diabetes, is
thought to act in this way (65). Also, fragments of the prion peptide
are believed to form or alter ion channels causing cytotoxicity, and
these can be considered in this group, because the prion peptide is a
naturally produced peptide in mammalian systems. Last, synthetic
peptides based on the sequences and properties of naturally occurring
cytotoxic peptides have been demonstrated to retain cytotoxic activity.
These can be distributed within the above three groups according to the
cytotoxic peptide from which they are derived (e.g., see Refs. 19, 22).
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STRUCTURE OF CYTOTOXIC PEPTIDES |
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Overall Characteristics
The minimum length of cytotoxic peptides found naturally is 23 amino acids, i.e., the magainins (24, 31), whereas shorter synthetic peptides with similar characteristics have also been demonstrated to have cytotoxic activity, i.e., prion fragment (58). These lengths correspond to the approximate minimum of 20 amino acids that is required forSequence and Structure
The amino acid sequences vary considerably among the cytotoxic peptides, but structurally related subgroups of peptides can be identified (see Table 5). One group contains positive residues distributed along the length of the molecule. This creates a molecule that is amphipathic, with hydrophobic and hydrophilic regions present. These molecules are predicted (and in some cases demonstrated) to form amphipathicJones (42) made prediction of the tertiary structure of NK-lysin using
multiple sequences and recognized supersecondary structural motifs.
Similarly, Liepinsh et al. (57) used the NMR structure of NK-lysin to
reveal folding in saposin. NK-lysin is viewed as the first
representative of a family of sequence-related proteins, such as
saposins, surfactant-associated protein B, pore-forming amoeba
proteins, and domains of acid sphingomyelinase, acyloxyacylhydrolase, and plant aspartic proteinases, for which a four--helix bundle motif
of cytolytic peptides is suggested (27).
Cationic molecules with evenly distributed positive residues.
Many of the cytotoxic peptides are thought to form structures composed
primarily of -helices when in a hydrophobic environment such as that
encountered in the lipid membrane. They are often compared with
melittin, one of the first cytotoxic molecules whose structure was
determined. Vogel and Jahnig (84) used Raman spectroscopy to determine
the three-dimensional shape of melittin in lipid membranes, finding
that the molecule formed a bent
-helix from residues 1 to 21. The
helix is divided into residues 1-11 and 12-21 that are bent
at an angle of 60° to each other. The COOH-terminal residues,
22-26, form a nonhelical hydrophilic domain Fig.
1 (see Ref. 84). The venom of the wolf
spider, L. carolinensis, contains two peptide toxins: lycotoxin
I and II. The amino acid sequences of lycotoxin I (25 amino acids) and
lycotoxin II (27 amino acids) are characterized by lysine residues
every four to five amino acids. Similar motifs are found in
adenoregulin, dermaseptins, and magainins (as determined by a GenBank
search). Both lycotoxin molecules are positively charged (lycotoxin I = +5; lycotoxin II = +6). The predicted secondary structure of these two
toxins is that of an amphipathic
-helix (87).
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Cysteine-rich molecules composed of both -helix and
-sheet domains.
Scorpion toxins, scorpion defensins, and insect defensins form a
second, structurally similar, group of cytotoxic molecules. Another
related peptide group are the thionins (50). The molecules have poor
sequence homology but are all cysteine rich and share a similar
cysteine motif. These cysteine molecules form disulfide bridges that
stabilize a common secondary structure composed of an antiparallel
triple-stranded
-sheet linked by two disulfide bridges to an
-helix; the
-sheet is linked via a third disulfide bridge to the
NH2-terminal region of the molecule (Fig.
2; see also Refs. 15-16, 50). This
structure forms the core of the molecules in this group. There have
been three groups of scorpion toxins identified.
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Molecules composed predominantly of -sheet
structures.
Yet another group of peptides with cytotoxic activity are
those composed predominantly of
-sheet structures. These include mammalian defensins and cryptdins, human amylin, and
PrP-(106-126). Of particular interest are those that were
predicted on the basis of their amino acid sequence to form
-helices
but that have been demonstrated to be composed of
-sheet when
active. This raises the question of whether other peptides predicted to
form
-helices in fact do so. Investigations of ion channel-forming
molecules in other areas has revealed that those forming
-sheets
represent an important structural class of ion channel-forming
molecules (6-9, 73).
Unknown sequence and/or structure. The amino acid sequence and/or secondary structure of a number of cytotoxic peptides has not been determined. Investigation of the structures of such molecules is needed if we are to gain a more comprehensive picture of the patterns of channel formation by cytotoxic peptides. Two peptides with antibacterial activity were isolated from the skin secretions of C. carpio (55). The 27-kDa protein isolated was found to be glycosylated, and its first 19 amino acid residues were determined. It did not show any similarity to known protein sequences. The 31-kDa peptide was nonglycosylated and able to form ion channels. It could not be sequenced because it was blocked at its NH2-terminus. NK-lysin, isolated from pig small intestines and believed to originate from cytotoxic T cells and NK cells, is 78 amino acid residues in length. It contains six cysteine residues, which form three disulfide bridges (3, 5, 17, 54). The molecule is much larger than the mammalian defensins and, despite the disulfide bridges, shows no sequence similarity to these molecules (3, 5).
Tertiary Structure: Models of Channel Formation
Many amphipathic molecules are commonly thought to form multimeric pores in membranes, with their hydrophilic faces forming the inside of the pore and their hydrophobic region interacting with the hydrophobic lipid membrane. This can be illustrated by a model for the formation of melittin channels. Vogel and Jahnig (84) proposed that melittin aggregated in tetramers, forming hydrophilic pores in membranes. Fluorescence quenching experiments were used to determine the orientation of melittin in the membrane. These experiments demonstrated that the COOH terminals of melittin molecules were located on the side of the membrane to which melittin was added (i.e., the outside of a bacterial cell membrane in vivo). The helical region of melittin has a hydrophilic and a hydrophobic face. Thus, to form an energetically favorable model of pore formation, melittin is required to form multimeric channels. In this model the hydrophilic faces of the molecules are aligned to form a hydrophilic pore while the hydrophobic faces interact with the hydrophobic regions of the lipid bilayer Fig. 5 (see Ref. 84).
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Cruciani et al. (24) proposed three structural models for a magainin
II-formed ion channel (Fig. 6). In the
models, the magainin II molecules are arranged in dimers of -helices
aligned to form antiparallel amphipathic units. Type 1 channels were
composed solely of peptide molecules arranged so that the polar side
chains of amino acids extended into the lumen, while the hydrophobic portions of the molecules formed the wall of the channel. Large numbers
of monomers were required to form channels of this type. The central
pore of the channel was calculated to be electropositive, making the
channel selective for anions. These channels are similar to those
described for melittin above. Type 2 channels were composed of both
peptides and lipids, with some of the channel wall being formed by the
hydrophilic heads of lipid molecules from the surrounding lipid
bilayer. The alkyl tails of lipid molecules involved in channel
formation extended into the hydrophobic region of the membrane and were
stabilized by aggregates of magainin II peptide on the surface of the
membrane, where hydrophobic lipid tails would otherwise come into
contact with the aqueous environment. The central pore of the channel
thus formed was calculated to be electronegative, consistent with
cationic selectivity. Type 3 channels also involved both lipid and
peptide molecules. In this case head groups from lipid molecules formed
the lining of the ion channel while the peptide molecules remained on
the surfaces of the membrane stabilizing the structure. Magainin II is
required on both sides of the membrane for this type of channel to
form. In this model the channel lumen was calculated to be
electronegative, consistent with cationic selectivity. The channel
models were dependent on the lipid composition of the membrane in which
the channel was formed. Cruciani et al. (24) based their models on
membranes composed of a combination of negatively and positively charged lipids. Type 2 structural models assumed that 24 out of the 27 lipid molecules involved in forming the pore were
palmitoyl-oleoyl-phosphatidylserine (PS), while type 3 models were
constructed using palmitoyl-oleoyl-phosphatidylethanolamine (PE) to
line the channel and PS to form the bilayer regions.
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Cruciani et al. (24) were then able to speculate on the type of channel
that was formed by magainin II in their experiments. Because the
channels that they found were cationic selective, type 1 channels were
not probable. They speculated that type 3 channels accounted for the
majority of conductance. However, because of the requirement for
peptide on both sides of the membrane for the formation of type 3 channels, these were not the initial channels formed. They proposed
that type 2 channels were formed first and then, as they broke down,
released some peptide on the other side of the membrane, allowing type
3 channels to form. Extending their work to other literature, Cruciani
et al. (24) speculated that the magainin I anionic selective channels,
found by Duclohier et al. (31), may have been type 1 channels. However,
this work was done using neutrally charged lipid membranes, and these
models of channel formation were formed on the basis of some negatively charged lipids in the membrane. They acknowledged that determining the
likely channel model was not straightforward. More complex models
involving lipids from the membrane-forming sections of the channel have
been suggested for the formation of some channels, in particular to
explain how cationic peptides could form a cationic-selective channel.
Models of channel formation for cytotoxic peptides not composed solely
of amphipathic -helices have not been investigated in such detail.
The family of scorpion toxins and scorpion defensin toxins are proposed
to interact with preexisting ion channels in the membrane. However, the
related peptide, insect defensin, itself forms ion channels (20),
suggesting that peptides from this structural group can form ion
channels. Further investigation will help to clarify this.
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BIOLOGICAL ACTIVITY OF CYTOTOXIC PEPTIDES: EVIDENCE FOR CYTOTOXIC ACTIVITY |
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By definition cytotoxic peptides have cytotoxic activity, mostly antimicrobial activity or hemolytic activity. This activity is demonstrated in assays of cytotoxicity against various cell types (bacterial, fungal, and mammalian cells, especially blood cells). The specific biological activity of each peptide depends on the environment in which it is found. Some peptides found in venom function as toxins and are used by organisms for defense or to subdue prey. Cytotoxic peptides found in antimicrobial secretions function primarily by providing defense against pathogens such as colonizing bacteria. Those produced by immune system cells are also involved in host defense. There is some evidence that molecules with a similar molecular activity are also involved in autocytotoxic conditions.
Lycotoxins I and II, from wolf spider venom, were shown to have antimicrobial activity against both prokaryotic and eukaryotic cells. Various microbes were incubated with the peptides to determine the specificity of their antimicrobial activity. Lycotoxin I was inhibitory at concentrations as low as 5 µM for one gram-positive species (Bacillus thuringiensis israelensis), whereas lycotoxin II was most active against gram-negative strains (Escherichia coli strain DH5 minimal inhibitory concentration 40 µM). Both peptides were active against yeast (Candida albicans) at a concentration of 40 µM. Magainin II was also assayed to provide comparison: in most instances the inhibitory activity exhibited by the lycotoxins was stronger than that exhibited by magainin II (87). Inhibition of growth occurred at a critical concentration, indicating self-aggregation of the peptide. Hemolytic activity was also assayed for lycotoxin I. Lycotoxin I was found to cause lysis of erythrocytes at concentrations >100 µM and was more active than magainin II in this respect (87). Pilosulin 1 causes cell lysis via disruption of membrane integrity in both dividing and nondividing blood cells (86). Lysis of erythrocytes was complete at a concentration of 40 µM, with partial lysis occurring at concentrations as low as 1.25 µM. White blood cells were differentially affected by pilosulin 1, with mononuclear cells being more susceptible to lysis than granulocytes. Lysis was rapid and usually complete, but results (for white blood cells) differed in normal individuals by up to fivefold. Pilosulin 1 also had a cytotoxic effect on Epstein-Barr virus (EBV)-transformed B lymphocytes [dose of toxin that will kill 50% of test subjects in standard time (LD50) = 0.4 µM] (86).
Lemaitre et al. (55) incubated various strains of bacteria with two ion-channel-forming peptides (27 and 31 kDa) isolated from carp (C. carpio) skin mucous. Both compounds were found to have antibacterial activity. Inhibition of bacterial growth occurred at concentrations of ~5 µg/ml (0.16-18 µM). Gram-positive and gram-negative bacterial strains were equally affected. However, Micrococcus luteus (gram positive) showed great sensitivity when exposed to the 27-kDa peptide at a concentration of only 0.5 µg/ml (0.018 µM) (55).
NK-lysin was shown to have antimicrobial activity against several
strains of gram-negative and gram-positive bacteria, the highest
activity being against E. coli and Bacillus megaterium. In assays for hemolytic activity against sheep red blood cells, no
hemolysis was detected at concentrations of NK-lysin of 170 µM. The
peptide also exhibited antitumor activity in assays against YAC-1 tumor
cells (lymphoma cells from the lung) (4). In addition, it has been
shown that NK-lysin (1-100 nM) potently and
reversibly stimulates insulin secretion in rat pancreatic islets and in
the cell line HIT T15 (89). However, the effect of NK-lysin was not
accompanied by changes in Ca2+ concentration. Furthermore,
the stimulatory activity of NK-lysin on insulin release was observed in
permeabilized islets under Ca2+-clamped conditions. These
findings indicate that NK-lysin action, although it may interact with
the membrane, is unlikely to be due to NK-lysin-formed
Ca2+-permeable channels.
RK-1, a novel agent related to the corticostatins/defensins and isolated from the kidney, was found to have antimicrobial activity. It was active against E. coli cultures at concentrations between 15 and 150 µg/ml (12). Magainin I and II show antibacterial activity against a range of microbes, including both gram-positive and gram-negative bacteria as well as fungi (90). Magainin II has between 5 and 10 times the antimicrobial activity of magainin I and is more abundant in the secretions of X. laevis. Magainins also have anti-protozoan activity, with lysis of the protozoan Paramecium caudatum occurring on exposure to magainin II at concentrations of 10 µg/ml (90).
Selected magainin analogs with amino acid substitutions aimed at
increasing the amphipathic -helical nature of the molecule have been
shown to have increased antimicrobial activity (19). The synthetic
peptides had the same spectrum of activity as the natural peptide but
were more active by one to two orders of magnitude. Whereas magainin I
does not cause hemolysis of erythrocytes at concentrations of 250 µg/ml, the altered analogs (except for analog H) were capable of
causing hemolysis at concentrations of 100 µg/ml. Dermaseptin was
tested for cytotoxic activity against fungal species (Aspergillus
fumigatus and Arthroderma simii) known to be pathogenic to
the frog P. sauvagii. The fungal species were inhibited at a
dermaseptin concentration of 10 µg/ml. Assays against a gram-positive
bacterial species (Bacillus subtilis) showed no inhibition
(67). The synthetic peptides that were constructed by Cornut et al.
(22) were assayed for hemolytic activity with the use of human red
blood cells. Hemolytic activity varied according to the length of the
peptide, with the longer peptides being more active. Activity was
recorded even at very low concentrations (LD50 = 4-6 × 10
8 µM). This corresponded to
an activity 6-10 times higher than that found for melittin (22).
On the basis of this cytotoxic activity, such peptides are suspected to
cause alterations in membrane permeability. Generally, it appears that
some peptides do this via the formation of ion channels, whereas others
may interact with ion channels already present in the membrane.
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CYTOTOXINS AS FORMERS OF ION TRANSPORT PATHWAYS: EVIDENCE FOR ION CHANNEL FORMATION |
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Ion Flux Experiments
Ion flux experiments can be used to help characterize the cytotoxic activity of peptides. These experiments can illustrate the interaction of these molecules with cell membranes and the consequent changes in membrane permeability and ion homeostasis. Cociancich et al. (20) demonstrated that the insect defensin A had antimicrobial activity and that this was mediated through the efflux of K+ from cells. They tested a synthetic peptide, corresponding to the sequence of defensin A from P. terranovae, for antimicrobial activity against M. luteus. Cells exposed to defensin A had immediately disrupted membrane permeability, resulting in loss of cytoplasmic K+. The effect of defensin was decreased in growth media of high ionic strength and maximal at pH 7.5. The cells maintained a membrane potential of 110 mV, indicating the peptide did not completely disrupt the membrane. Instead, Cociancich et al. (20) suggested, the defensin formed an ion channel that resulted in K+ transport.Lycotoxin I was found to cause an efflux of Ca2+ from rat brain synaptosomes and to dissipate the membrane potentials of insect muscle cells (87). Addition of lycotoxin I to Ca2+-loaded rat synaptosomes resulted in dissipation of the Ca2+ gradient, mediated through an efflux of Ca2+ from the synaptosome, and the prevention of Ca2+ sequestration. Similarly, the membrane potentials of insect muscle cells were dissipated when the cells were exposed to lycotoxin I at a concentration of 3 µM. In combination with the antimicrobial data, these factors constitute indirect evidence that lycotoxins act as pore-forming peptides (87). However, detailed characterization of these potential pores, including ion channel kinetics, selectivity, and conductance, was not completed.
Florio et al. (36) report that the PrP-(106-126) fragment induces apoptosis in GH3 cells via dose-dependent inactivation of the L-type voltage-sensitive Ca2+ channels. The blockage of these channels resulted in blockade of the increase in cytosolic Ca2+ following depolarization and resulted in cell death with features of apoptosis. They speculated that alterations in Ca2+ homeostasis may be the trigger for apoptosis. In contrast, Lin et al. (58) found that PrP-(106-126) in planar lipid bilayers induced the formation of ion channels that were permeable to common physiological ions. It was proposed that the channels would cause leakage of ions across the membrane of cells, resulting in disturbance of ion balance. This could lead to a large metabolic demand being placed on the cells with attempts to correct the ion balance. Disturbance of ion balance could also lead to ionic toxicity and might trigger apoptosis. Discharge of membrane potential, due to ion imbalance, could alter cellular Ca2+ levels (via voltage-dependent Ca2+ channels, N-methyl-D-aspartate receptors, or alteration in Na+/Ca2+ exchange mechanisms), which may trigger apoptosis (58, 69).
More recently, Lin et al. (58) used a combined immunofluorescence
labeling with an antibody raised against the NH2-terminal domain, and atomic force microscopy, to image the structure of AP-reconstituted phospholipid vesicles, whose
45Ca2+ uptake was measured to assess the
Ca2+ permeability across the vesicular membrane. Their
findings suggest that globular and fresh A
P-(1-40) forms
Ca2+-permeable channels. For example,
45Ca2+ uptake was inhibited by a monoclonal
antibody raised against the NH2-terminal region of A
P
and by Zn2+, a known blocker of A
P-formed channels (58).
Villus Enterocyte Volume Assay
The villus enterocyte volume assay is used to assess the activity of peptides on villus enterocytes. Because these cells use movement of ions across the cell membrane to regulate their volume, cell volume can be used as an indicator of ion movement (59, 60). The movement of water in response to ion flux contributes to any volume change. A change in cell volume on incubation with a peptide suggests that the peptide has altered the cell membrane permeability and ion balance. This assay has been used to demonstrate membrane permeabilization effects of peptides belonging to the corticostatin/mammalian defensin family of molecules (cryptdins, RK-1, corticostatic peptides/defensins).MacLeod et al. (60) demonstrated the effects of the family of corticostatic peptides and defensins on villus enterocytes. The peptides, when incubated in isosmotic conditions with enterocytes from guinea pig jejunums, caused a reduction in cell volume. They suggested that the reduction in volume was due to activation of L-type Ca2+ channels (60). By using the villus enterocyte volume assay, it was shown that RK-1, like the corticostatin/defensins, causes enterocyte shrinkage of epithelial cells in isotonic media. This shrinkage occurred in both the presence and absence of Na+ and was prevented by the use of Ca2+-free media. Niguldipine prevented the volume reduction caused by RK-1. Activation of dihydropyridine-sensitive Ca2+ channels is implicated in the shrinkage of the cells (12).
The cryptdins, or enteric defensins, are secreted by cells lining the
mammalian gut and have been found to form anion-conductive channels in
human intestinal T84 cells in vitro (56). Use of the villus
enterocyte volume assay to assess ion movement across the cell membrane
showed that the cryptdins elicited a Cl secretory
response. The secretory response was not attributable to receptors or
ion channels already present in the cells and was correlated with the
formation of new anion-permeable channels in the cell membrane. The
pores formed were permeable to carboxyfluorescein.
Electrical Properties
The short-circuit current (Isc) method was used to investigate the actions of cryptdins. Cryptdins 2 and 3 caused ClMost experiments to characterize ion channels formed by cytotoxic
peptides have used the patch-clamp or lipid bilayer techniques (Table
1). Ion channel experiments directly
illustrate the capacity of peptides to form ion-permeable channels.
Relatively few peptides known to have cytotoxic activity have been
proven to form ion channels in such experiments. Those that do form ion
channels include insect defensin (20), melittin (71, 72), C. carpio peptides (55), the magainins (24, 31), mammalian defensin rabbit NP-1 (43), AP-(1-40) (9), amylin (65), and
PrP-(106-126) (58). Some of these channels have also been further
characterized to determine their conductance, current-voltage
relationships, and ion selectivities.
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The characteristics of ion channels formed by cytotoxic peptides vary
considerably. For example, magainin I forms anion-selective channels
(31) and magainin II forms cation-selective channels (24). Rabbit NP-1,
a mammalian defensin, forms voltage-dependent, weakly anion-selective
channels (43). AP formed cation-selective channels (6, 9), and human
amylin was also found to form voltage-dependent channels that are
permeable to Na+, K+, Ca2+, and
Cl
(65). Prion peptide segment PrP-(106-126),
which was reported to block L-type voltage-sensitive Ca2+
channels (36), was found to form nonselective ion channels (58). Insect
defensin was shown to form cation-selective voltage-dependent channels
in giant liposomes (20), and C. carpio peptides form ion
channels of differing selectivities (55). The details of these
investigations and the characterization of the ion channels appear
below and in Tables 1 and 2.
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ION CHANNEL CHARACTERISTICS OF CYTOTOXIC PEPTIDES |
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Conditions Influencing the Interaction of Cytotoxic Peptides With Membranes
Various factors, e.g., lipid composition, voltage, pH, and Ca2+, influence the interaction of cytotoxic peptides with cell membranes (e.g., see Ref. 40). Most of the cytotoxic peptides are positively charged, and thus it is expected that the negative phospholipids may affect any interaction between them. Specifically, the effects of different levels (or even the absence) of the negatively charged phospholipid (PS) in bilayers on the channel activity need to be examined. The studies of the interaction of cytotoxic peptides with membranes could both reveal the conditions that may make the cell membranes susceptible to enhanced interaction of cytotoxic peptides and indicate how this susceptibility can be overcome. It is already known that voltage and acidic conditions are required for the domains of some peptides to dock onto and interact with the bilayer to form ion channels, as suggested for antimicrobial colicin E1 (63) and Bcl-2-formed channels (64). The role of acidic solutions in peptide aggregation andThe conductance properties and the experimental conditions for the cytotoxic peptide-formed channels are shown in Table 1. The phospholipid composition (Table 1) of bilayers used to investigate ion channel formation by cytotoxic peptides can be important. Although some peptides (e.g., NP-1) have been shown to form ion channels in membranes composed of a wide range of lipids and to have no specific lipid requirements, many peptides require a certain content of negatively charged lipids in the membrane. In some instances the characteristics of the channels formed in membranes of different composition are different (e.g., magainin I).
Kagan et al. (43) used membranes composed of PE, phosphatidylcholine (PC), and PS in a ratio of 2:2:1 (wt/wt/wt) when testing the ability of NP-1 to form ion channels. In addition to forming ion channels in this type of membrane, they found that NP-1 was able to form ion channels in membranes composed of different mixtures of these lipids, indicating that membrane composition did not affect activity (43).
For ion channels formed by magainin I, the channel amplitude was altered by membrane composition. Membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (7:3) favored conductance levels of 80 pS, considerably lower than the conductance levels determined in membranes composed of POPC/1,2-dioleoyl-3-phosphatidylethanolamine (7:3; 366 and 683 pS) (31). Magainin II ion channel formation requires the presence of negatively charged lipids in the membrane (24). Ion channel formation, as measured by increased membrane conductance, was demonstrated in membranes composed of PS/PC or PS/PE, but not in those containing only PE or PE/PC combinations. Cruciani et al. (24) speculated that the type of lipids present in the membrane helped determine the ion selectivity of the magainin II ion channel. Thus when magainin II interacts with membranes containing negatively charged lipids, ion channels selective for cations are generated. Conversely, membranes that do not contain negatively charged lipids are unable to support ion channel formation. Ion channel formation by magainin I was similarly dependent on the lipid composition of the membrane: membranes composed of PE/PS supported channel formation, whereas those composed of PE alone or PE/PC did not induce channel formation, indicating a requirement for negatively charged lipids in target membranes. Membrane composition altered the channel-forming activity of amylin. Membranes that had a high net negative surface charge induced the highest channel-forming activity. Membranes that contained 40% negatively charged phospholipids were approximately six times more sensitive to amylin channel formation than those containing only 20% negatively charged amino acids (65). Increasing membrane rigidity by including cholesterol causes a reduction in the membrane's sensitivity to amylin.
In some investigations, the reversibility of ion channel formation has been tested. The association of amylin with the membrane appears to be irreversible: extensive washing of the side of the membrane that contained the amylin failed to reduce or eliminate the current (65). Similarly, washing of the compartment containing the prion peptide fragment did not affect conductance, indicating that the channels formed were irreversibly associated with the membrane (58).
For several peptides, channel formation occurs only when the membrane
is held at a negative voltage. This reflects the state of the membrane
as it would "appear" to such peptides in vivo. For
example, ion channels formed from magainin I appeared only when the
membrane was held at a negative voltage (31). Similarly, Kagan et al.
(43) found that the steady-state conductance could be induced after
negative voltages (70 to
90 mV) were applied to the
membrane for 15-30 min, and increased exponentially with increasingly negative voltage. The channels formed by
A
P-(25-35) are voltage dependent, opening when the opposite
side of the membrane is made negative with respect to the
A
P-(25-35)-containing side and closing when the polarity of the
voltage is reversed (66). This is taken to indicate that
A
P-(25-35) channels would open at the resting membrane
potential of neurons if the peptides were located in an extracellular
or endosomal/lysosomal compartment.
The variations in conductance and kinetic properties of cytotoxic
peptide-formed channels, e.g., AP-formed channels (6, 9, 40) reflect
the differences in 1) peptide properties, e.g., isoforms, the
ratio of
-helices to
-sheets, homogeneity, and aggregation;
2) bilayer properties, e.g., phospholipids' composition, presence of solvent, bilayer capacitance, and stability; 3)
experimental solutions, e.g., ionic composition, ionic concentrations
and gradients, buffers, pH, and Ca2+ levels; and
4) recording conditions, e.g., voltage, gain,
recording duration, and sampling rates.
Conductance and Current-Voltage Relations
The ability of a peptide to form ion channels can be further investigated by the demonstration of an increase in membrane conductance. In addition, the current-voltage relationship for the peptide-associated membranes can be determined (Table 1). Lemaitre et al. (55) isolated two hydrophobic peptides with channel-forming properties from the skin mucus of carp (C. carpio). The two peptides induced strong current fluctuations in lipid bilayers (7:3 PC/PE). The 27-kDa peptide gave a conductance value of 900 pS in 1 M KCl (at +50 mV) and the 31-kDa peptide gave a conductance value of 500 pS (1 M KCl, and at +40 mV). Lemaitre et al. (55) state that these conductances were higher than those found previously for toxins or outer membrane proteins from bacteria (41) and higher than those of insect defensins (72); conductances previously determined for antimicrobial peptides from amphibians are closer in value (31).Kagan et al. (43) found that single-channel conductance values were heterogeneous, ranging between 10 and 1,000 pS. Conductance increased with NP-1 concentration, suggesting that a multimer of NP-1 may form the ion channel. Conductance also consistently increased with time, which may be due to the formation of larger channels with an increased number of the NP-1 molecules per channel. Magainin I ion channels were shown to exhibit two possible conductance levels (366 and 683 pS). Each occurred at approximately equal rates but tended not to occur in the same experiment (31). Conductance was dependent on peptide concentration, with increased macroscopic conductance occurring with increased peptide concentration. Greater concentrations of peptide resulted in increased current amplitude. This is very likely to be due to the recruitment of more subunits of the channel-forming peptide. The current-voltage relationships for channels of both conductance levels were nonsaturable and ohmic (31). Cruciani et al. (24) found that the ion channels formed by magainin II gave heterogeneous conductance values (1-300 pS). They suggested that this wide variation was due to variable channel structure occurring with dynamic incorporation and reaggregation of molecules within the membrane. However, the unitary conductance of the single-channel conductance is reported to be between 1 and 2 pS. The current-voltage relationship for channels formed by magainin II showed rectification at both negative and positive membrane voltages. This was more marked at positive voltages, with greater current flow recorded at positive voltages than at negative voltages of the same magnitude (24).
To provide evidence that ion transport that occurred in the presence of
insect defensin from P. terranovae was due to the formation of
ion channels by the peptide, Cociancich et al. (20) demonstrated that
insect defensin formed ion channels in giant liposomes. Peptide samples
were incorporated into giant liposomes composed of asolectin, and
patch-clamp techniques were used to determine the single-channel
conductance of ion channels thus formed. Insect defensin was found to
form channels of heterogeneous conductance values (ranging from 100 to
200 pS). The channels also had varying open and closed times. The
current through the insect defensin-formed channels increased at high
absolute membrane voltages. At potentials more positive than +27 mV and
more negative than 45 mV, the frequency of current transitions
between the mainly open state and the closed state also increased.
The channels formed by PrP-(106-126) (58) were found to have
heterogeneous conductance values, with the most common single-channel conductance values being 20, 40, and 60 pS (membrane voltage held at
+50 mV). The channels were voltage dependent and the current-voltage relationship was linear. Lin et al. (58) stated that the concentration at which ion channels formed (20 µM) was similar to that which causes neurotoxicity (37) and also comparable to the concentrations required for channel formation by other ion-channel-forming peptides (58). Channel activity was increased at acidic pH and also after aging
of the peptide under adverse conditions (detailed below in
CYTOTOXIC CHANNELS UNDER ADVERSE CONDITIONS).
Amylin was shown to cause an increase in membrane
conductance, with single-channel conductance determined as 7-8 pS.
The current-voltage relationship for the open state of the channel was
ohmic (65).
The properties of AP-formed channels are summarized in Table 2. The
conductance of the A
P-formed channels can be as high as 5 nS (9),
with multiconductance properties (Fig. 7)
(see also Refs. 7, 44, 73). A
P-(1-40) channel activity
resulting from the incorporation of the peptide from the solution into
an inside-out excised patch showed spontaneous transitions between different current levels, which is characteristic of multiconductance channels.
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The steady-state conductance of the cell membrane of bullfrog
sympathetic neurons after the application of AP-(25-35) was examined (77). The current was linear between
110 and +50 mV, and the reversal potential was close to 0 mV. There was a time lag
between the time of application of A
P-(25-35) and the observed increase in conductance. The time lag was shorter when higher concentrations of A
P-(25-35) were used. The magnitude of the conductance increase, however, was not dependent on the concentration of A
P-(25-35).
Recently, Hirakura et al. (40) reported that
AP- (1-42) incorporation into anionic planar bilayer
membrane also forms slightly cation-selective, voltage-independent
ion channels with multiple conductance levels at neurotoxic
concentrations in acidic solutions. The channels show substantial
irregularity of activity, and the size of conductances and the
length of open lifetimes depend on solvent history. These properties
are in agreement with the suggestion that these channels are very
likely to be formed by aggregates of A
P-(1-42). The
pharmacology of A
P-(1-42)-formed channels is in agreement with
other A
P-formed channels, in that the channels are reversibly
blocked by Zn2+ in a voltage-independent manner.
Cation/Anion Permeability and Selectivity Sequence
Cytotoxic peptides and other toxins often exert their effects by inducing nonselective or weakly selective anion or cation channels that depolarize membrane potential and dissipate ionic and water gradients, leading to cell lysis and death. Some of these peptides may also function by allowing the movement of Ca2+ across the membrane, leading to changes in the Ca2+ homeostasis that underlies muscle contraction and relaxation, cell volume regulation, and salt secretion of epithelial cells and neuron synapses.The channels formed from cytotoxic peptides may be expected to be
selective for anions over cations because of the cationic nature of
most of these peptides. However, both anionic and cationic channels, as
well as nonselective channels, have been reported (Table
3). Interestingly, more molecules have been
shown to form cation-selective channels than either nonselective or
anion-selective channels. This has implications for the models of
channel formation proposed, suggesting that the more simplistic models
that predict anion selectivity may not reflect the actual structure of
the channels that form.
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Channels formed from rabbit NP-1 showed weak anion selectivity,
favoring Cl over Na+ and K+,
but this was not exclusive
(PCl/PNa = 2.4:1) (43).
Similarly, as expected from its overall positive charge, the magainin I
molecule forms anion-selective channels in artificial planar membranes (PCl/PK = 3) (31). In contrast
to the results obtained for magainin I by Duclohier et al. (31),
magainin II, also a positively charged peptide, was found to form
cationic selective channels (PNa,
PK/PCl = 5:1) (24). The ion
channel formed by magainin II showed no difference in conductance for
different monovalent cations (Na+, K+,
Li+) or monovalent anions (e.g., Cl
,
I
) tested. Cruciani et al. (24) also reported
results for magainin I, which indicated that it also formed cation
selective channels, contradicting previous findings (31).
Cociancich et al. (21) showed that the antimicrobial activity of insect defensins was caused by efflux of K+ from the cytoplasm of cells. In addition, they demonstrated that the peptide formed ion channels in giant liposomes. These results imply that the channels formed by the insect defensin were responsible for the K+ movement and thus selective for cations, in particular K+. They do not, however, report data that directly demonstrate the cationic selectivity and sequence.
Channels formed by PrP-(106-126) exhibited cationic selectivity
(PNa/PCl = 2.5) and were
permeable to most physiological ions. The sequence of ion permeability
was Ca2+ > Na+ > K+ > Li+ > Rb+ > Cs+ > Cl. Lin et al. (58) state that the channels were
relatively nonselective and sufficiently large to cause cell death via
membrane disruption. This leads to the loss of the membrane potential
and to changes in membrane permeability that result in altered ion
balance and, in particular, altered Ca2+ balance, which is
a potential trigger for apoptosis.
The two peptides isolated by Lemaitre et al. (55) from the secretions
of C. carpio skin showed different ion selectivities. The
27-kDa peptide showed weak cationic selectivity
(PCl/PK ratio = 0.6), whereas
the 31-kDa peptide was nonselective
(PCl/PK = 1.0). In addition to
the nonselective channel formed by the 31-kDa peptide isolated by
Lemaitre et al. (55), channels formed by amylin were relatively
nonselective. These channels were demonstrated to be permeable to
Na+, K+, Ca2+, and
Cl (65). The A
P channel is permeable to
K+, Cs+, Na+, and Li+
in the presence of 1 mM Ca2+. The permeability sequence of
amyloid channels, PCs > PLi > PCa
PK > PNa, is thought to be typical of Ca2+
channels (9, 73). Also, as expected, Ca2+ was able to block
the conduction of Cs+ through the A
P-(1-40)
channels. The A
P-(25-35) channel showed a slight preference for
the transport of cations over anions. The permeability ratio was
Ca2+:K+:Na+:Cl
= 5.4: 1.6: 1.4: 1 (66).
Kinetics
Very little is known about the kinetics of channels formed by cytotoxic peptides. The channels formed by insect defensin in giant liposomes (20) showed variable kinetics, with heterogeneous values for the open and closed times as well as the single-channel conductance values. Single events of channels formed by mammalian defensin NP-1 were also heterogeneous, but this was not investigated further (43). The channels formed by magainin I were rare and short lived. Duclohier et al. (31) report data for one channel only, indicating that the open probability was 0.08 for the channel opening at the 360-pS level. The mean lifetime of the open state of this channel was 100 ms. These data are rather incomplete, given that they refer to a single-channel experiment only. The kinetics of channels formed by melittin have not been extensively investigated. However, it has been shown that frequency of opening and lifetime of the pore increases with voltage (72). Kawahara et al. (44) found that the probability of the channel being open (Po) of an APharmacology
Another area in which little research has been undertaken is that of the pharmacology of the channels formed by cytotoxic peptides. Further research would obviously be required, since knowledge of organic and inorganic blockers and inhibitors of channels would not only help to further characterize the channels but could possibly lead to treatments for conditions caused by such peptides. The efflux of K+, caused by the action of defensin on M. luteus cells, was blocked by prior treatment of the cells with divalent cations. In addition, exposure of cells already affected by defensin to divalent cations resulted in cessation of the K+ efflux (20). This suggests that divalent cations block channels already formed in addition to preventing new channels from forming. Cociancich et al. propose that the formation of ion channels is blocked by preventing the initial interaction of the peptide with the membrane. This occurs via the interaction of divalent cations with membrane lipids, which causes the negative charge of the membrane to be masked. They propose that inactivation of already formed channels occurs either via a direct interaction of divalent cations with channels or via changes in the interactions of the lipids with the peptide (mediated by the presence of divalent cations) that result in changes in the ion channel structure (20).High ionic concentration has been shown to reduce the channel-forming
activity of amylin (65) and insect defensins (20). High ionic
concentrations may mask the negative charge on membranes in a manner
similar to that reported for divalent cations and insect defensin. For
peptides thought to interact with ion channels already in the target
membrane, the action of pharmaceutical agents can help to identify the
specific ion channels involved (28, 75). This possibility has not been
fully investigated in this report, which instead emphasizes peptides
that form ion channels. It is important to note that the channel
hypothesis for a peptide-induced cytotoxicity is not inconsistent with
other cytotoxic effects via modification of intrinsic ion channels
(51), changes in Ca2+ homeostasis (38), and oxidative
stress (83, 88). However, two examples are of note because of differing
ideas about their actions in the literature. Florio et al. (35) showed
that the action of PrP-(106-126) was a result of its actions on
L-type voltage-sensitive Ca2+ channels. The prevention of
its action by nicardipine, which blocks L-type voltage-sensitive
Ca2+ channels, gave support, as did the prevention of its
action by the use of Ca2+-free conditions (35, 36). In
light of this finding, it would be interesting to investigate the
action of nicardipine on prion peptide-formed channels (58). Mammalian
defensins were found to form ion channels by Kagan et al. (43).
RK-1, a molecule thought to be related to the mammalian defensins, was
found to cause a decrease in cell volume in the villus enterocyte
volume assay. This action was prevented by treatment of the cells with niguldipine, which blocks dihydropyridine-sensitive L-type
Ca2+ channels, therefore implicating the L-type
Ca2+ channels in the action of this peptide (12). Other
defensins have been shown to act in a similar manner in this assay
(60), but the two actions (cell volume reduction via actions on
preexisting channels and channel formation) do not seem to have been
reconciled in the literature. Ion channel blockers, organic and
inorganic, have also been used to examine the inhibition of the
AP-formed channels (Table 4). The weak
base Tris is able to block the A
P-(1-40) channel (9). Although
1-2 mM Tris completely blocked the large channels (<400 pS), it
had very little effect on the giant channels (>400 pS). The L-type
Ca2+-channel-blocking drug nitrendipine is ineffective in
blocking A
P-(1-40) channels. Similarly, the compound Cognex
(tetrahydroacridine), an inhibitor of acetylcholinesterase, was
ineffective (at 100 µM) in blocking Cs2+ permeation of
A
P-(1-40) channels (73). Unlike Tris, aluminum inhibits A
P
channel current irreversibly (micromolar range). The blockade of the
A
P channel current by aluminum and Tris depends on transmembrane
potential and the dose of aluminum (9). Similarly, the blockade of the
A
P channel (<400 pS) is dependent on Zn2+
concentration. The block is characterized by an increase in the frequency of flickering. However, Zn2+-induced channel
block is reversible by the Zn2+ chelator
o-phenanthroline (8). It is suggested that the uniform frequency of the transitions between conductance levels indicates that
the Zn2+-A
P interactions occur at the mouth of the A
P
channel where the electric field is thought to be relatively constant
(8). The A
P-(25-35) channel is also blocked reversibly with
micromolar concentrations of Cu2+ or Cd2+ (66).
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SPECIALIZED REGIONS OF PROTEIN STRUCTURE AND PROPERTIES OF FORMED ION TRANSPORT PATHWAYS |
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Only a small amount of research has been completed on the regions of the cytotoxic peptides that are important for channel formation. The NH2 terminus was found to be the important region of pilosulin 1 for cytotoxic activity. This was determined by incubation of pilosulin 1 fragments with EBV-transformed cells. Results indicated that partial peptides, corresponding to amino acid residues 1-22 and 11-56, retained some cytotoxic activity, although this was reduced compared with pilosulin 1. Other peptide fragments (23-56, 37-56, and 47-56) had no cytotoxic activity, indicating that pilosulin 1 requires the NH2 terminus for cytotoxicity. Partial peptide 1-22 was shown to interact with unilamellar membranes, whereas peptide 23-56 did not, suggesting that the NH2 terminus is required for membrane association rather than lytic activity (86). Because fragment 1-22 had reduced cytotoxic activity, the domain responsible for cell lysis is likely to extend further than residue 22.
Cuervo et al. (26) determined that the first 12 residues of the magainin molecules were necessary for antimicrobial activity. Adding to this work, Zasloff et al. (90) used truncated forms of the magainin II molecule to determine the amino acids necessary for antimicrobial activity. They found that removing amino acids from the NH2 terminus of the peptide reduced antimicrobial activity. The first three amino acids could be removed with minimal effect on activity, but shortening the peptide to 19 amino acids in length, by removing the lysine at position 4, resulted in markedly reduced antimicrobial activity. Further truncation of magainin II resulted in virtually inactive molecules. They suggested that the loss of activity correlated with an inability of the truncated peptide to span the membrane. In addition, Zasloff et al. (90) investigated the activity of a peptide that had a single amino acid (serine) removed from the COOH terminal. This truncated form also exhibited reduced antimicrobial activity. It would be interesting to determine whether the reduced activity of the truncated peptides was due to the nature of the particular amino acids removed or solely to the reduced length of the peptide.
In the cytotoxic peptides discussed, the important amino acids that
have been identified are essentially involved in the secondary structure. In particular, positively charged residues (lysine, arginine) are important in providing the overall cationic charge of the
peptide (Table 5). They also
play a significant role in the amphipathic nature of many of the
cytotoxic peptides, a feature thought to be important in channel
formation. In particular, as mentioned previously, the arginine residue
at position 15 in cryptdins 2 and 3 is thought to be important for the
biological activity of these molecules: position 15 is not occupied by
an arginine in the inactive peptides cryptdin 1 and 6 (56). There
appears to be little information on Ca2+ binding sites that
could be important for formation and regulation of cytotoxic
peptide-formed channels (see Ref. 48). However, there are findings that
may indicate that the AP-(1-40) channel gating could become
sensitive to the transmembrane potential in the presence of divalent
cations (44).
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As noted in relation to the structure of cytotoxic peptides, the
disulfide bridge plays an important role in the stabilization of the
secondary structures of cytotoxic peptides. In particular, disulfide
bridges associated with particular consensual structural motifs are
found in insect defensins, scorpion defensins, and scorpion toxins
(Table 5). Similarly, the mammalian defensins are characterized by a
structural motif stabilized by disulfide bonds. Experiments involving
the alteration of amino acid residues and consequent peptide features,
including channel formation, have been largely limited to investigating
the features of truncated peptides. In addition, a small amount of work
has been completed that involves the alteration of residues to increase
the -helical content of the peptides. This work is described above
(19) in Sequence and Structure.
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CYTOTOXIC CHANNELS UNDER ADVERSE CONDITIONS |
---|
Few of the channels formed by cytotoxic peptides have been investigated
under varying conditions. This is another area of interest, as the
environmental conditions required for activity and the limits placed on
the peptides by naturally occurring environments can help us understand
the biological activity of these peptides. Environmental parameters
that may be of interest and that have been investigated for some
peptides include low oxygen levels and low pH. Another interesting
parameter, perhaps worth further investigation with other peptides, is
the effect of age on the peptides, e.g., AP and prions (see
below). In conditions of high salt concentration, amylin
has reduced channel-forming activity (channel-forming activity is 100 times higher in 10 mM KCl than in 1 M KCl) (65). Similarly for insect
defensin, high ionic concentration seems to reduce channel-forming
activity. As in conditions of high ionic concentration, the
K+ efflux, thought to result from channel formation, was
reduced (20). As mentioned above, high ionic concentration may act to "mask" the charge of the membrane, reducing the interaction of the peptide with the lipid bilayer and hence reducing channel formation.
Amylin exhibits no change in activity with changes in pH (65). In contrast, changes in pH did affect channel formation by PrP-(106-126): acidic conditions (pH 4.5) increased ion-channel-forming activity. At this pH, ion channels were formed at concentrations as low as 1 µM, with characteristics different from those of channels formed at higher pH values. The most common single-channel conductances recorded at this pH were 20, 100, and 120 pS (58). For insect defensin, the maximal K+ efflux from cells treated with the peptide was recorded when pH = 7.5 (20). Aging of the PrP-(106-126) fragment increased its ion-channel-forming activity (58). Fragments were aged by incubation at room temperature in 100 mM NaCl for up to 9 days. Nine-day-old fragments showed ion channel formation at concentrations of 0.1 µM, a 200-fold increase in activity. The distribution of single-channel conductance values for such samples were different from those of nonaged samples with peaks at 10, 20, 30, 40, 50, and 60 pS. Three-day-old fragments showed a 20-fold increase in channel activity (58).
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PHYSIOPATHOLOGICAL SIGNIFICANCE OF CYTOTOXIC PEPTIDES AS ION-CHANNEL-FORMING PROTEINS |
---|
Little is known about the ion channels formed by these peptides to determine their role in biological systems. However, it is known that these peptides can disrupt ion homeostasis across the membrane and that this leads to cell death. The formation of large nonselective pores could result in a complete loss of ion gradients across the membrane, and it can be envisaged that this would lead to cell death via a combination of pathways, including loss of gradients for ATP production. However, ion channels that are selective for either anions or cations must have a more specific result, which could involve the subsequent activation of other channels in the membrane and loss of ion homeostasis as above, or a more specific action such as a triggering of apoptosis.
The properties of the significant domains and individual residues of the channels can be used as criteria to investigate how these channels function under adverse conditions. Pathological conditions often lead to depolarization of cell membranes and subsequently cause cell death in vivo. Voltage-depolarizing protocols (mimicking membrane depolarization under pathological conditions) can be used to indicate how the cytotoxic peptide-formed channel may function under pathological conditions e.g., hypoxia, low pH, and Ca2+ overload. Modulation of the cytotoxic peptide-formed channels, containing cysteine residues, under hypoxia-reperfusion is based on the fact that SH groups and S-S bridges of several ion channels are regulated by oxidation and reduction, oxygen-reactive species, and low oxygen (45). Effects of solutions with different oxygen levels, H2O2, and oxidizing and reducing agents on the function of ion channels can be examined by perfusing the solution in the cis and trans chambers (cytoplasmic and luminal sides of channel proteins) with solutions of different concentrations of O2 and N2, redox agents. This could indicate the role of S-S in the function of different cytotoxic peptide-formed channels and its physiological regulation under the reduced cell environment or increased levels of reactive oxygen species (ROS), providing insight into the role of these peptides in cell function under ischaemic conditions of the heart, kidney, and intestines.
One of the early cytosolic changes occurring under hypoxia of epithelial and muscle cells is the lowering of cytoplasmic pH, which depresses force output in muscle (30, 34) and electrolyte regulation in epithelial cells (85), probably via modifications in the mechanism(s) involved in Ca2+ homeostasis. Whether Ca2+ is involved in conferring cytotoxicity on a cytotoxic peptide-formed channel is not known and it needs to be determined. This is because of the possibility that these channels may become functionally active only under physiological conditions where Ca2+ levels are high, e.g., during cardiac and skeletal muscle contractions, volume regulation, and pathological conditions that lead to a Ca2+ overload, e.g., ischemic heart and muscle fatigue. Cytotoxic peptide-formed channels that could be regulated with Ca2+ may modify a cascade of physiological functions that are dependent on the Ca2+-activated channels, e.g., vasodilation and salt secretion. Such accumulated knowledge can indicate the likely conditions for channel formation in vivo and under pathological conditions. Knowledge of the biophysical and regulatory properties of the cytotoxic-peptide formed channels allows the evaluation of the physiological significance and possible pathological functions of these peptides. These can be verified by testing the in vitro and in vivo toxicity of these peptides, e.g., measuring muscle contraction and relaxation, cell volume regulation of cultured epithelial cells, and/or liposomes. Pharmacological agents used to modulate these channels can then be used to rectify the cytotoxic effects of these peptides in vivo.
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CONCLUSIONS |
---|
The peptides discussed here have several characteristics in common: they are small, generally cationic, often amphipathic peptides. They have cytotoxic activity, which is often manifested as antibacterial activity but which can also be antifungal or anti-mammalian cell activity. These peptides can mediate their effects by interacting with cell membranes and forming ion channels, which are examinable with electrophysiological techniques. The channels can be then characterized in terms of their biophysical, pharmacological, and functional properties. The biophysical and pharmacological investigations of these small peptide-formed channels could 1) clarify conditions needed for peptide-bilayer interaction and ion channel formation (bilayer properties and conditions required for peptide interaction and incorporation, differences in the incorporation of these peptides, and structural characteristics to identify the domains that interact with lipid membrane); 2) characterize the biophysical properties (channel type and its ion concentration dependency, the channel's selectivity and conductance properties, and the voltage dependency of the channel's conductance and kinetic properties); and 3) characterize the pharmacological properties (fundamental mechanisms of agonist-induced activation and antagonist-induced channel block, channel regulation by cytosolic factors and second messengers, the symmetry or asymmetry of the transmembrane channel-forming peptide, and the domains important in channel formation and regulation). Although these small ion-channel-forming peptides could provide relatively simple structural models, it is still difficult to predict the structural requirements for ion channel formation. There are no models for the sequence of events leading to channel formation, e.g., If a multimer forms the channel, when is this multimer itself formed: before the peptide is incorporated into the membrane or once the peptide molecules are within the membrane (see Ref. 20)? In conclusion, the investigations of these peptides could enhance our understanding of their structure-function relationship and elucidate the molecular events underlying their interactions with biological membranes and the role of such interactions both in major physiological mechanisms and in cytotoxic and pathological conditions.
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ACKNOWLEDGEMENTS |
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We thank Dr. W. Armarego and H. Wood for numerous discussions, suggestions, and critical reading of the manuscript. The assistance of P. Farrelly, S. Pradhananga, C. Henry, and A. Culverson is greatly appreciated.
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FOOTNOTES |
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J. I. Kourie is supported by National Health and Medical Research Council Project Grant 970122 and The Australian Research Council Grant F99123.
Address for reprint requests and other correspondence: J. I. Kourie, Membrane Transport Group, Dept. of Chemistry, The Faculties, The Australian National Univ., Canberra City, ACT, 0200 Australia (E-mail: joseph.kourie{at}anu.edu.au).
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