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Current Protein & Peptide
Science
ISSN: 1389-2042
Current Protein and Peptide
Science
Volume 7, Number 6, December 2006
Contents
The Multi-Purpose Amphiphilic α-Helix
– A Historical Perspective
Guest Editors: David Phoenix and Frederick Harris
Editorial Pp. 471-472
David Phoenix and Frederick Harris
Combinatorial Synthesis and Directed Evolution
Applied to the Production of α-Helix
Forming Antimicrobial Peptides Analogues Pp. 473-478
Mariana S. Castro, Eduardo M. Cilli and Wagner Fontes
[Abstract]
Host Defense Peptides and Lipopeptides: Modes of Action
and Potential Candidates for the Treatment of Bacterial and
Fungal Infections Pp. 479-486
Yechiel Shai, Arik Makovitzky and Dorit Avrahami
[Abstract]
Anticancer α-Helical
Peptides and Structure / Function Relationships Underpinning
Their Interactions with Tumour Cell Membranes Pp.
487-499
Sarah R. Dennison, Michelle Whittaker, Frederick Harris
and David A. Phoenix
[Abstract]
Biological Applications of the Receptor Mimetic Peptide
Mastoparan Pp. 501-508
Sarah Jones and John Howl
[Abstract]
Applications of Type I Antifreeze Proteins: Studies
with Model Membranes & Cryoprotectant Properties
Pp. 509-522
Steven R. Inglis, Jennifer J. Turner and Margaret M. Harding
[Abstract]
Tilted Peptides: The History Pp. 523-527
Annick Thomas and Robert Brasseur
[Abstract]
Oblique Orientated α-Helices
and Their Prediction Pp. 529-537
Frederick Harris, Abel Daman, James Wallace, Sarah R.
Dennison and David A. Phoenix
[Abstract]
Amphipathic Helices as Mediators of the Membrane Interaction
of Amphitropic Proteins, and as Modulators of Bilayer Physical
Properties Pp. 539-552
Rosemary B. Cornell and Svetla G. Taneva
[Abstract]
Surface-Active Helices in Transmembrane Proteins
Pp. 553-560
Joseph P.R.O. Orgel
[Abstract]
Membrane Interactive α-Helices
in GPCRs as a Novel Drug Target Pp. 561-575
Wataru Nemoto and Hiroyuki Toh
[Abstract]
Abstracts
[Back to top]
Editorial
David Phoenix and Frederick Harris
Proteins play a fundamental role in membrane dependent
processes and due to the inherently amphiphilic nature of
the bilayer, such proteins must accommodate both polar and
non-polar environments. In response, membrane interactive
proteins adopt amphiphilic secondary structures, which can
be subdivided into several general classes but it is generally
accepted that amphiphilic α-helices
form the major example of these classes. Members of this latter
structural type may possess primary amphiphilicity, which
is exhibited by most transmembrane α-helices,
or secondary amphiphilicity, which is generally associated
with α-helices
that are active at a lipid / membrane interface [1].
The secondary amphiphilicity of α-helices
is characterised by an ordered spatial segregation of hydrophobic
and hydrophilic amino acid residues about the α-helical
long axis and, historically, was first reported within the
molecules of myoglobin and haemoglobin during the mid 1960s
[2]. The ubiquitous occurrence and clear functional importance
of these α-helical
structures was soon realized and, over the subsequent decades,
led to a series of theoretical approaches designed to enable
their identification from sequence information alone. These
approaches were generally based on the fact that the secondary
amphiphilicity of α-helices
is reflected in the primary structure of a protein by the
periodic occurrence of doublets or triplets of polar or apolar
residues [3]. The earliest of the techniques used to identify
this residue periodicity was developed in the late 1960s and
were graphical with the major example being the α-helical
wheels of Schiffer-Edmundson [4]. Over the next few decades,
it became apparent that there was a need to formally quantify
the amphiphilicity of protein α-helices,
which led to the development of measures of amphiphilicity
such as the Amphipathic Index (AI) of Cornette et al.,
[5] and the Molecular Hydrophobic Potential (MHP) of Brasseur
[6]. Undoubtedly though, the most commonly used measure of
amphiphilicty developed within this period was the Hydrophobic
Moment (<
μH
>) of Eisenberg [7], which was developed not long
after by this author to give Hydrophobic Moment Plot methodology
[8]. This methodology attempted to broadly classify membrane
interactive α-helices
as either transmembrane or active at the interface (surface-active)
and numerous authors have adapted the methodology to characterize
the structure / function relationships of subclasses of these
α-helices
[1]. Probably the most used of these adapted methodologies
is the taxonomy of Segrest et al. [9] which subclassifies
membrane interactive amphiphilic α-helices
as those of apolipoproteins (class A), lytic peptides (class
L), hormones (class H) and transmembrane proteins (class M).
The MHP of Brasseur [6] has been used as a basis to make similar
subclassifications of amphiphilic α-helices
[1] and most recently has been used to aid the identification
of oblique orientated α-helices
[10].
Since the first description of amphiphilic α-helices,
they have formed the basis of numerous papers, reviews, conferences
and books. A major contribution to the literature of these
α-helices
was made by publication of “The Amphipathic Helix”
(ISBN: 0849349265) in 1993, which was edited by Richard Epand
and provided a comprehensive overview of the major α-helical
classes then known. This Hot Topics issue of CPPS provides
an update on some of these α-helical
classes and introduces a number of such classes that have
been discovered since.
Amphiphilic α-helical
defence peptides were first reported in the late 1980s and
are effectors of innate immunity that generally exert antimicrobial
activity through permeabilising the membranes of target organisms.
These peptides are attractive propositions for development
as novel antimicrobial agents and, in this capacity, attempts
to optimize their lytic activity and target specificity by
the use of combinatorial synthesis and directed evolution
are reviewed here by Mariana De Castro et al. Moreover,
based on the lessons learnt from structure / function studies
on these defence peptides, lipopeptides are currently being
studied for development as potent agents against pathogenic
fungi and yeast, reviewed here by Yechel Shai et al.
Amphiphilic α-helical
defence peptides have also been found to show potent anticancer
activity and progress in the understanding of this activity
is reviewed by Sarah Dennison et al. Functionally
related to defence peptides are α-helical
peptide venoms such as mastoparan (MP), which is known to
bind and modulate G-proteins in addition to a variety of other
intracellular targets. MP, along with its analogues and chimaera,
has proved a crucial tool in probing diverse biological phenomenon,
particularly G-protein function, and is reviewed here by Sarah
Jones and John Howl. Another class of α-helical
proteins that show the potential for biological application
are antifreeze proteins (AFPs), some of which are able to
stabilize membranes and thereby function as cryoprotectants.
The development of AFPs in this capacity is hampered by the
fact that it is currently not possible to predict whether
a particular AFP will stabilize or destabilize a given lipid
system. However, some progress in this direction has been
made and is reviewed here by Steven Inglis et al.
Around the same time as defence peptides were discovered,
oblique orientated α-helices
were first reported in viral proteins, promoting the fusion
of host and viral membranes. The subsequent identification
of these α-helices
in a diverse array of proteins is described here by Annick
Thomas and Robert Brasseur whilst Frederick Harris et
al., review theoretical techniques developed to identify
these structures. In the late 1990s, a number of amphitrophic
proteins, which bind reversibly to membranes via
amphiphilic α-helices,
were first described in detail and progress in the characterization
of these protein – membrane anchors is reviewed by Rosemary
Cornell et al. More recently in 2004, amphiphilic
α-helices
within the structures of transmembrane proteins (TM) were
reported to perform membrane anchoring / support functions
and the latest developments in these studies are reviewed
by Joseph Orgel. One of the most important classes of TM proteins
is that formed by G-protein coupled receptors (GPCRs), which
act as major targets for pharmacological drugs, and only recently
has it become generally accepted that oligomerisation is crucial
to the function of GPCRs. The role of TM α-helical
interfaces in facilitating the oligomerisation of GPCRs and
the potential significance of these interfaces in pharmaceutical
drug design is reviewed by Wataru Nemoto and Hiroyuki Toh.
REFERENCES
[1] Phoenix, D.A. and Harris, F. (2002) Mol.
Mem. Biol., 19, 1-10.
[2] Perutz, M.F., Kendrew, J.C. and Watson, H.C. (1965)
J. Mol. Biol., 13, 669-677.
[3] Phoenix, D.A., Harris, F., Daman, O.A. and Wallace, J.
(2002) Curr. Prot. Peptide Sci.,
3, 201-221.
[4] Schiffer, M. and Edmundson, A.B. (1967).
Biophys. J., 7, 121-135.
[5] Cornette, J.L., Cease, K.B., Margelit, H., Spouge, J L.,
Berzofsky, J.A.D and De Lisi, C. (1987) J.
Mol. Biol., 195, 659-685
[6] Brasseur, R (1991). J. Biol. Chem.,
24, 16120-16127.
[7] Eisenberg, D., Weiss, R.M. and Terwilliger, T.C. (1982)
Nature, 299, 371-373.
[8] Eisenberg, D., Schwarz, E., Komaromy, M. and Wall, R.
(1984) J. Mol. Biol., 179, 125-142.
[9] Segrest, J.P., De Loof, H., Dohlman, J.G. Brouillette,
C.G. and Anantharamaiah, G.M. (1990) Prot.
Struct. Funct. Genet., 8, 103-117.
[10] Rahman, M., Lins, L., Thomas-Soumarmon., T. and Brasseur,
R. (1997) J. Mol. Model, 3, 203-215.
David Phoenix and Frederick Harris
Guest Editors
Current Protein & Peptide Science
Faculty of Science
University of Central Lancashire
Preston, PR1 2HE, England,
UK
E-mail: daphoenix@uclan.ac.uk
[Back to top]
Combinatorial Synthesis and Directed Evolution Applied to
the Production of α-Helix
Forming Antimicrobial Peptides Analogues
Mariana S. Castro, Eduardo M. Cilli and Wagner Fontes
Antimicrobial peptides (AMPs) are effector molecules of innate
immune systems found in different groups of organisms, including
microorganisms, plants, insects, amphibians and humans. These
peptides exhibit several structural motifs but the most abundant
AMPs assume an amphipathic α
helical structure. The α-helix
forming antimicrobial peptides are excellent candidates for
protein engineering leading to an optimization of their biological
activity and target specificity. Nowadays several approaches
are available and this review deals with the use of combinatorial
synthesis and directed evolution in order to provide a high
throughput source of antimicrobial peptides analogues with
enhanced lytic activity and specificity.
[Back to top]
Host Defense Peptides and Lipopeptides: Modes of Action
and Potential Candidates for the Treatment of Bacterial and
Fungal Infections
Yechiel Shai, Arik Makovitzky and Dorit Avrahami
Endogenous peptide antibiotics (termed also host-defense or
antimicrobial peptides) are known as evolutionarily old components
of innate immunity. They were found initially in invertebrates,
but later on also in vertebrates, including humans. This secondary,
chemical immune system provides organisms with a repertoire
of small peptides that act against invasion (for both offensive
and defensive purposes) by occasional and obligate pathogens.
Each antimicrobial peptide has a broad but not identical spectrum
of antimicrobial activity, predominantly against bacteria,
providing the host maximum coverage against a rather broad
spectrum of microbial organisms. Many of these peptides interact
with the target cell membranes and increase their permeability,
which results in cell lysis. A second important family includes
lipopeptides. They are produced in bacteria and fungi during
cultivation on various carbon sources, and possess a strong
antifungal activity. Unfortunately, native lipopeptides are
non-cell selective and therefore extremely toxic to mammalian
cells. Whereas extensive studies have emerged on the requirements
for a peptide to be antibacterial, very little is known concerning
the parameters that contribute to antifungal activity. This
review summarizes recent studies aimed to understand how antimicrobial
peptides and lipopeptides select their target cell. This includes
a new group of lipopeptides highly potent against pathogenic
fungi and yeast. They are composed of inert cationic peptides
conjugated to aliphatic acids with different lengths. Deep
understanding of the molecular mechanisms underlying the differential
cells specificity of these families of host defense molecule
is required to meet the challenges imposed by the life-threatening
infections.
[Back to top]
Anticancer α-Helical
Peptides and Structure / Function Relationships Underpinning
Their Interactions with Tumour Cell Membranes
Sarah R. Dennison, Michelle Whittaker, Frederick Harris
and David A. Phoenix
Cancer is a major cause of premature death and there is an
urgent need for new anticancer agents with novel mechanisms
of action. Here we review recent studies on a group of peptides
that show much promise in this regard, exemplified by arthropod
cecropins and amphibian magainins and aureins. These molecules
are α-helical
defence peptides, which show potent anticancer activity (α-ACPs)
in addition to their established roles as antimicrobial factors
and modulators of innate immune systems. Generally, α-ACPs
exhibit selectivity for cancer and microbial cells primarily
due to their elevated levels of negative membrane surface
charge as compared to non-cancerous eukaryotic cells. The
anticancer activity of α-ACPs
normally occurs at micromolar levels but is not accompanied
by significant levels of haemolysis or toxicity to other mammalian
cells. Structure / function studies have established that
architectural features of α-ACPs
such as amphiphilicty levels and hydrophobic arc size are
of major importance to the ability of these peptides to invade
cancer cell membranes. In the vast majority of cases the mechanisms
underlying such killing involves disruption of mitochondrial
membrane integrity and / or that of the plasma membrane of
the target tumour cells. Moreover, these mechanisms do not
appear to proceed via receptor-mediated routes but
are thought to be effected in most cases by the carpet / toroidal
pore model and variants. Usually, these membrane interactions
lead to loss of membrane integrity and cell death utilising
apoptic and necrotic pathways. It is concluded that that α-ACPs
are major contenders in the search for new anticancer drugs,
underlined by the fact that a number of these peptides have
been patented in this capacity.
[Back to top]
Biological Applications of the Receptor Mimetic Peptide
Mastoparan
Sarah Jones and John Howl
The receptor mimetic and mast cell degranulating peptide mastoparan
(MP) translocates cell membranes as an amphipathic α−helix,
a feature that is undoubtedly a major determinant of bioactivity
through the activation of heterotrimeric G proteins. Chimeric
combinations of MP with G protein-coupled receptor (GPCR)
ligands has produced peptides that exhibit biological activities
distinct from their composite components. Thus, chimeric peptides
such as galparan and M391 differentially modulate GTPase activity,
display altered binding affinities for appropriate GPCRs and
possess disparate secretory properties. MP and MP-containing
chimerae also bind and modulate the activities of various
other intracellular protein targets and are valuable tools
to manipulate and study enzymatic activity, calcium homeostasis
and apoptotic signalling pathways. In addition, charge delocalisation
within the hydrophilic face of MP has produced analogues,
including [Lys5, Lys8,Aib10]MP,
that differentially regulate mast cell secretion and/or cytotoxicity.
Finally, the identification of cell penetrant variants of
MP chimerae has enabled the effective intracellular delivery
of non-permeable biomolecules and presents an opportunity
to target novel intracellular therapeutic loci.
[Back to top]
Applications of Type I Antifreeze Proteins: Studies
with Model Membranes & Cryoprotectant Properties
Steven R. Inglis, Jennifer J. Turner and Margaret M. Harding
Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs),
found in the body fluids of many species of polar fish allow
them to survive in waters colder than the equilibrium freezing
point of their blood and other internal fluids. Despite their
structural diversity, all AF(G)Ps kinetically depress the
temperature at which ice grows in a non-colligative manner
and hence exhibit thermal hysteresis. AF(G)Ps also share the
ability to interact with and protect mammalian cells and tissues
from hypothermic damage (e.g., improved storage of human blood
platelets at low temperatures), and are able to stabilize
or disrupt membrane composition during low temperature and
freezing stress (e.g., cryoprotectant properties in stabilization
of sperm and oocytes). This review will summarize studies
of AFPs with phospholipids and plant lipids, proposed mechanisms
for inhibition of leakage from membranes, and cryoprotectant
studies with biological samples. The major focus will be on
the α-helical
type I antifreeze proteins, and synthetic mutants, that have
been most widely studied. For completeness, data on glycoproteins
will also be presented. While a number of models to explain
stabilization and destabilization of different lipid systems
have been proposed, it is currently not possible to predict
whether a particular AFP will stabilize or destabilize a given
lipid system. Furthermore the relationship between the antifreeze
property of thermal hysteresis and membrane stabilization
is unknown. This lack of detailed knowledge about how AFPs
function in the presence of different types of materials has
hampered progress toward the development of antifreezes for
cold storage of cells, tissues, and organs.
[Back to top]
Tilted Peptides: The History
Annick Thomas and Robert Brasseur
Nature has selected peptide motifs for protein functions.
It is clear that specific sequence motifs can identify families
of enzymes. These sequence motifs are one dimensional signatures
and nature has also developed two dimension motifs which cannot
be read in the one dimension of sequence language but can
be detected in the three dimensional properties of a secondary
structure. One of such motifs is tilted peptides. They do
not correspond to any consensus of sequence but correspond
to a consensus motif where hydrophobicity balance is used
as a functional device. In the nineteen eighties, the first
tilted peptide was deciphered from the sequence of a virus
fusion protein by molecular modelling. It was described as
a protein fragment hydrophobic enough to insert into a membrane
but too short to span it. The fragment exhibited an asymmetric
distribution of hydrophobicity along the helix long axis and
hence, was unable to lie parallel or perpendicular to a membrane
surface but adopted an orientation in between. Hydrophobicity
motif was a very new and very challenging concept and tilted
peptides were rapidly found to be involved in several mechanisms
of virus fusion. They were also found to be involved in protein
secretion and future studies could establish their involvement
in the destabilization of 3D protein structures and in the
α to
β transconformations,
which drive the generation of amyloid deposits.
[Back to top]
Oblique Orientated α-Helices
and Their Prediction
Frederick Harris, Abel Daman, James Wallace, Sarah R.
Dennison and David A. Phoenix
Oblique orientated α-helices
possess hydrophobicity gradients, which allow the parent α-helices
to penetrate the membrane at a shallow angle, thereby destabilising
membrane lipid organisation and promoting a range of biological
processes. These α-helices
occur in a variety of membrane interactive proteins and a
number of techniques have been developed to guide their identification
using sequence data alone. Hydrophobicity profiling, which
provides a one-dimensional analysis of sequence data, identified
only 30% of known tilted peptides in a control dataset and
was thus of limited predictive use. In contrast, extended
hydrophobic moment plot methodology and amphipilic profiling
which take residue distribution into account and provide two-dimensional
analysis of primary structural data, were found to be good
indicators of tilted peptide structure. Amphiphilic profiling
identified 67% of tilted peptides in the control dataset and
showed that potentially, approximately 40% of transmembrane
α-helices
possess tilted peptide structure. However, it has been shown
that extending these simple methods to take into account the
three-dimensional spatial distribution of residues gives no
clear additional benefit to identifying tilted peptides.
[Back to top]
Amphipathic Helices as Mediators of the Membrane Interaction
of Amphitropic Proteins, and as Modulators of Bilayer Physical
Properties
Rosemary B. Cornell and Svetla G. Taneva
The amphipathic helix (AH) motif is used by a subset of amphitropic
proteins to accomplish reversible and controlled association
with the interfacial zone of membranes. Functioning as more
than mere membrane anchoring domains, amphipathic helices
can serve as autoinhibitory domains to suppress the protein
activity in its soluble form, and as sensors or modulators
of membrane curvature. Thus amphipathic helices can both respond
to and modulate membrane physical properties. These and other
features are illustrated by the behavior of CTP: phosphocholine
cytidylyltransferase (CCT), a key regulatory enzyme in PC
synthesis. A comparison of the physico-chemical features of
CCT’s AH motif and 10 others reveals similarities and
several differences. The importance of these parameters to
the particulars of the membrane interaction and to functional
consequences requires more systematic exploration. The membrane
partitioning of amphitropic proteins with AH motifs can be
regulated by various strategies including changes in membrane
lipid composition, phosphorylation, ligand-induced conformational
changes, and membrane curvature. Several amphitropic proteins
that control budding or tubule formation in cells have AH
motifs. The insertion of the hydrophobic face of these amphipathic
helices generates an asymmetry in the lateral pressure of
the two leaflets resulting in an induction of positive curvature.
Curvature induction or stabilization may be a universal property
of AHA proteins, not just those involved in budding, but this
possibility requires further demonstration.
[Back to top]
Surface-Active Helices in Transmembrane Proteins
Joseph P.R.O. Orgel
Amphipathic surface-active helices enable peripheral proteins
to perform a variety of important cellular functions such
as: lipid association and transport, membrane perturbation
and disruption in programmed cell death or antimicrobial activity,
and signal transduction. Amphipathic helices that adopt a
surface-active membrane location are also found in transmembrane
proteins. Since they possess similar amino acid composition
and therefore chemical and physical properties, it seems intuitively
obvious that the specific role of these surface seeking, or
horizontal helices in membrane spanning proteins in some ways
parallel those of their cousins in peripheral proteins. This
review compares research literature and data from both proteins
sets (peripheral proteins and transmembrane) to examine this
assumption. Furthermore, since the occurrence of surface-active
/ seeking helices in transmembrane protein structure is often
omitted from comment in the literature, a brief survey of
their apparent roles in transmembrane protein / lipid stabilization,
microenvironment enclosure and signal transduction is offered
here.
[Back to top]
Membrane Interactive α-Helices
in GPCRs as a Novel Drug Target
Wataru Nemoto and Hiroyuki Toh
G-Protein Coupled Receptors (GPCRs) are one of the most important
targets for pharmaceutical drug design. Over the past 30 years,
mounting evidence has suggested the existence of homo and
hetero dimers or higher-order complexes (oligomers) that are
involved in signal transduction and some diseases. The number
of reports describing GPCR oligomerization has increased,
and in 2003, the organization of mouse rhodopsin into two-dimensional
arrays of dimers was determined by an atomic force microscopic
analysis. The analysis of the mouse rhodopsin complex has
enabled us to discuss the oligomerization based on structural
data. Although many unsolved problems still remains, the idea
that GPCRs directly interact to form oligomers has been gradually
accepted. One of the recent findings in the GPCR investigations
is the clarification of the mechanisms of GPCR oligomerization
at a molecular level.
Most of these studies have suggested the importance of transmembrane
α-helices
for GPCR oligomerization. In this review, we will first summarize
the importance of GPCR oligomerization and the functions of
GPCRs. Then, we will explain the involvement of transmembrane
α-helices
in the oligomerization and a drug design strategy that targets
these regions for GPCR oligomerization. Considering the current
drug design methods, which are based on the modification of
the protein-protein interactions of soluble regions of proteins,
a “peptide mimic approach” that targets the transmembrane
α-helices
constituting the interfaces would be promising in drug discovery
for GPCR oligomerization. For that purpose, we must know the
positions of the interfaces. However, problems specific to
membrane proteins have made it difficult to identify the positions
of the interfaces experimentally. Therefore, information about
the interfaces predicted by bioinformatics approaches is valuable.
At the end of this review, several bioinformatics approaches
toward interface prediction for oligomerization are introduced.
The benefits and the pitfalls of these approaches are also
discussed.
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