Current Protein and Peptide Science - Volume 7, Issue 6, 2006

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 a-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 a-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 a-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.......

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.

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.

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.

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.

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 noncolligative 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.

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 αto β transconformations, which drive the generation of amyloid deposits.

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 onedimensional 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 a-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.

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.

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.

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.