Frederick Harris

Amphiphilic α-Helical Antimicrobial Peptides and Their Structure/Function Relationships

To facilitate microbial membrane invasion, amphiphilic α-helical antimicrobial peptides (α-AMPs) show a spatial segregation of hydrophobic and hydrophilic residues about the α-helical long axis. Here we discuss potential mechanisms by which these peptides are able to disrupt membrane structure and the structural characteristics, which are required for function.

Anticancer α-Helical Peptides and Structure / Function Relationships Underpinning Their Interactions with Tumour Cell Membranes

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 alpha-helical defence peptides, which show potent anticancer activity (alpha-ACPs) in addition to their established roles as antimicrobial factors and modulators of innate immune systems. Generally, alpha-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 alpha-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 alpha-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 alpha-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.

On the selectivity and efficacy of defense peptides with respect to cancer cells

Here, we review potential determinants of the anticancer efficacy of innate immune peptides (ACPs) for cancer cells. These determinants include membrane‐based factors, such as receptors, phosphatidylserine, sialic acid residues, and sulfated glycans, and peptide‐based factors, such as residue composition, sequence length, net charge, hydrophobic arc size, hydrophobicity, and amphiphilicity. Each of these factors may contribute to the anticancer action of ACPs, but no single factor(s) makes an overriding contribution to their overall selectivity and toxicity. Differences between the anticancer actions of ACPs seem to relate to different levels of interplay between these peptide and membrane‐based factors.

Photodynamic therapy based on 5-aminolevulinic acid and its use as an antimicrobial agent.

Exogenous 5‐aminolevulinic acid (ALA) is taken up directly by bacteria, yeasts, fungi, and some parasites, which then induces the accumulation of protoporphyrin IX (PPIX). Subsequent light irradiation of PPIX leads to the inactivation of these organisms via photodamage to their cellular structures. ALA uptake and light irradiation of PPIX produced by host cells leads to the inactivation of other parasites, along with some viruses, via the induction of an immune response. ALA‐mediated PPIX production by host cells and light irradiation result in the inactivation of other viruses via either the induction of a host cell response or direct photodynamic attack on viral particles. This ALA‐mediated production of light‐activated PPIX has been extensively used as a form of photodynamic therapy (PDT) and has shown varying levels of efficacy in treating conditions that are associated with microbial infection, ranging from acne and verrucae to leishmaniasis and onychomycosis. However, for the treatment of some of these conditions by ALA‐based PDT, the role of an antimicrobial effect has been disputed and in general, the mechanisms by which the technique inactivates microbes are not well understood. In this study, we review current understanding of the antimicrobial mechanisms used by ALA‐based PDT and its role in the treatment of microbial infections along with its potential medical and nonmedical applications.

The hydrophobic moment and its use in the classification of amphiphilic structures (Review)

Amphiphilic f -helices play a major role in membrane dependent processes and are manifested in the primary structure of a protein by the periodic appearance of hydrophobic residues. Based on these periodic sequences, the hydrophobic moment was introduced, , which essentially treats the hydrophobicity of amino acid residues as a two-dimensional vector sum and provides a measure of amphiphilicity within regular repeat structures. To identify putative amphiphilic f -helix forming sequences, hydrophobic moment analysis assumes an amino acid residue periodicity of 100° and scans protein primary structures to find the 11-residue window with maximal . Taken with the windows mean hydrophobicity, < H0 >, hydrophobic moment plot analysis uses the coordinate pair, [, ] to classify f -helices as either surface active, globular or transmembrane. More recently, this latter analysis has been extended to recognize candidate oblique orientated f -helices. Here, the hydrophobic moment is reviewed and data to query the logic of using a fixed window length and a fixed residue angular periodicity in hydrophobic moment analysis are provided. In addition, problems associated with the use of such analysis to predict f -helix structure/function relationships are considered.

The Prediction of Amphiphilic α-Helices

A number of sequence-based analyses have been developed to identify protein segments, which are able to form membrane interactive amphiphilic α-helices. Earlier techniques attempted to detect the characteristic periodicity in hydrophobic amino acid residues shown by these structure and included the Molecular Hydrophobic Potential (MHP), which represents the hydrophobicity of amino acid residues as lines of isopotential around the α-helix and analyses based on Fourier transforms. These latter analyses compare the periodicity of hydrophobic residues in a putative α-helical sequence with that of a test mathematical function to provide a measure of amphiphilicity using either the Amphipathic Index or the Hydrophobic Moment. More recently, the introduction of computational procedures based on techniques such as hydropathy analysis, homology modelling, multiple sequence alignments and neural networks has led to the prediction of transmembrane α-helices with accuracies of the order of 95perc ent and transmembrane protein topology with accuracies greater than 75percent. Statistical approaches to transmembrane protein modeling such as hidden Markov models have increased these prediction levels to an even higher level. Here, we review a number of these predictive techniques and consider problems associated with their use in the prediction of structure / function relationships, using α-helices from G-coupled protein receptors, penicillin binding proteins, apolipoproteins, peptide hormones, lytic peptides and tilted peptides as examples.

Calpains and Their Multiple Roles in Diabetes Mellitus

Abstract:  Type 2 diabetes mellitus (T2DM) can lead to death without treatment and it has been predicted that the condition will affect 215 million people worldwide by 2010. T2DM is a multifactorial disorder whose precise genetic causes and biochemical defects have not been fully elucidated, but at both levels, calpains appear to play a role. Positional cloning studies mapped T2DM susceptibility to CAPN10, the gene encoding the intracellular cysteine protease, calpain 10. Further studies have shown a number of noncoding polymorphisms in CAPN10 to be functionally associated with T2DM while the identification of coding polymorphisms, suggested that mutant calpain 10 proteins may also contribute to the disease. Here we review recent studies, which in addition to the latter enzyme, have linked calpain 5, calpain 3, and its splice variants, calpain 2 and calpain 1 to T2DM‐related metabolic pathways along with T2DM‐associated phenotypes, such as obesity and impaired insulin secretion, and T2DM‐related complications, such as epithelial dysfunction and diabetic cataract.

Are Oblique Orientated α-Helices Used by Antimicrobial Peptides for Membrane Invasion?

Oblique orientated α-helices are highly specialised protein structural elements that penetrate membranes at a shallow angle and are used to promote membrane destabilisation by a number of protein classes. Here, the use of extended hydrophobic moment methodology shows that the amphibian extrudates, aurein 1.2 and citropin 1.1, may use oblique orientated α-helices in their antimicrobial action and that such use may be shared by other antimicrobial peptides. This appears to be the first systematic analysis of these peptides for the possession of oblique orientated a-helical structure.

Use of hydrophobic moment plot methodology to aid the identification of oblique orientated a-helices

A number of a-helix forming peptides have been reported which appear to promote membrane fusion and other biological events related to the disruption of a hydrophobic/hydrophilic interface, due to the presence of a hydrophobicity gradient along the helical long axis. When a-helices from this class were analysed according to hydrophobic moment plot methodology a linear association was found to exist between the mean hydrophobic moment, < lH > , and the corresponding mean hydrophobicity, < H0 > . This association was described by the least squares regression line: < lH > =0.508 Ð 0.422 < H0 > and, here, a methodology to aid the prediction of oblique orientated a-helices is presented, based on a 99% prediction band around this regression line. This methodology is intended to provide an initial identification of candidates for further investigation by other techniques such as the molecular hydrophobic potential and laboratory based experimentation, not to assign function.A number of alpha-helix forming peptides have been reported which appear to promote membrane fusion and other biological events related to the disruption of a hydrophobic/hydrophilic interface, due to the presence of a hydrophobicity gradient along the helical long axis. When alpha-helices from this class were analysed according to hydrophobic moment plot methodology a linear association was found to exist between the mean hydrophobic moment, , and the corresponding mean hydrophobicity, . This association was described by the least squares regression line: =0.508-0.422 and, here, a methodology to aid the prediction of oblique orientated alpha-helices is presented, based on a 99% prediction band around this regression line. This methodology is intended to provide an initial identification of candidates for further investigation by other techniques such as the molecular hydrophobic potential and laboratory based experimentation, not to assign function.

The in vitro retardation of porcine cataractogenesis by the calpain inhibitor, SJA6017

Calpain inhibitors show the potential to serve as non-surgical alternatives in treating diabetic cataract and other types of these disorders. Here, we have tested the recently developed calpain inhibitor, SJA6017, for its ability to inhibit cataractogenesis in porcine lenses. These lenses were incubated in increasing levels of extralenticular calcium (Ca2+; 5–30 mM). Atomic absorption spectroscopy was used to determine total internal lens Ca2+ and a correlation between porcine lens Ca2+ uptake and levels of lens opacification were found with a total internal lens Ca2+ level of 5.8 μM Ca2+ g−1 wet lens weight corresponding to the onset of catarctogenesis. A total internal lens Ca2+ level of 8.0 μM Ca2+ g−1 wet lens weight corresponded to cataract occupying approximately 70% of the lens cell volume. This degree of cataract was reduced by approximately 40%, when SJA6017 (final concentration 0.8 μM) was included in the extralenticular medium, suggesting that the Ca2+-mediated activation of calpains may be involved in the observed opacification. Supporting this suggestion atomic absorption spectroscopy showed that the effect of SJA6017 (final concentration 0.8 μM) on lens opacification was not due to the compound restricting porcine lens Ca2+ uptake. The results indicate that calpain-induced cataractogenesis is dependent on extracellular Ca2+ and the calpain inhibitor SJA6017 (0.8 μM) had no significant effect on Ca2+ uptake by lens. Its inhibitory effect on lens opacification may be due to a direct action on the activity of calpain. (Mol Cell Biochem 261: 169–173, 2004)