Multidimensional signatures in antimicrobial peptides.

Much research on host defenses against infection has concentrated on the amino acid sequences of antimicrobial peptides in the belief that the order of the acids and their replication reflect how they work against aberrant cells. Now researchers at the University of California, Los Angeles, (UCLA) suggest that the shape the sequences are arranged in may be a critical part of how these peptides work. A new report indicates that host defense systems across the spectrum of life rely on a universal core structure integral to many natural antimicrobial peptides. This core motif may play a key role in preventing or limiting infection, an insight that could accelerate a major advancement in antimicrobial drug development. “It has been generally accepted that there is a wide diversity in amino acid sequences and sources of antimicrobial peptides,” says study co-investigator Michael Yeaman, a professor of medicine at the David Geffen School of Medicine at UCLA. “But there hasn’t been as much insight into the similarities that might exist that link all of these diverse groups of molecules.” The gamma (γ)-core motif—so called because it resembles the Greek letter—may be that missing link, providing a key ingredient in the signature of antimicrobial peptides. Yeaman and coauthor Nannette Yount, a molecular biologist at the Los Angeles Biomedical Research Institute, say the γ-core alone can have antimicrobial activity, but also appears to provide a scaffold on which critical modules are configured to create molecules that hunt down microbial pathogens and destroy them in diverse tissue contexts without injury to the host. The duo studied the amino acid sequences and three-dimensional structures of over 500 antimicrobial peptides, and found the γ-core structure in molecules as diverse as pea defensins, fruit fly drosomycin, pig protegrin, and human hepcidin. Such molecules share the multidimensional signature of antimicrobial peptides. In a paper published 11 May 2004 in Proceedings of the National Academy of Sciences, the authors wrote, “This striking multidimensional signature is conserved among disulfide-containing antimicrobial peptides spanning biological kingdoms, and it transcends motifs previously limited to defined peptide subclasses.” But the sequence, composition, and biochemistry of the amino acids that make up the signature still play a major role, says Yeaman. “We feel that some of the universality identified here may have been missed previously because to identify this signature, we had to look at amino acid sequences in both forward and reverse orientation, and that is not typically done,” he says. “The broad conservation of the multidimensional signature identified may have been missed if we only performed amino acid sequence searches and alignments in a conventional way.” There are other critical aspects of the γ-core motif as well, Yeaman says. “The amino acid sequence is configured in three-dimensional space so that the γ-core has certain characteristics. For example, electrostatic charge tends to be placed in one part of this motif and hydrophobicity in another; disulfide linkages are also conserved. These hallmark features of the γ-core motif rely on both composition and three-dimensional structure.” Yeaman and Yount are now translating the motif into peptide mimetics and small molecules, and are designing so-called modular anti-infectives with customized payloads of drugs that attach to the γ-core motif. These compounds are at different stages of development—some are in the design phase, while some have been tested and proven to have antimicrobial efficacy. Still others are being optimized based on data generated in the lab as well as in initial ex vivo studies. “We are trying to develop entirely new types of ‘smart’ antibiotics that recognize and act against harmful microbes, particularly those that have become resistant to most all conventional drugs,” Yeaman says. The work has captured the attention of researchers in the drug development industry. “It’s the structure that defines the signature,” says Steve Projan, vice president of biological technologies at Wyeth Research in Cambridge, Massachusetts, and a consultant to the American Society for Microbiology, based in Washington, D.C. “That structure may be more important than sequence of amino acids. Even if the amino acids are different, it is the overall structure that defines the activity of the molecule.” However, Projan admits, “I’ll be skeptical about the impact of this work until we have a molecule that works by [these] rules and a molecule that also works in an infection model.” Yeaman suggests that learning how nature has evolved antimicrobial agents may allow scientists to use the γ-core motif or mimetics thereof as the scaffold that will guide the right peptide or molecule to the right target. “Nature has done much of the designing,” he says. “We are capitalizing on the experiments that nature has performed over millions of years [and] trying to integrate the results of that process in new antibiotics.”

Fibril Formation of hsp10 Homologue Proteins and Determination of Fibril Core Regions: Differences in Fibril Core Regions Dependent on Subtle Differences in Amino Acid Sequence

High-molecular-weight glutenin subunits (HMW-GS) play an important role for the baking quality of wheat. The ancient wheats emmer and spelt differ in their HMW-GS pattern compared to modern common wheat and this might be one reason for their comparatively poor baking quality. The aim of this study was to elucidate similarities and differences in the amino acid sequences of two 1Bx HMW-GS of common wheat, spelt and emmer. First, the sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) system was optimized to separate common wheat, spelt and emmer Bx6 and Bx7 from other HMW-GS (e.g., 1Ax and 1By) in high concentrations. The in-gel digests of the Bx6 and Bx7 bands were analyzed by untargeted LC-MS/MS experiments revealing different UniProtKB accessions in spelt and emmer compared to common wheat. The HMW-GS Bx6 and Bx7, respectively, of emmer and spelt showed differences in the amino acid sequences compared to those of common wheat. The identities of the peptide variations were confirmed by targeted LC-MS/MS. These peptides can be used to differentiate between Bx6 and Bx7 of spelt and emmer and Bx6 and Bx7 of common wheat. The findings should help to increase the reliability and curation status of wheat protein databases and to understand the effects of protein structure on the functional properties. Graphical abstract.

Co-operativity and calcium/magnesium binding to troponin C and muscle calcium binding parvalbumin: An hypothesis

Human immunodeficiency virus type 1 (HIV‐1) shows a very narrow host range limited to humans and chimpanzees. Experimentally, HIV‐1 does not infect Old World monkeys, such as rhesus (Rh) and cynomolgus (CM) monkeys, and fails to replicate in activated CD4 positive T lymphocytes obtained from these monkeys. In contrast, simian immunodeficiency virus isolated from a macaque monkey (SIVmac) can replicate well in both Rh and CM. In 2004, tripartite motif 5α (TRIM5α) was identified as a host factor which plays an important role in the restricted host range of HIV‐1. Rh and CM TRIM5α restrict HIV‐1 infection but not SIVmac, while in comparison, anti‐viral activity of human TRIM5α against those viruses is very weak. TRIM5α consists of the RING, B‐box 2, coiled‐coil and SPRY (B30.2) domains. The RING domain is frequently found in E3 ubiquitin ligase and TRIM5α is degraded via the ubiquitin‐proteasome pathway during HIV‐1 restriction. TRIM5α recognises the multimerised capsid (viral core) of an incoming virus by its α‐isoform specific SPRY domain and is believed to be involved in innate immunity to control retroviral infection. Differences in amino acid sequences in the SPRY domain of TRIM5α of different monkey species were found to affect species‐specific restriction of retrovirus infection, while differences in amino acid sequences in the viral capsid protein determine viral sensitivity to restriction. Accurate structural analysis of the binding surface between the viral capsid protein and TRIM5α SPRY is thus required for the development of new antiretroviral drugs that enhance anti‐HIV‐1 activity of human TRIM5α. Copyright © 2009 John Wiley & Sons, Ltd.

Evolution of vertebrate neuropeptides

In Singapore, both influenza A and B viruses circulate in significant proportions throughout the year, with bi-annual peaks corresponding to the monsoon seasons. This statistic is not shared in most temperate countries where influenza is only prevalent during winter. Exploiting this unique situation in Singapore, we studied the characteristics of non-tissue culture adapted influenza B clinical specimens collected from SAF servicemen between the years of 2004 and 2009. A total of 81 clinical specimens were received and 66 tested positive for influenza B upon diagnostic PCR. Growth kinetics of these clinical specimens could not be studied as none of these specimens could replicate in tissue culture. Forty-six of these clinical specimens yielded sequence information for further analysis. Lineage identity of these specimens were determined through phylogenetic analysis, followed by a comparison of the amino acid sequences between the HA, NA, NB, NS1, NEP and BM2 proteins. Molecular cloning of the NB, NS1 and BM2 genes of representative strains were carried out to determine if differences in sequence would translate into phenotypic differences. Phylogenetic analysis show that majority of the specimens were re-assortants with the HA belonging to the Victoria lineage and the NA from the Yamagata lineage. Only one specimen had both these gene segments from the Yamagata lineage. This finding has resulted in us determining several vaccine mismatches over the six years in focus. The NS1 phylogenetic tree showed that seven of the specimens isolated in 2004 clustered closely to B/Lee/40 than the rest. This finding provides an interesting look into the separation of the influenza B lineages. Analysis of the amino acid sequences reveal that majority of the clinical specimens harboured a glycosylation site at position 211 of the HA protein, which was previously not dominant. No mutations conferring drug resistance were noticed among the HA and NA proteins analysed. The 7 specimens clustering with B/Lee/40 in the NS phylogenetic tree had almost identical amino acid sequences to B/Lee/40 NS1 and NEP proteins, differing from the other clinical specimens. We found an inability of these clinical specimens to replicate well in tissue culture, a trait shared with the reference strain B/Lee/40. In an attempt to exploit the differences in amino acid sequences within the clinical specimens, the NB, BM2 and NS1 genes of representative sequences were cloned into a mammalian expression vector. Basic molecular characterisation indicates that the NB protein, which has an unknown role, localised primarily in the cis-Golgi and is present throughout the Golgi. This is similar to the staining pattern displayed by the BM2 protein, suggesting a similarity in the function. SDS-PAGE analysis reveal that both these proteins are modified by the cell and further elucidation is required to determine the kinetics and the function of these modifications. The bulk of the work presented in this thesis revolves around the molecular characterisation of the…

Antimicrobial peptides: has their time arrived?

Although the bioactivities of bovine lactoferrin have been extensively investigated, little is known about deer milk lactoferrin bioactivity and its amino acid sequence. This research investigated the amino acid sequence of deer lactoferrin and the antimicrobial activities of two lactoferrin-encrypted peptides; lactoferricin (Lfcin) and lactoferrampin (Lfampin). Deer lactoferrin was found to have a molecular weight of 77.1 kDa and an isoelectric point of 7.99, which are similar to that of bovine lactoferrin, 78 kDa and pI 7.9. Deer lactoferrin contains 707 amino acids, one amino acid less than bovine lactoferrin, and has 92% homology with bovine lactoferrin. Deer lactoferricin exhibited strong antimicrobial activity against E. coli American Type Culture Collection (ATCC) 25922 and L. acidophilus ATCC 4356. The antimicrobial activities of deer and bovine Lfcin and Lfampin were compared. Based on MIC, deer Lfcin was found to be a more effective inhibitor of L. acidophilus ATCC 4356 than bovine Lfcin, but bovine Lfcin and Lfampin were more effective against E. coli ATCC 25922 than deer Lfcin and Lfampin. The deer Lfcin sequence differed at seven amino acids from bovine Lfcin and this decreased the net positive charge and increased the hydrophobicity. Deer Lfampin contained two differences in amino acid sequence compared to bovine Lfampin which decreased the net positive charge. These amino acid sequence differences likely account for differences in antibacterial activity. Positive charge and hydrophobic residues provide the amphipathic character of these helical peptides, and are considered important for binding of antimicrobial peptides. In silico modelling of deer Lfcin indicated an identical α-helical structure compared to bovine Lfcin.

Myoglobin isolated from red muscle of the shark H. portusjacksoni was purified by ion-exchange chromatography on sulfopropyl-Sephadex and gel-filtration. Amino acid analysis and sequence determination showed 148 amino acid residues. The amino terminal residue is acetylated as shown by mass spectrographic analysis of N-terminal peptides. There is a deletion of four residues at the amino terminal end as well as one residue in the CD interhelical area relative to other myoglobins. The complete amino acid sequence has been determined following digestion with trypsin, chymotrypsin, pepsin and staphylococcal protease. Sequences of the purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed approximately 85 differences from mammalian, monotreme and bird myoglobins. The date of divergence of the shark H. portusjacksoni from these other orders was estimated at 450 +/- 16 million years, based on the number of amino acid differences between species and allowing for multiple mutations during the evolutionary period. This estimate agrees well with similar estimates made using alpha- and beta-globin sequences, in contrast to widely differing estimates of dates of divergence for monotremes using the same three globin chains. Compared with myoglobins from species previously studied, there are many more differences in amino acid sequences, and in many positions residues are found that are more characteristic of alpha- and beta-globins, suggesting a conservation of residues over a long period of evolutionary time. There are fewer stabilizing hydrogen bonds and salt-linkages than in other myoglobins.

Human lysozyme has a structure similar to that of hen lysozyme and differs in amino acid sequence by 51 out of 129 residues with one insertion at the position between 47 and 48 in hen lysozyme. The backbone dynamics of free or (NAG)3-bound human lysozyme has been determined by measurements of 15N nuclear relaxation. The relaxation data were analyzed using the Lipari-Szabo formalism and were compared with those of hen lysozyme, which was already reported (Mine S et al.. 1999, J Mol Biol 286:1547-1565). In this paper, it was found that the backbone dynamics of free human and hen lysozymes showed very similar behavior except for some residues, indicating that the difference in amino acid sequence did not affect the behavior of entire backbone dynamics, but the folded pattern was the major determinant of the internal motion of lysozymes. On the other hand, it was also found that the number of residues in (NAG)3-bound human and hen lysozymes showed an increase or decrease in the order parameters at or near active sites on the binding of (NAG)3, indicating the increase in picosecond to nanosecond. These results suggested that the immobilization of residues upon binding (NAG)3 resulted in an entropy penalty and that this penalty was compensated by mobilizing other residues. However, compared with the internal motions between both ligand-bound human and hen lysozymes, differences in dynamic behavior between them were found at substrate binding sites, reflecting a subtle difference in the substrate-binding mode or efficiency of activity between them.

The prion protein (PrP(C)) is essential for susceptibility to transmissible spongiform encephalopathies. A specific conformer of this protein (PrP(Sc)) is, according to the 'protein only' hypothesis, the principal or only component of the infectious agent, designated prion. Transmission of prions between species is often inefficient, resulting in low attack rates and/or prolonged incubation times and is ascribed to a 'species barrier' caused by differences in the amino acid sequence of PrP between recipient and donor. In this report, we demonstrate that these differences in amino acid sequence result in presentation of distinct peptides on major histocompatibility complex class II molecules. These peptides result in activation of specific CD4+ T cells which leads to the induction of an effective immune response against foreign PrP as demonstrated by antibody production. Therefore, CD4+ T cells represent a crucial component of the immune system to distinguish between foreign and self PrP.

Properties of ribulose-P 2-carboxylases from wheat, maize, tobacco, spinach and pea and one photosynthetic bacterium (Rhodospirillum rubrum) were compared. Electrophoresis showed no differences in mass of the large subunit polypeptides of the higher plant carboxylases. The small subunit polypeptides from spinach, pea and tobacco carboxylases had similar molecular weights of about 14000. This value is smaller than that of the small subunit of wheat and larger than that of the small subunit of maize (carboxylase). The purified carboxylases differed in their content of a rapidly activating form of the enzyme and in their sensitivity to Mg 2+. The differences were not easily fitted into discrete categories so that no clear classification was possible on the basis of activation characteristics. Relative activities of the various enzymes as carboxylases and oxygenases were quantified as specificity factors. The carboxylase from R. rubrum, was much less effective as a carboxylase compared to an oxygenase than carboxylases from the higher plants. Specificity factors for the higher plant enzymes were between 77 and 104. The highest values were recorded for the carboxylase of wheat and the lowest for carboxylases of maize and tobacco. The differences in specificity factors between carboxylases from wheat, maize and tobacco are discussed in relation to differences in amino acid sequences of the large subunit polypeptide. Four differences in residues within the active site may be relevant with respect to the observed differences in enzyme properties. Mild trypsinolysis very rapidly decreased the activity of carboxylases from maize, wheat and spinach but pea, barley and tobacco carboxylases were inactivated more slowly and the enzyme from R. rubrum was not affected at all.

Polyproteins comprise long polypeptides that are post-translationally cleaved into proteins of different function, or tandemly repetitive polypeptides which are processed into multiple versions of proteins which are presumed to have the same function. In the latter case the individual units of the polyprotein can differ substantially in sequence. Identity of function between the different units therefore cannot be assumed. Here we have examined the ABA-1 polyprotein allergen of the parasitic nematode Ascaris suum and found it to contain units which show a 50% difference in amino acid sequence. The parasite therefore produces at least two radically different forms of the allergen encoded within the polyprotein array. In fluorescence-based ligand-binding assays, recombinant polypeptides representing the two forms (designated ABA-1A1 and ABA-1B1) showed similar binding affinities for a range of fluorescent active-site probes [retinol, dansylundecanoic acid, dansyl-DL-alpha-amino-octanoic acid, cis-parinaric acid (cPnA)] and for the non-specific hydrophobic surface probe 8-anilinonaphthalene-1-sulphonic acid. However, the molecular environments in the active sites are markedly different, as indicated by disparate fluorescence emission peaks and intensities of bound probes. CD showed that the proteins have similar secondary structures but differ in susceptibility to chemical denaturation/unfolding by guanidinium chloride. Both retain a single conserved tryptophan residue in a characteristic non-polar environment, as revealed by extreme fluorescence blue shift. Thus the gross differences in sequence of the two proteins are not reflected in their ligand-binding specificities but in their binding-site environments.

Structural Plasticity in Globins: Role of Protein Dynamics in Defining Ligand Migration Pathways.