Protein and Peptide Letters - Volume 19, Issue 6, 2012

This issue of Protein & Peptide Letters includes 13 papers contributed by members of the Editorial Advisory Board (EAB) of the journal. These individuals play an important role in reviewing submissions to the journal and in promoting PPL to their colleagues around the world. One of the 12 issues each year features papers from their laboratories. This issue deals with manuscripts that were submitted during 2011, but with peer review and revisions, the accepted papers are only being published now. We will work to improve the speed of the process in future EAB member issues. First, Ajoy Basak's laboratory has presented a paper titled “Proprotein Convertase Subtilisin Kexin9 (PCSK9): A Novel Target For Cholesterol Regulation”. This is a member of the mammalian subtilase family and is involved in degradation of the low density lipoprotein receptor. This work is important in the potential development of a new target for cholesterol lowering drugs. The second paper is from the laboratory of David Phoenix titled “Effect of Amidation on the Antimicrobial Peptide Aurein 2.5 from Australian Southern Bell Frogs”. In addition to demonstrating a 2.5-fold enhancement of activity against Klebsiella pneumonia by the C-terminally amidated peptide, this paper explores the surface properties, helical content, and monolayers using Langmuir techniques.....

Proprotein Convertase Subtilisin Kexin9 (PCSK9), originally called Neural Apoptosis-Regulated Convertase1 (NARC1), is the latest member of mammalian subtilase super-family. Since its discovery in 2003, it has drawn significant attention because of its function in the degradation of Low Density Lipoprotein Receptor (LDL-R). LDL-R removes circulating LDL-cholesterol (LDL-C) in the blood. Increased level of PCSK9 functional activity will lead to an accumulation of cholesterol in the blood - a high risk factor for cardiovascular disease. This is confirmed by PCSK9 knock out and transgenic animals, various biochemical and clinical studies involving “gain and loss of function” genetic mutations of PCSK9 found in various subset of populations. Owing to this finding, development of strategies for inhibition of PCSK9 function has drawn significant research interest for therapeutic intervention of hypercholesterolemia. Thus PCSK9 is a target for the development of new cholesterol lowering drugs.

Aurein 2.5 is a naturally C-terminally amidated amphibian antimicrobial peptide. C-terminal amidation can increase efficacy and hence a comparison was made between aurein 2.5-CONH2 and its nonamidated analogue. Amidation of the C-terminal carboxyl of aurein 2.5 enhanced antimicrobial activity 2.5- fold against Klebsiella pneumonia. Our results demonstrate that both peptide analogues had high surface activities (23 mN m-1for aurein 2.5-COOH and 26 mN m-1 aurein 2.5-CONH2). Circular dichroism measurements suggest that the helical content of the amidated form, in the presence of trifluoroethanol, was significantly enhanced (33.66 % for aurein 2.5-COOH and 60.89 % aurein 2.5-CONH2). The interaction of aurein 2.5 with bacterial cell membrane mimics was investigated using Langmuir monolayers. Aurein 2.5-CONH2 induced stable surface pressure changes in monolayers formed from K. pneumonia (circa 4.7 mN m-1), however, lower surface pressure changes were observed for aurein 2.5- COOH (circa 3.8 mN m-1). The data shows that in the case of aurein 2.5, amidation is able to enhance antibacterial activity and it is proposed that the increase in effectiveness is due to stabilization of the α-helical structure at the membrane interface.

The Saccaromices cerevisiae D-serine dehydratase is a pyridoxal 5'-phosphate dependent enzyme that requires zinc for its function. It catalyses the conversion of D-serine into pyruvate and ammonia with the K(m) and k(cat) values of 0.39 mM and 13.1 s(-1) respectively. In this work, a new methodology for monitoring D-serine is presented. Our results show that this enzyme could be successfully used as a biological probe for detection of D-serine via fluorescence spectroscopy.

The increase in bacterial resistance to current antibiotics has led to the development of new active molecules. We have isolated the antimicrobial peptide Ctx-Ha from the skin secretion of the frog Hypsiboas albopunctatus. The aim of the present work was to elucidate the mechanism of action of this new antimicrobial peptide. The sequence similarity with Ceratotoxin, the pore size, and the pore-like release of carboxyfluorescein from vesicles indicated that Ctx(Ile21)-Ha has a mechanism of action based on the barrel- stave model. In a second part of this work, we synthesized three analogues to provide information about the relationship between the peptide’s structure and its biological activity. Ctx(Ile21)-Ha-VD 16, Ctx(Ile21)- Ha-VD 5,16 and Ctx(Ile21)-Ha-I9K were designed to disrupt the peptide’s helical structure and change the hydrophobicity/ hydrophilicity and amphipathicity of the apolar face in order to uncouple the antimicrobial activity of Ctx(Ile21)-Ha from its hemolytic activity. To evaluate the effects of the amino acid substitutions on peptide conformation, secondary structure was accessed using CD measurements. The peptides presented a high amount of α-helical structure in the presence of TFE and LPC. The CD data showed that destruction of the amphipathic α-helix by the replacing isoleucine by lysine is less harmful to the structure than D-amino acid substitutions. Biological tests demonstrated that all peptides have activity. Nevertheless, the peptide Ctx(Ile21)-Ha-I9K showed the highest value of therapeutic index. Our findings suggest that these peptides are potential templates for the development of new antimicrobial drugs. These studies highlight the importance of single amino acid modification as a tool to modulate the biological activity of antimicrobial peptides.

The binding of fisetin with human serum albumin (HSA) has been studied at different pH using UV-Vis, FTIR, CD and fluorescence spectroscopic techniques. The binding constants were found to increase with the rise in pH of the media. The negative ΔH° (kJ mol-1) and positive ΔS° (J mol-1 K-1) indicate that fisetin binds to HSA via electrostatic interactions with an initial hydrophobic association that result in a positive ΔS°. In presence of potassium chloride (KCl) the binding constants were found to be decrease. The α-helical content of HSA increased after binding with fisetin as analyzed from both CD and FTIR methods. The site marker displacement studies using fluorescence anisotropy suggest that fisetin binds to the hydrophobic pocket (Site 1, subdomain IIA) of HSA which is in good accordance with the molecular docking study. The change in accessible surface area (ASA) of residues of HSA was calculated to get a better insight into the binding.

Hsp70 is a highly conserved protein that refolds misfolded proteins and has numerous housekeeping functions which regulate apoptosis and other cell activities. Hsp70 consists of a nucleotide binding domain which binds ATP and a substrate binding domain that binds misfolded proteins. The substrate binding domain contains a peptide binding pocket which is covered by a helical lid. In humans, there are three major cytosolic Hsp70 isotypes, Hsp70-8, Hsp70-1 and Hsp70-2. Leukemic and numerous other cancer cells have a greater amount of Hsp70-1 and -2, which help the cancer cells inhibit apoptosis in response to stress. This review summarizes the structure and role of Hsp70 proteins in cancer survival.

Polybia paulista wasp venom possesses three major allergens: phospholipase A1, hyaluronidase and antigen-5. To the best of our knowledge, no hyaluronidase from the venom of Neotropical social wasps was structurally characterized up to this moment, mainly due to its reduced amount in the venom of the tropical wasp species (about 0.5% of crude venom). Four different glycoproteic forms of this enzyme were detected in the venom of the wasp Polybia paulista. In the present investigation, an innovative experimental approach was developed combining 2-D SDS-PAGE with in-gel protein digestion by different proteolytic enzymes, followed by mass spectrometry analysis under collision-induced dissociation CID) conditions for the complete assignment of the protein sequencing. Thus, the most abundant form of this enzyme in P. paulista venom, the hyaluronidase-III, was sequenced, revealing that the first 47 amino acid residues from the N-terminal region, common to other Hymenoptera venom hyaluronidases, are missing. The molecular modeling revealed that hyaluronidase-III has a single polypeptide chain, folded into a tertiary structure, presenting a central (β/α)5 core with alternation of β-strands and α-helices; the tertiary structure stabilized by a single disulfide bridge between the residues Cys189 and Cys201. The structural pattern reported for P. paulista venom hyaluronidase-III is compatible with the classification of the enzyme as member of the family 56 of glycosidase hydrolases. Moreover, its structural characterization will encourage the use of this protein as a model for future development of “component-resolved diagnosis”.

Microtubule cytoskeleton is a dynamic structure involved in the maintenance of eukaryote cell shape, motion of cilia and flagellum, and intracellular movement of vesicles and organelles. Many antibodies against tubulins have been described, most of them against the C-terminal portion, which is exposed at the outside of the microtubules. By generating a novel set of monoclonal antibodies against the cytoskeleton of Trypanosoma cruzi, a flagellate protozoan that causes Chagas’ disease, we selected a clone (mAb 3G4) that recognizes β-tubulin. The epitope for mAb 3G4 was mapped by pepscan to a highly conserved sequence motif found between α-helices 11 and 12 of the C-terminus of β-tubulin in eukaryotes. It labels vesicular structures in both T. cruzi and mammalian cells, colocalizing respectively with a major cysteine protease (Cruzipain) and lysosome associated protein (LAMP2) respectively, but it does not label regular microtubules on these cellular models. We propose that the epitope recognized by mAb 3G4 is exposed only in a form of tubulin associated with endosomes.

The information of protein subcellular localization is vitally important for in-depth understanding the intricate pathways that regulate biological processes at the cellular level. With the rapidly increasing number of newly found protein sequence in the Post-Genomic Age, many automated methods have been developed attempting to help annotate their subcellular locations in a timely manner. However, very few of them were developed using the protein-protein interaction (PPI) network information. In this paper, we have introduced a new concept called “tethering potential” by which the PPI information can be effectively fused into the formulation for protein samples. Based on such a network frame, a new predictor called Yeast-PLoc has been developed for identifying budding yeast proteins among their 19 subcellular location sites. Meanwhile, a purely sequence-based approach, called the “hybrid-property” method, is integrated into Yeast-PLoc as a fall-back to deal with those proteins without sufficient PPI information. The overall success rate by the jackknife test on the 4,683 yeast proteins in the training dataset was 70.25%. Furthermore, it was shown that the success rate by Yeast- PLoc on an independent dataset was remarkably higher than those by some other existing predictors, indicating that the current approach by incorporating the PPI information is quite promising. As a user-friendly web-server, Yeast-PLoc is freely accessible at http://yeastloc.biosino.org/.

In a previous study, we reported that truncation of HP (2-20) (derived from the N-terminal region of Helicobacter pylori Ribosomal Protein L1 (RPL1)) at the N- (residues 2-3) and C-terminal (residues 17-20) truncated fragments to give HP (4-16) induces increased antibiotic activity against several bacterial strains without hemolysis. In this study, to develop novel short antibiotic peptides useful as therapeutic drugs, an analogue was designed to possess increased hydrophobicity by Trp substitution in position 2 region of HP (4-16). Synthetic HP (4-16)-W showed an enhanced antimicrobial and antitumor activity. The antimicrobial activity of this peptide and others was measured by their growth inhibitory effect upon S. aureus, B. subtilis, S. epidermidis, E. coli, S. typimurium, P. aeruginosa, C. albicans, T. beigelii and S. cerevisiae. None of the peptides exhibited hemolytic activity against human erythrocyte cells except melittin as a positive control. Its antibiotic activity suggests that HP (4-16)-W is an excellent candidate as a lead compound for the development of novel antibiotic agents.

Soybean urease has been investigated extensively to reveal the presence of histidine residue (s) in the active site and their potential role in the catalysis. The spectrophotometric studies using diethylpyrocarbonate (DEP) showed the modification of 11.76 ± 0.1 histidine residues per mole of native urease. Therefore, the results are indicative of the presence of twelve histidine residues per urease molecule. It is presumed that the soybean urease, being a hexameric protein possess two histidine residues per subunit. Correlation plot showed that the complete inactivation of soybean urease corresponds to the modification of 1.97 histidine residues per subunit. Further, double logarithmic plot of kapp versus DEP concentration has resulted in a linear correlation and thereby demonstrating that only one of the two histidine residues per subunit is catalytically essential. Significant protection has been observed against inactivation when urea or acetohydroxamate (AHA) is incubated with DEP treated urease. The studies have demonstrated the presence of one histidine residue at the active site of soybean urease and its significance in catalysis.

Background: Neutrophils have an impressive array of microbicidal weapons, and in the presence of a pathogen, progress from a quiescent state in the bloodstream to a completely activated state. Failure to regulate this activation, for example, when the blood is flooded with cytokines after severe trauma, causes inappropriate neutrophil activation that paradoxically, is associated with tissue and organ damage. Acidic proteomic maps of quiescent human neutrophils were analyzed and compared to those of activated neutrophils from severe trauma patients. The analysis revealed 114 spots whose measured volumes differed between activated and quiescent neutrophils, with 27 upregulated and 87 downregulated in trauma conditions. Among the identified proteins, grancalcin, S100-A9 and CACNB2 reinforce observed correlations between motility and ion flux, ANXA3, SNAP, FGD1 and Zfyve19 are involved in vesicular transport and exocytosis, and GSTP1, HSPA1 HSPA1L, MAOB, UCH-L5, and PPA1 presented evidence that activated neutrophils may have diminished protection against oxidative damage and are prone to apoptosis. These are discussed, along with proteins involved in cytoskeleton reorganization, reactive oxygen species production, and ion flux. Proteins such as Zfyve19, MAOB and albumin- like protein were described for the first time in the neutrophil. In this work we achieved the identification of several proteins potentially involved in inflammatory signaling after trauma, as well as proteins described for the first time in neutrophils.

Successful approaches of de novo protein design suggest a great potential to create novel structural folds and to understand natural rules of protein folding. For these purposes, smaller and simpler de novo proteins have been developed. Here, we constructed smaller proteins by removing the terminal sequences from stable de novo vTAJ proteins and compared stabilities between mutant and original proteins. vTAJ proteins were screened from an α3β3 binary-patterned library which was designed with polar/ nonpolar periodicities of α-helix and β-sheet. vTAJ proteins have the additional terminal sequences due to the method of constructing the genetically repeated library sequences. By removing the parts of the sequences, we successfully obtained the stable smaller de novo protein mutants with fewer amino acid alphabets than the originals. However, these mutants showed the differences on ANS binding properties and stabilities against denaturant and pH change. The terminal sequences, which were designed just as flexible linkers not as secondary structure units, sufficiently affected these physicochemical details. This study showed implications for adjusting protein stabilities by designing N- and C-terminal sequences.

Although it is known that neutrophils infiltrate damaged sites immediately after tissue injury, the endogenous factors that induce their acute transmigration and activation have not been thoroughly investigated. For the candidates of those factors, we recently discovered two novel neutrophil-activating cryptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial proteins. In addition, many unknown neutrophil-activating peptides other than MCT-1 and MCT-2 were also observed during their purification. Here, we isolated and purified a novel neutrophil-activating peptide from porcine hearts, which we showed by structural analyses to have an identical primary structure to porcine mitochondrial cytochrome c (68-85). We named this novel functional octadecapeptide as mitocryptide-CYC (MCT-CYC). Structure-activity relationships of cytochrome c on β-hexosaminidase (β-HA) release from neutrophilic-differentiated HL- 60 cells demonstrated that peptides derived from the C-terminal part of cytochrome c induced β-HA release and that cytochrome c (70-85) was the most potent cryptide among them. Since cytochrome c is known to be involved in the apoptotic process, our results suggest that cryptides, including MCT-CYC, derived from mitochondrial cytochrome c are possible factors that induce scavenging of toxic debris produced from apoptotic cells by neutrophils.

In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.