Protein and Peptide Letters - Volume 20, Issue 8, 2013

Alginate is a biofilm exopolysaccharide secreted by the opportunistic pathogen Pseudomonas aeruginosa that acts to prevent the diffusion of antibiotics toward the bacterial cell membrane. Cationic antimicrobial peptides (CAPs) have been increasingly recognized as a viable alternative for prospective antimicrobial agents. The D-isomer chiral counterparts of active L-isomer CAPs tend to show slightly greater antimicrobial activities because bacteria lack proteases to hydrolyze the unnatural D-isomers. Using an enantiomeric pair of synthetic CAPs designed in our laboratory (L-4Leu in the sequence KKKKKKALFALWLAFLA–NH2 and its D-analog D-4Leu), we studied the binding and interactions of Lvs. D-isomers of CAPs with alginate using circular dichroism and Raman spectroscopic techniques. We found that the peptide D-4Leu underwent a more rapid structural transition over time from an initial alginate-induced α-helical conformation to a less soluble β-sheet conformation than L-4Leu, indicating that the D-isomer of this peptide has a relatively greater affinity for alginate. Through Raman spectroscopy it was observed that Raman modes at 1297 cm-1 and 1453 cm-1 wavenumbers were found to differ between the spectra obtained from the insoluble complexes formed between L-4Leu vs. D-4Leu and alginate. These modes were tentatively assigned to CH, and CH3 deformation modes, respectively. Our findings reveal previously undetected subtleties in the binding of this diastereomeric pair of peptides in the microenvironment of a biofilm exopolysaccharide, and provide guidelines for future development of antimicrobial peptides.

This work developed an alternative approach targeting the evaluation of the aggregation propensity of the (1- 42) β-amyloid peptide (Alzheimer’s disease) and some segments, either attached to a polymer during their synthesis or when free in solution. The solvation behavior of peptide-resins was gauged by measuring the swelling of beads in a microscope and the degree of chain motion through EPR spectra of previously labeled resins with an amino acid-type probe. In terms of comparative solvent dissociation power towards aggregated structures, the findings revealed greater values of peptide-resin swelling, peptide chain mobility and solubility when in strong electron donor dimethylsulfoxide than in strong electron acceptor trifluoroethanol. Otherwise, the weakest chain-chain disruption power was verified for acetonitrile, an internally neutral solvent in terms of Lewis acid/base properties. In complement, fluorescence and light scattering experiments depicted that the 15-35 region plays an essential role in the amyloid peptide fibril formation capacity.

Glycosyltransferases (GTs) are a ubiquitous group of enzymes that catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Nucleotide-sugars, lipid phosphate sugars and phosphate sugars can act as activated donor molecules while acceptor substrates involve carbohydrates, proteins, lipids, DNA and also, numerous small molecules (i. e. antibiotics, flavonols, steroids). GTs enzyme families are very ancient. They are founded in all the three domains of life and display three different folds (named GT-A, GTB and GT-C) which are a variant of a common α/β scaffold. In addition, several GTs contain an associated non-catalytic carbohydrate binding module (CBM) that could be critical for enzyme activity. This work reviews the current knowledge on the GTs structures and functions and highlights those enzymes that contain CBMs, particularly starch binding domains (SBDs). In addition, we also focus on A. thaliana starch synthase III enzyme, from the GT-5 family. This protein has a GT-B fold, and contains in its N-terminal region three in tandem SBDs, which are essential for the regulation of enzyme activity.

Brugia malayi is a threadlike nematode cause’s swelling of lymphatic organs, condition well known as lymphatic filariasis; till date no invention made to effectively address lymphatic filariasis. In this analysis we a have predicted suitable antigenic peptides from Brugia malayi antigen proteinfor peptide vaccine design against lymphatic filariasis based on cross protection phenomenon as, an ample immune response can be generated with a single protein subunit. We found MHC class II binding peptides of Brugia malayi antigen protein are important determinant against the diseased condition. The analysis shows Brugia malayi antigen protein having 505 amino acids, which shows 497 nonamers. In this assay, we have predicted MHC-I binding peptides for 8mer_H2_Db (optimal score- 15.966), 9mer_H2_Db (optimal score- 15.595), 10mer_H2_Db (optimal score- 19.405), 11mer_H2_Dballeles (optimal score- 23.801). We also predicted the SVM based MHCII-IAb nonamers, 51-FQQIDPLDA, 442-FAAIACLVH, 206-YLNPFGHQF, 167-WYVIMAACY, 367-YAMIVIRLL, 434- LVITTAANF, 176-LDSYCLWKP, 435-VITTAANFA, 364-WPGYAMIVI (optimal score- 13.963); MHCII-IAd nonamers, 52-QQIDPLDAE, 171-MAACYLDSY, 239-QWRSVILCN, 168-YVIMAACYL, 3-QYLSVHSLS, 322-EILLHAKVV, 417- LGIIASFVS, 396-KAIFLAHFG, 167-WYVIMAACY, 269-LALHCINVI, 93-FINKAAPKQ, 259-NCIIVLKAF, 79- QGVLLIIPR, 22-TILQRSQAI, 63-RGFVYGNVS, 109-NISSLAFET,(optimal score- 16.748); and MHCII-IAg7 nonamers 171-MAACYLDSY, 73-KIVNGAQGV, 259-NCIIVLKAF, 209-PFGHQFSFE, 102-SCDTLLKNI, 25-QRSQAIRIV, 444- AIACLVHLF, 88-SLVNGFINK, 252-FPRHQLLNC, 471-RFVLANDNE, 52-QQIDPLDAE, 469-HRRFVLAND, 457- SNRHYFLAD, 362-KSWPGYAMI, 476-NDNEGEDFE, 370-IVIRLLQAL (optimal score- 19.847) which represents potential binders from Brugia malayi antigen protein. The method integrates prediction of MHC class I binding proteasomal Cterminal cleavage peptides and Eighteen potential antigenic peptides at average propensity 1.063 having highest local hydrophilicity. Thus a small antigen fragment can induce immune response against whole antigen. This approach can be applied for designing subunit and synthetic peptide vaccines.

Synthesis of a series of urea and thiourea derivatives of glycine and proline conjugated to 2,3-dichlorophenyl piperazine has been reported. The structures were confirmed by physical and spectroscopical measurements followed by characterization of antiglycation activity. All synthesized compounds were able to inhibit protein glycation, particularly halogen containing derivatives without preference of oxygen or sulphur at the urea function. The best analogues are nearly 20 fold (< 5 µM) more potent than the reference standard, rutin (41.9 µM).

Myoglobin is an α-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues impairs the productive folding of the protein making the polypeptide chain highly prone to aggregate forming amyloid fibrils at physiological pH and room temperature. The role played by tryptophanyl residues in driving the productive folding process was investigated by providing structural details at low resolution of compact intermediate of three mutated apomyoglobins, i.e., W7F, W14F and the amyloid forming mutant W7FW14F. In particular, we followed the hydrogen/deuterium exchange rate of protein segments using proteolysis with pepsin followed by mass spectrometry analysis. The results revealed significant differences in the N-terminal region, consisting in an alteration of the physico-chemical properties of the 7-11 segment for W7F and in an increase of local flexibility of the12-29 segment for W14F. In the double trypthophanyl substituted mutant, these effects are additive and impair the formation of native-like contacts and favour inter-chain interactions leading to protein aggregation and amyloid formation at physiological pH.

A series of Ile-containing elastin-derived peptide-analogs, (Ile-Pro-Gly-Val-Gly)n (n=7–10) possessing remarkable and reversible coacervation property were newly synthesized. In comparison with the known elastin-derived peptideanalogs, which were so-called polypeptides, the obtained 35 to 50 mer peptides, (IPGVG)n (n=7–10) were significantly low molecular sized-polypeptides. However, they clearly exhibited coacervation property as same as the polypeptides did. Because of their low molecular size, spectrographic analyses of (IPGVG)n (n=7–10) became feasible to carry out. As results of secondary structural analyses by CD and FT-IR, it was found that the coacervation property of the peptides is clearly attributed to the ordered secondary-structures, mainly, type II β–turn.

Protein methylation is one of the most important post-translational modifications. Typically methylation occurs on arginine or lysine residues in the protein sequence. In the biological system, methylation is catalyzed by enzymes, and should be involved in modification of heavy metals, regulation of gene expression, regulation of protein function, and RNA metabolism. Thus the prediction of methylation sites plays a crucial role. As we know, traditional experiment approaches to predict the sites are accurate, but that are always labor-intensive and time-consuming. Thus, it is common to see that computational methods receive increasingly attentions due to their convenience and fast speed in recent years. In this study, we develop a computational approach to predict the performance of methylarginine and methyllysine sites. First, a new encoding schema as called the CKASSP is used in our method. Then, the support vector machine (SVM) algorithm is used as a predictor. Experimental results show that our method can obtain average prediction accuracy of 87.46%, sensitivity of 99.09%, specificity of 86.89% for arginine methylation sites, and average prediction accuracy of 88.78%, sensitivity of 93.75%, specificity of 81.79% for lysine methylation sites as well, which is better than those of other state-of-art predictors. The online service is implemented by java 1.4.2 and is freely available at http://202.198.129.219:8080/cksaap_methsite.

The extracellular thermolysin like protease (TLP) was purified and characterized from Thermoactinomyces thalpophilus MCMB-380 (Genbank Accession No. EF397000). The enzyme was purified to homogeneity by successive ultra filtration steps using 50 kDa and 10 kDa membrane filters followed by anion exchange chromatography. The molecular mass and isoelectric point of the enzyme were found to be 34.4 kDa and 9.5, respectively. The proteolytic activity was inhibited by EDTA and the enzyme required Ca2+ to show the full activity as well as thermostability. The T50 of the enzyme at 80 °C was 1 h and the activation energy was estimated to be 11.02 Kcal / mol. Atomic absorption spectrophotometric analysis revealed the presence of Zn2+ ion in the protein core indicating that it is a metalloprotease. This protease has commercial potential in catalyzing the condensation reaction of two amino acids for production of the dipeptide aspartame, an artificial sweetener. The one hour time-frame is significantly faster than that of the enzyme thermolysin from Bacillus thermoproteolyticus. Moreover the TLP was stable at 80°C for one hour which makes it industrially robust. The Zn2+ ion in the T. thalpophilus protease appears to be necessary for maintaining the active conformation of the enzyme molecule.

Apelin was identified as natural ligand for APJ, a G protein-coupled receptor. APJ is expressed in spinal cord and dorsal root ganglion. This study was designed to investigate the effects and mechanisms of intrathecally (i.t.) administered apelin-13 on nociceptive response in formalin test and tail-flick test. In formalin test, i.t. injection of apelin-13 (0.3– 3 nmol/mouse) had no effect on the nociceptive response in either acute phase (0– 10 min) or interphase (10– 20 min), but significantly produced hyperalgesic effect in late phase (20– 30 min) at the dose of 3 nmol/mouse. The APJ receptor antagonist apelin-13(F13A) and GABAA receptor antagonist bicuculline methiodide, but not opioid receptor antagonist naloxone, significantly blocked the hyperalgesia caused by apelin-13 in late phase, indicating that i.t. apelin-13- induced hyperalgesia was mediated by APJ and GABAA receptor, rather than opioid receptor. However, in tail-flick test, i.t. injected apelin-13 (1 and 3 nmol/mouse) induced a significant antinociceptive effect, which was significantly antagonized by apelin-13(F13A) and naloxone, suggesting APJ and opioid receptor were involved in the antinociception of spinal apelin-13.

Various chemical modifications can reduce chaperone activity of α-crystallin (α-Cry) and the loss of which has been implicated in the development of cataract diseases. The side chains of lysine residues are the target of both glycation and homocysteinylation, and lysine modification by the two reactions may similarly affect the structure and function of α- Cry. In this study, α-Cry was incubated with homocysteine thiolactone (HCTL), resulting in significant protein homocysteinylation, as determined with Ellman’s assay. Homocysteinylation of α-Cry resulted in the reduction in surface hydrophobicity and alpha-helix to beta-sheet transition, as observed respectively with fluorescence and circular dichroism (CD) spectroscopy. The structural alteration of homocysteinylated α-Cry was accompanied by protein aggregation, including the formation of amyloid fibrils as detected by thioflavin T (ThT) fluorescence and Congo red (CR) absorption spectroscopy. The mobility shifts of homocysteinylated α-Cry on reducing and non-reducing SDS-PAGEs suggest that disulfide cross-linking in addition to lysine modification, also plays a role in aggregation of this protein. The chaperone activities of α-Cry, namely to prevent aggregation, to assist refolding and to restore activity of thermally stressed α-glucosidase (α-Gls) were reduced after homocysteinylation. Overall, this study suggests that similar to non-enzymatic glycation, homocysteinylation of α-Cry is a risk factor for the development of cataract disorders, for instance during hyperhomocysteinemia which is linked to the various ocular pathological disorders.

We characterized the formation of amyloid fibers by two peptides derived from the CsgA sequence: R5 (133- 151) corresponding to the whole repeating unit R5 and a truncated form of this peptide called R5T (134-143). In the presence of either of the two peptides: an increase in the fluorescence intensity of Thioflavin T was observed; a shift of the absorbance of Congo red was measured; spontaneous formation of amyloid fibers was observed by polarized light as well asatomic force microscopy imaging. Large-size aggregates were observed with R5 while R5T formed fagots of individualized fibers. The infrared spectroscopy analysis revealed the presence of a greater number of intermolecular bonds for R5. In conclusion, a 10 aminoacids peptide derived from the R5 sequence was sufficient for the spontaneous formation of amyloid fibrils but not to form large-size aggregates of fibers.

Vascular endothelial growth factor-A (VEGF) is a potentially ideal angiogenic agent in tissue repair, however, various side effects still limit its application in clinical practice. If VEGF could be localized and activated in a specific region, its side effects would be minimized. A VEGF variant was designed by fusing the peptide VEGF183 (132-158), which contains plasmin and matrix metalloproteinases (MMPs ) cleavage sites, as well as extracellular matrix (ECM) binding sequences to the COOH-terminus of plasmin-resistant VEGF165 (designated as VEGF192). These were then expressed in Pichia pastoris and mouse breast cancer EMT-6 cells. Its stimulation of dermal vessel permeability in rats, mitogenic activity in cultured human umbilical vein endothelial cells (HUVECs), affinity for ECM, as well as its half-life in rats were compared with those of VEGF165. The results show that VEGF192 has weaker vessel permeabilization activity and mitogenic activity for HUVECs only at lower concentrations. It also has a longer half-life and a higher ECM-binding affinity compared with those of VEGF165. However, the plasmin-cleaved VEGF192 could stimulate HUVEC proliferation in a dose-dependent manner. Different functional peptide combinations should have potential applications for VEGF modifications and VEGF192 might be used in tissue engineering and the treatment of ischemia-related diseases.

Chemokines are members of the superfamily of cytokines involved in: (i) cell migration to sites of infection; or (ii) cellular stress during an immune response. Human CXCL14/BRAK is a monocyte-selective chemokine expressed in all normal tissues, but is also involved in the development of several cancers. We describe the expression, structural characterization and biological activity of an N-terminal truncated mutant of CXCL14, ΔCXCL14, where the first eleven residues and the two disulphide bridges were removed. We designed this species in order to analyse the biological importance of the disulphide bonds and the flexible N terminus of CXCL14 for its protein folding, stability and function. The mutant ΔCXCL14 is biologically active, as suggested by the in vitro assays with migration of pancreatic cancer cells, but also its structure is not well-fixed, as suggested by fluorescence, CD and NMR. We conclude that the disulphide bridges are important in maintaining the structure of this chemokine, but they are not necessary for the biological activity of CXCL14 species.