Current Proteomics - Volume 15, Issue 4, 2018

Background: Few HIV-1fusion and replication inhibitors were developed, with limited clinical applications because of their short half-life, drug resistance and cross-reactivity with preexisting antibodies in HIV-infected patients. Objective: These limitations call for new strategies in the development of next anti-HIV-1 drugs. Among anti-gp41HIV-1 inhibitors, short-peptides exhibit high antiviral activity but the mechanism of action at molecular level has not been sufficiently addressed. Method: We report potent QSAR (Quantitative Structure-Activity Relationship) models, used for biological activity prediction of novel short HIV-1 gp41 inhibitor peptides in order to: (i) validate the anti-HIV-1 activity of MT-sifuvirtide, MT-SC34EK, MT-C34 and HP23, expressed as IC50fusion and IC50replication; (ii) predict inhibitory activity of SC24EK and its MT-derivative expressed as IC50resistant HIV-1 NL4-3 variant; (iii) propose new derivatives DMT-SC22EK, DMT-SC29EK and DMT-sifuvirtide through addition of aspartic acids by induced-mutagenesis; (iv) use molecular similarity established by fingerprint models to correlate molecular spatial features with predicted biological activity of newly generated inhibitors over parent compounds. Results: We obtained good QSAR statistic parameters, demonstrating that our QSAR models are able to predict biological activity of new HIV-1 inhibitors with suitable accuracy. Conclusion: Despite acknowledged drawbacks of a reduced dataset, our results may enhance the evaluation of biological activity of new and classical synthetic peptides as anti-HIV-1agents and represent a good start for further studies in developing new antiviral drugs.

History: The freshwater cyanobacteria genus Microcystis includes some of the dominating cyanobacteria species in algae blooms and is distributed worldwide. Method: We collected three types of Microcystis, M. aeruginosa (FACHB 912), M. flos-aquae (FACHB 1028), and M. ichthyoblabe (FACHB 1294), from Lake Taihu in China to analyze their secretome in culture medium using shotgun proteomics. Observations: We identified 51, 92, and 44 unique proteins in M. aeruginosa, M. flos-aquae, and M. ichthyoblabe, respectively. The largest fraction of proteins had an unknown and hypothetical function. Results: Only five (MAE27990, MAE06090, MAE59990, MAE47530, MAE36530) of these proteins were expressed in all three Microcystis species. Gas vesicle structural proteins were present in the secretome of both M. aeruginosa and M. flos-aquae, but not in that of M. ichthyoblabe. Conclusion: This comprehensive dataset provides a novel insight into the composition and function of cyanobacterium secretome. We also analyzed proteins found in our proteomic experiment that were related to Microcystis buoyancy motility.

Objective: The aim of this study was to conduct a comparative proteome quantitation of Vibrio parahaemolyticus in response to cold stress, demonstrating specifically characteristic changes at proteomic level, and to analyze the possible potential risks based on cold-stressed strains. Method: Comparative proteome quantitation analysis based on Stable Isotope Labeling by Amino Acid in Cell Culture (SILAC) is combined with high resolution mass spectrometry, which minimizes errors and contributes to the higher accuracy compared with chemical or label-free approaches. Results: A total of 1182 proteins were identified, among which 601 were quantified. Catalase/peroxidase HPI, aromatic amino acid aminotransferase and 16S rRNA methyltransferase were significantly upregulated in spite of the inconsistency of some proteins expression and its corresponding mRNA expression. Glutamine synthetase, asparagine synthetase and enzymes related to pentose phosphate pathway increased as well. Some proteins associated with glycolysis, tricarboxylic acid cycle, transcription and translation were downregulated. Some proteins related to DNA synthesis were almost unchanged. Conclusion: The results demonstrated that most upregulated proteins enhance the stress resistance and self-adaptation. We assume that there may be correlation between upregulation and potential risks on seafood. Most downregulated proteins contribute to energy conservation and self-protection in order to resist various negative stresses. This study provides information on specific differences of proteome and offers some new clues to the differentiation and analysis of V. parahaemolyticus under cold stress.

Background: Wounding is the major trigger factor for agarwood formation in Aquilaria malaccensis. To date, the mechanism of agarwood formation in Aquilaria has been extensively explored through anatomy and genomics approaches; however, at the protein level, it is yet to be reported. Objective: A comparative proteomics analysis was carried out to identify proteins responsive to wounding in A. malaccensis, using two-dimensional gel electrophoresis (2-DE) coupled with Matrix- Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Method: Proteins were extracted from A. malaccensis tree stems at 0, 6, 12, and 24 h after wounding and separated by 2-DE, sequenced using MALDI-TOF MS and identified through UniProt/Swiss-Prot protein database. Results: Changes in differentially displayed proteins were observed. A total of 15 protein spots were identified reproducible under 2-DE, of which only two protein spots showed differences in expression affected by the wounding treatment in a time-dependent experiment. They were predicted as Malate Synthase (MS) and the reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) quinone oxidoreductase subunit 2 B. Both proteins were directly and indirectly related to wounding treatments in plants and may be related to agarwood formation mechanism. Conclusion: As Aquilaria proteome is not available to date, this first report on differential protein expression in response to wounding could serve as a useful information towards understanding agarwood biosynthesis at the protein level.

Background: Channa striata is a freshwater snakehead fish that is abundantly available in South East Asia. Proteins available in the C. striata's mucus have been confirmed to contribute towards wound healing enhancement, yet the biologically active proteins within are poorly understood. Objective: The main objective was to identify the potential proteins in the C. striata's mucus that possibly contributed to the wound healing enhancement. Besides, the post-translational modifications (PTMs) were included as well to complement the protein list for better understanding of those proteins. Method: Proteins were fractionated using liquid fractionation system prior to mass spectrometry (MS) analysis. LTQ-Orbitrap Velos Pro mass spectrometer was utilised to identify the proteins available in the sample. The data generated were then compared with the Uniprot Actinopterygii database to identify the proteins and PTMs available. Results: Fifty-three and 120 unique proteins in the crude mucus sample and fractionated sample were identified respectively. Interesting proteins such as histones, ribosomal proteins, protein S100, heat shock protein, proteolytic enzymes, heparin cofactor II and group of uncharacterized proteins were identified and discussed thoroughly. Besides, 39% of the proteins identified were post-translational modified. Methylation, hydroxylation, acetylation, ubiquitin and biotinylation were the PTMs detected. Conclusion: The proteins and PTMs profiling of the C. striata mucus serve as a preliminary report and foundation for future in-depth exploration of the species. These results serve as a fundamental preliminary report on the mucus of C. striata, which provides insights for harvesting the bio-active proteins for drug production and medication purposes in the future.

Background: Semenogelin II protein plays a major role in human fertility. Semenogelin II (SGII) is a seminal plasma protein that participates in sperm clotting or gelling in binding with Zn+ ions. Proteolysis of this protein is done by a prostate-specific antigen, and further releases the entrapped sperms after ejaculation. Aim and Objective: To identify the presence of semenogelin II protein in the seminal plasma of various categories and to characterize the protein 3D structure by in silico methods. Methods: Total 137 semen samples have been collected from the Bangalore Assisted Conception Center, Andrology Department, for this research. SDS-PAGE was done to identify the presence of semenogelin II protein. In silico characterization was performed by structural modelling using the ITASSER server. In addition, the structural refinement and protein behaviour were studied by MD simulations using Gromacs. Results: In this research, we identified the presence of semenogelin II in normospermia and oligospermia categories and not in the case of asthenospermia and oligoasthenospermia. This result suggested that the absence of this protein in these categories correlated with motility issues. The structural model of human semenogelin II was generated from the template 4UXV (cytoplasmic domain of the bacterial cell-division protein EzrA) based on the C-score. The predicted model was evaluated by the Ramachandran plot with 86.4% in favoured regions and the overall model quality with a quality index score -3.03, matching with experimental structures. Moreover, MD simulations results showed that the native structure was stable from trajectory analysis. Conclusion: Our study concludes the presence of semenogelin II protein in normospermia and the absence in motility-related infertile categories. Furthermore, the plausible structure of the protein was characterized by in silico methods that can be used for further structure and functional annotation. Thus, semenogellin-II can be a potent marker for the diagnosis of human male infertility.

Aims: Secretomics will lead to an increase in understanding of how cells combine the concerted action of secreted protein networks with their internal and external environments. Methods: Shotgun proteomics were used to analyze Synechocystis sp. PCC 6803 secreted proteins and results showed the identification of unique proteins, 13 in normal medium and 7 in phosphate-depleted BG-11 medium, respectively. Results: Six proteins were secreted under both normal and phosphate-depleted conditions (slr0513, slr1667, sll0654, sll1694, sll1578, sll1009), but only one secreted protein (Sll1694) was commonly detected by shotgun proteomics in the present study and previous gel-based studies. Both the number of secreted proteins and their classifications decreased in phosphate-depleted conditions. The peptide count of hypothetical protein codified by slr1667 decreased significantly under phosphate-depleted conditions, whereas alkaline phosphatase protein codified by sll0654 increases significantly under phosphate- depleted conditions. Results of quantitative real-time PCR showed that the transcript level of alkaline phosphatases (sll0654) was higher in phosphate-depleted BG-11 medium than in normal medium, and the transcript level of proteins related to twitching motility decreased significantly. Conclusion: This comprehensive data set provides novel insights into the composition and function of the cyanobacterium secretome and improves understanding of the biological processes at work between cyanobacterium and its environment.

Background: Constitutive Triple Response 1 (CTR1) is a Raf-like kinase belonging to the Mitogen-activated protein kinase kinase kinase family with central roles in cell signaling. Objective: This study was carried out in order to test if the naturally occurring plant lipids, phosphoglycerides, affect the activity of CTR1. Methods: The three-dimensional structures of the protein kinase domain of CTR1 from coffee and various species of Solanaceae, including tomato, potato, eggplant and pepper, were generated by the homology modeling technique of structure prediction using Modeller. CTR1 protein kinase sequences from the selected species were used for structure prediction in reference to the highly similar Arabidopsis CTR1 as structure template. These models were validated for their three-dimensional conformation after necessary refinements to allow the residues to fall in acceptable ranges according to the Ramachandran plot as well as the confinement of spatial arrangements as per allowed Errat and Verify3D scores. Protein-lipid molecular dockings were performed using AutoDockTools. Results: The generated models were found to possess high similarities at secondary as well as tertiary levels. Refinements and optimization of the models resulted in validated protein structures for all the homologs with scores in acceptable ranges. Docking of the AtCTR1 protein structure with phosphoglycerides produced significant interactions forming both hydrogen bonds as well as hydrophobic interactions with the active site residues of CTR1 which are also involved in various important cellular functions. Conclusion: The strong lipid-protein interactions suggest that the lipids by binding to the active site of CTR1 can affect the roles it plays in various pathways it is involved in.