Current Proteomics - Volume 14, Issue 4, 2017

Background: At the cellular level, normal chromosome segregation is ensured by the intrinsic mechanics of mitosis and the proper functioning of the error-checking spindle assembly checkpoint (SAC). Protein Mad1 (the mitotic arrest-deficient), an important SAC component, plays a crucial role in avoiding cellular aneupoidy, a state leading to genetic diseases such as cancer or bipolar disorder. Objective: To clarify the role of aneuploidy in genetic diseases, a number of wild type (wt) and mutant spindle checkpoint proteins have been studied, but till now the process is not well understood. Method: Here, we report a number of 32 Mad1 mutants (8 already known to induce aneuploidy or Mad1 dimer destabilization and 24 de novo mutants designed by us) comprising mutation in the carboxi-terminal domain (CTD) represented by residues 598-718. Their molecular features (electronic, steric, and also the descriptors derived directly from amino acids sequence: counts of atom and bound types, dihedral angles) were calculated and compared by structure-activity relationships methods (SAR) in order to elucidate their possible contribution to aneuploidy. Results: Our results suggest that some molecular descriptors of Mad1-CTD mutants and wt Mad1, like accessible solvent surface areas and its derivatives, could be important for predicting aneuploidy induced by Mad1 improper function. Conclusion: It was found that molecular descriptors of Mad1 wt and mutants evaluated here are important resources for upcoming computational studies focused on aneuploidy, provided kinetic data about Mad1- kinetocore and/or Mad1-Bub1 interactions

Background: RNA binding Proteins (RBPs) influence the structure and function of RNA and play a very important role in many biological processes like transcription, translation, cell cycle and gene silencing mechanisms. Objective: Structural studies of spliceosome machinery and proteins involved in gene silencing mechanisms are of prime importance as it is poorly understood in Neurospora crassa. In view of this, the present study was undertaken for in silico identification of RBPs in N. crassa and making their catalog based on the function. Method: Briefly, by in silico approach, the comparative proteomics strategy was used to identify the RBPs. The network analysis was performed based on the STRING interactions, docking studies between protein and RNA by NPdock whereas ClusPro was used to infer protein-protein docking. Results: A total of 313 RBPs in N. crassa were identified and pathway analysis has revealed about 98 RBPs which are involved in different known pathways based on the function. Based on the function 12 RBPs were found to be observed in one of the most critical mechanisms, the post transcriptional gene silencing (PTGS) in Neurospora. Majority of the RBPs were involved in spliceosome mechanism and mRNA related pathways, hence the RBPs involved in these pathways were mapped and modeled by merging the interlinking pathways viz., splicing, RNA transport and degradation. From the interaction analysis, the hub protein was found to be NCU06318. This hub protein was docked with its best interaction partner NCU11357(a cell cycle control protein). The PAZ domain containing PTGS proteins which can associate with dicer like proteins were observed in qde-2 and hence were docked with dcl-2. Conclusion: The protein-protein interactions were depicted as strong non-covalent interactions which indicates that these proteins are associated in the regulation. In this study, a detailed report of the protein (RBP) and snRNA structures with their interactions are presented which enables us to understand the role of RBPs in spliceosome pathway and gene silencing mechanisms of N. crassa.

Background: Methamphetamine is a well-known psychostimulant, which exerts its influence on the central nervous system. To identify the changes caused by this drug at the molecular level, several proteomic studies were performed, their results were published in available literature. Objective: We summed up the results of available proteomic data on the topic of methamphetamine influence on central nervous system published up to date. Our goal was to reveal proteins, which were indicated several times by different proteomics studies, and thus might be considered as validated. Method: We used bioinformatic analysis tools to estimate the interactions between proteins. It was used: WEB-based GEne SeT AnaLysis Toolkit (WebGestalt), KEGG Database, Gene Ontology (GO) analysis and STRING database. Results: As a result, a list of 80 proteins was generated. After bioinformatic analysis of their functions and interactions it found out that, that many of them are engaged in energy metabolism, mainly in glycolysis/gluconeogenesis, TCA cycle, and respiratory electron transport. Conclusion: Such meta-analysis of the results generated by various laboratories might be the first step to broaden our knowledge about molecular pathways involved in methamphetamine addiction and neurotoxicity.

Background: The ribosome binding site (RBS) containing Shine-Dalgarno (SD) sequence in prokaryotes plays an important role in identification of the translation initiation site within mRNA by ribosome. Objective: Our study aimed to improve the target protein production under translational level in prokaryotes by engineering of RBSs. Method: Two RBSs were designed in Streptomyces coelicolor M145, Sco-RBS* with an SD sequence which is completely complementary to 3'-end of 16S RNA and Sco-RBS0 with an SD sequence which is completely non-complementary to 3'-end of 16S RNA. The enhanced green fluorescent protein (EGFP) was used as a reporter, whose gene was transcribed and translated under the control of Sco- RBS*, Sco-RBS0, and a native RBS Sco-RBSactI in recombinant S. coelicolor strains. Results: Replacement of Sco-RBSactI with Sco-RBS* resulted in increase of both EGFP production and the ratio of EGFP to EGFP mRNA by 2.67-fold and 6.07-fold, respectively. Replacement of Sco- RBSactI with Sco-RBS0 resulted in decrease of both EGFP and the ratio of EGFP to EGFP mRNA by 4.35-fold and 2.18-fold, respectively. Conclusion: We provided a method to increase protein production at the translational level in Streptomyces.

Background: Water buffalo (Bubalus bubalus) are domesticated animals of economic importance in many Asian countries, whereas the basic helix-loop-helix (bHLH) transcription factors (TFs) have been analyzed in the genomes of many eukaryote organisms. However, the bovine bHLH TFs have not been characterized and their specific functions remain unknown. Objective: To better characterize and functionally understand the bovine bHLH TFs, we systematically analyzed and characterized putative bHLH proteins in the ongoing genome project of water buffalo. Method: We carried out multiple BLAST searches, phylogenetic analysis, conserved site analysis, enrichment analyses of gene ontology and pathway annotations, and other functional annlyses with KOBAS, Blast2GO, and DAVID Bioinformatics Resources. Results: Among the identified 105 bHLH TFs, phylogenetic analyses classified 103 bHLH TFs into 43 families with 60, 77, 25, 20, and 9 protein sequences in the higher-order groups A, B, C, E and F, respectively. The remaining 8 protein sequences of 3 putative bHLH TFs were categorized as “orphan proteins” and annotated as SOHLH2, MGA, and TCFL5. Furthermore, these bovine bHLH proteins were characterized as significantly enriched in 361 significant (corrected P value ≤ 0.05) and 215 very significant (corrected P value ≤ 0.001) Gene Ontology (GO) annotations as well as 18 significantly (corrected P value ≤ 0.05) and 12 very significant (corrected P value ≤ 0.001) enriched cellular pathways and other categories. Conclusion: The present research provides new data and a good reference for further functional and evolutionary investigations on mammalian development using water buffalo as a model animal.

Background: Halophiles, which thrive under saline/hypersaline habitats and retain their structural and functional integrity under high salt conditions, present an interesting model system for understanding their global proteome under different salinity. Objective: The present work compares the intracellular proteome profiles of two moderately halophilic Gram positive Bacillus sp. EMB9 and Gram negative Marinobacter sp. EMB5 in different salt concentrations. Method: Moderately halophilic bacterial cells were incubated in the presence of varying NaCl concentration and their growth profile monitored. Their responses towards the altered saline environment were further evaluated using a trizole-based protein extraction followed by 2D gel electrophoresis. The change in protein spot intensities between the control and treated samples was analyzed using the Image Master Platinum 7.0 software and was a measure of the protein expression levels. Results: Notable global changes in the intracellular bacterial proteome were observed when grown under optimal (5%, w/v) and low NaCl concentrations. Protein expression levels decreased by 21% and 32% under low salinity in case of Bacillus sp. EMB9 and Marinobacter sp. EMB5 respectively. Of the total number of 235 protein spots detected in case of Bacillus sp. EMB9, 91 were commonly expressed, 12 new protein spots were observed while 41 proteins spots were completely suppressed. In case of Marinobacter strain, 83 total protein spots were detected, of which 20 were commonly expressed, 36 new protein spots appeared and expression of 27 protein spots was suppressed under low salinity. The role of suppressed proteins could be specific to the presence of higher salinity, and associated with the regulation of saline/environmental stress. Proteins exclusively expressed in low salinity may be responsible for survival at low salt concentration. Conclusion: The study provides preliminary understanding on the protein diversity in moderately halophilic Gram positive and negative bacteria under varying salinity. The work is deemed to add to the knowhow of osmoadaptive strategies in moderately halophilic bacteria.

Background: Solubility of recombinant therapeutic enzymes is one of the major purpose their production in bacterial host The factors such as metabolic system, a cellular stress response of the bacterial host and formation of inclusion bodies cause undesirable expression results. One method to overcome this problem is secretion of recombinant protein into the extracellular growth medium by selection of proper signal peptides. Reteplase (Retavase) is a serine protease simulating endogenous tissue plasminogen activator (t-PA), fibrin-specific plasminogen activator, playing role in thrombolysis and fibrinolysis (clot lysis) by converting plasminogen to plasmin. Objective: The aim of this study was to evaluate several signal peptides in order to selection the proper one which suitable for excretory production of the reteplase. Method: The presence and cleavage site location in signal peptides was predicted by SignalP 4.1. The prediction of secretory pathway of signal peptides was performed using high accurate database. Several critical features of the signal peptide were evaluated applying various online web-servers such as ProtParam and Solpro with high sensitivity and specificity. Finally, the selected signal peptides were compared and the one which was appropriate candidate for high yields production of secretory reteplase in the E. coli host was defined. Results: Among total 30 signal peptides, 6 of them, CHIS_BACSU, MPT53_MYCTU, PET_ECOLX, PIC_ECOL6, PIC_ECLOX, TSH_ECOLX were omitted. CPLR_DESHA, AGAR_ALTAT, CDGT_BACS2, PAG_BACAN could be considered as proper candidate for efficient extracellular production of reteplase in E. coli. Conclusion: Theoretical analysis and evaluation of signal peptides by using computational tools can be acceptably carried out, but these results must be established via practical effort.