Current Proteomics - Volume 14, Issue 3, 2017

Background: Polyploid breeding is a powerful approach for Populus genetic improvement because polyploid trees have valuable characteristics, including better timber quality and a higher degree of stress resistance compared with their full-sib diploids. However, the genetic mechanism underlying this phenomenon remains unknown. Objective: To better understand the proteomic changes between Populus allotriploids and diploids, we examined the proteomic profiles of allotriploid and diploid Populus by iTRAQ labeling coupled with two-dimensional liquid chromatography and MALDI-TOF/TOF mass spectrometry (MS). Method: iTRAQ labeling coupled with two-dimensional liquid chromatography and MALDITOF/TOF mass spectrometry (MS). Results: Between the Populus allotriploid and the full-sib diploid, 932 differentially expressed proteins (DEPs) were identified. These DEPs were primarily involved in stress, defense, transportation, transcriptional and/or translational modification, and energy production. The pathway analysis indicated that most of the DEPs were implicated in carbohydrate transport and metabolism, nitrogen metabolism and glycolysis, and the ribosome assembly pathway. These data suggest high protein divergence between Populus allotriploids and diploids, allotriploids and diploids, and rapid changes during hybridization. Conclusion: The results provide new data for further understanding of the mechanisms of polyploid trees that generally display increased height growth compared with their full-sib diploids.

Background: Pullulanase is broadly used in saccharification process for glucose, maltose, maltotriose, and fructose production. The in silico investigation can be a useful approach to screen an enzyme with suitable characteristics for application in industry. Objective: The aim of this study was an in silico characterization and functional analysis of pullulanases from different Bacillus species. Method: A total number of 38 amino acid sequences of pullulanase were selected from different Bacillus species, and were investigated from the view of physiochemical properties, phylogenetic relation, and domain architecture. Results: Computational analyses showed that the pI values of the pullulanases were in the range of 4-7, which indicated that the enzyme is active in acidic to neutral environments. The pI value greater than 7 belongs to pullulanase of Bacillus psychrosaccharolyticus and Bacillus flexus (8.19 and 8.55, respectively), which indicated the basic properties of the enzymes in these two species. Their aliphatic indexes were in the range of 68-92. The pullulanase of Bacillus vireti had the highest aliphatic index and thermostability value. The enzymes half-lives were more than 16 h, except for Bacillus subtilis and Bacillus amyloliquefaciens (<5 h). All species had several domains with common α-amylase catalytic motif containing conserved Glu/Asp residues. Four domains were observed in the tertiary structure of Bacillus vireti pullulanase that were built by Swiss-model server. Two domains, CBM and α-amylase, were conserved among the studied species. Conclusion: Our study revealed that the Bacillus vireti pullulanase had the higher thermostability and half-life compared to the other species. Therefore, pullulanase of Bacillus vireti is suggested as a suitable candidate for industrial usages. In this case, investigating the physiochemical characterstics, phylogenetic relation, secondary and tertiary structures of the enzyme can help us to improve the properties of enzyme that is important for industrial applications.

Background: Meta-prediction seeks to combine the strengths of multiple predicting programs with the hope of improving the predicting performance surpassing that of all existing predictors. As increasing numbers of predicting programs have been developed in this field, there is an urgent need for effective meta-prediction strategies. Objective: In this paper, we apply meta-prediction for the identification of protein-protein interaction (PPI) sites which is still a challenge task in computational biology. Method: In this paper, six PPI sites predictors, CRF, SSWRF, DC-RF-RUS-PF, LORIS, SPPIDER and SPRINGS, are integrated to construct meta-predictors using several methods, including unweighted voting, unreduced weighted voting, reduced unweighted voting and weighted voting strategies. Results: PPIsMeta, the meta-predictor produced by using weighted voting strategy with parameters selected by restricted grid search, performs the best for predicting the PPI sites. Its accuracy and Matthew's Correlation Coefficient reach up to 70.6% and 0.26, respectively. Compared to the best individual element predictor (SSWRF), the predictor proposed in this study achieves a significant improvement in Matthew's Correlation Coefficient of 0.036, and an improvement in accuracy of 5.8%. Conclusion: The experimental results demonstrate that PPIsMeta is a powerful tool for predicting PPI sites. Compared with the existing methods, we find that our tool shows greater robustness in accuracy and Matthew's Correlation Coefficient. PPIsMeta is available to the public at http://59.73.198.144:8080/EPuL/download.jsp.

Background: The fibroblast growth factor (FGF) family is essential to tissue development and stem cell differentiation; therefore, there is increasing interest in the clinical application of FGFs, resulting in increasing demand for active recombinant human FGFs (rhFGFs). Objective: To improve the production of rhFGF18, we determined the optimal culture conditions required to maximize rhFGF18 protein expression E. coli. Method: Competent cell type, inducer concentration, induction temperature, and induction time for the optimal expression of rhFGF18 were analysed. Results: The rhFGF18 protein expression was highest in BL21 competent cells at an L-arabinose induction concentration of 0.1% (w/v), an induction temperature of 10 °C, and an induction time of 6 h under the control of an araBAD promoter introduced using the pBAD-HisA vector. Conclusion: Under optimal culture conditions, the rhFGF18 protein could induce osteogenic differentiation by increasing the alkaline phosphatase activity and mineralization activity. The approaches described herein may be useful for producing active rhFGF18 to meet the increasing demands for its pharmacological application, allowing for its future clinical use.

Background: The relevance of bioinformatics is being realized in the era of genomics, assisting in genome-wide identification and characterization of putative gene families of different enzymes for diverse industrial applications. Therefore, in the present manuscript, Mn peroxidases protein sequences from different fungal sources were subjected to bioinformatics assessment for homology search, multiple sequence alignment, phylogenetic tree construction and motif search. Objectives: The available protein sequences representing enzymes is being utilized to decipher the sequence-structure-functional relationship with the aid of bioinformatics tools. In-silico analysis of protein sequences of several industrially important enzymes with an aim to improve the catalytic efficiency, thermostability, structural prediction and validation have been reported in recent years. Method: A total of 78 full length protein sequences representing manganese peroxidases from diverse fungal sources have been retrieved from GenBank and subjected to in-silico analysis for homology search, physio-chemical attributes, multiple sequence alignment, phylogenetic tree and motif assessments. Multiple accessions from same fungal sources like 9 accessions of Puncularia strigoso zonata were included to get an insight into the structural and functional diversity of the Mn Peroxidases. Results: The physio-chemical attributes showed variability in the amino acid residues ranging from 341 to 613, molecular weight from 31939.035 to 58082.9 daltons while the pI ranging from 3.8 to 5.39. Thermostability and hydrophilic nature of these proteins was predominantly observed as revealed by comparatively high value of aliphatic index and negative value of GRAVY respectively. The phylogenetic tree revealed distinct clusters for different fungal genus and multiple accessions representing the same genus were grouped together indicating sequence level similarity. Conclusion: Five motifs were uniformly observed among all the sequences and these revealed similarities to Plant_ peroxidase_family. The plant peroxidase-like superfamily is reported to be prevalent in three kingdoms of life and know to carry out a variety of biosynthetic and degradative functions.

Objectives: Hypertension exerts a chronic tensile stretch on the endothelial lining of the inner layer of blood vessels and is thought to cause cellular injury leading to numerous pathologies. Studies have indicated that tensile stretch could alter the phenotype and cellular functions of the cell, depending on the stretch magnitude applied. However, the molecular mechanisms of the cellular injury under these conditions remain unclear. Method: In order to investigate the protein changes potentially involved in hypertension, human cerebral microvascular endothelial cells were subjected to a simulated physiological (5%) and pathological (20%) cyclic stretch for a period of 2 hr or 18 hr on fibronectin-coated silicone cubes followed by a quantitative label-free proteomics experiment on cell lysates. Result: Data analysis demonstrated that proteins involved in structural activity were significantly up-regulated in the 20% condition at 2 hrs notably microtubule actin cross-linking factor 1 [MACF1 (+24.6 fold)] and tubulin alpha chain 3 [TUBAL3 (+8.0 fold)]. Similarly, proteins that have been previously observed to be altered in clinical aneurysm formation such as titin [TTN (+60.4)] and apolipoprotein B-100 [APOB (+21.3 fold)]) were also found to be up-regulated at 20% stretch for 18 hrs. Protein interaction network analysis suggested that the signaling pathway involving nuclear factor-kappa B (NFkB) may the main protein network affected by shorter stretch conditions. Compensatory inflammatory processes may have commenced during early exposure to stretch. Conclusion: This study provides a basis for understanding early and long-term molecular changes that may lead to vascular dysfunction as a consequence of pathological stretch.

Objectives: Cyclophilin D (CypD) is a chief regulatory protein of the necroptosis pathway involved in various neurological disorders, and ablation/inhibition of this protein confers neuroprotection. Current in silico drug design strategies employ multiple structures of a protein target since they enable the identification of diverse inhibitor molecules. However, structure-based drug design against a protein target becomes challenging if it contains numerous known structures with varying ligand interactions. Considering all these structures for virtual screeing of database compounds would be inappropriate in view of the computational resources that might be demanded. Therefore, identifying appropriate structures with varied binding site conformations is of utmost importance in order to identify inhibitors with diverse scaffolds. Method: In the present study, clustering of a large number of CypD structures was carried out by comparing the pharmacophores derived from their binding sites. A representative structure from each cluster was adopted to build an ensemble pharmacophore that was further employed in dual ensemble screening of database compounds. Results: Two compounds that exhibited better docking scores, compared to the already reported CypD inhibitors, formed stable complexes and desirable interactions with the protein during molecular dynamics (MD) simulation.

Background: Hepatic cirrhosis is associated with a variety of hemodynamic and hydrodynamic abnormalities, including renal dysfunction with salt and water retention leading to ascites and dilutional hyponatremia. Objective: In the present study, a proteomic analysis of the kidney of rats subjected to bile duct ligation (BDL) has been carried out in order to identify the proteins altered under this condition. Method: The experiments were performed at the fourth week after BDL, and the protein mixtures were separated by two-dimensional gel electrophoresis using the differential in-gel system(DIGE). An internal standard composed of equal amounts of the above 12 extracts was labeled with Cy2, and loaded onto six gels. Results: More than 4,000 proteins were labeled and numbered, and around 60 proteins were differentially expressed in the kidneys of BDL rats as compared to the controls, as analyzed by De-Cyder software. The respective spots were excised and digested with trypsin for MALDITOF/TOF peptide mass fingerprinting analysis. Some of the proteins down-regulated in the kidney of the BDL rats were Ezrin, Moesin and Regucalcin, whereas Annexin-4, Cathepsin B and Heat Shock Proteins were significantly up-regulated. Discussion: The relationship of these proteins to the systemic and renal abnormalities seen in BDL rats is discussed.

Objectives: The main goal of this study was to decipher the role of CSA homologues in the DNA damage repair machinery of Arabidopsis thaliana. Method: Bioinformatics tools were used in this work to analyse DNA Damage-Binding proteins; MSA, Phylogenetic tree construction, 3-D structure prediction, domain analysis, subcellular localization prediction, interactome analysis and docking sites. Result: The pairwise alignment from Clustal Omega showed 92% similarity between AtCSA1A and AtCSA1B proteins, whereas 34 mismatches have been detected. The study constructed a phylogenetic tree using AtCSA1A and AtCSA1B orthologs from different plant species. It depicts the conservation of the protein throughout the plant kingdom. Furthermore, Prosite online tool available on ExPASy site was used to make the sequence logo [32]. These results showed conserved region of CSA and gave additional information on the quantity of certain residues in gene positions. The 3D structure analysis of AtCSA1A and AtCSA1B was visualized using PyMol and validated with Ramachandran plots. AtCSA1A consisted of 2 alpha helices and 24 beta sheets, while AtCSA1B was bulkier with 4 alpha helices and 26 beta sheets. Domain analysis revealed that both AtCSA1A and AtCSA1B contain five WD-40 repeat (WDR) domains. WDR, also known as beta transduction repeat is made up of 40 amino acids' sequences and usually terminates with Trp-Asp [WD] residues. Interactome analysis revealed that both AtCSA1A and AtCSA1B interact with DDB1A, CUL4, ERCC1, CHR8, UVH3, UVH6, XPB1 and XPB2 proteins. The docking site analysis of AtCSA1A interaction with ERCC1 and DDB1A; and ERCC1, CHR8, DDB1A interaction with AtCSA1B was identified. Significantly, residues ‘LATASADSRVKLWDVR’, were found, also known as WDxR motif that are involved in the interaction of AtCSA1A and ERCC1 proteins. Additionally ‘LATASADSRVKL’ motif was found in the docking sites between AtCSA1A-DDB1A, AtCSA1B-CUL4 and AtCSA1B-DDB1A. Conclusion: Two homologues of Cockayne Syndrome A were present in the Arabidopsis thaliana. AtCSA1A and AtCSA1B played important role in the DNA repair mechanism. This study hypothesized on the potential additional roles of CSA proteins in the DNA repair. 3D structure analysis, identification of docking sites and interactome analysis of AtCSA1A and AtCSA1B proteins reported in this study, revealed the complexity of DNA repair mechanism in plants. However, additional experimental investigations are needed to confirm these interactions and their physiological relevance.