Current Proteomics - Volume 17, Issue 2, 2020

Urolithiasis, which is the presence of stones in the urinary tract, has long been linked with a higher risk of causing chronic kidney diseases and associated illnesses, such as diabetes-affecting 12% of the world population. This clinical condition arises due to the supersaturation of urine and alterations in the expression of cellular and urinary proteins. The renal stone mineral composition has been well understood and incorporated as a routine part of stone removal, however, the protein composition, an essential fraction of the stone matrix has been inadequately understood and not adeptly established. Stone proteomics consists of a number of techniques including crystal analysis using X-ray diffractometry and IR spectroscopy, sample purification, identification and characterization of proteins using high throughput mass spectrometric methods. However, not many studies have utilized the data obtained from these experiments to assign functional significance to associated identified proteins. Protein network analysis using bioinformatic tools such as STRING to study protein-protein interactions will enable researchers to get better insight into stone formation mechanics. Hence, a comprehensive proteomic study of kidney stone matrix will help in deciphering protein-crystal pathways generating novel information useful for clinical application.

Background: Amidases are ubiquitous enzymes and biological functions of these enzymes vary widely. They are considered to be synergistically involved in the synthesis of a wide variety of carboxylic acids, hydroxamic acids and hydrazides, which find applications in commodity chemicals synthesis, pharmaceuticals agrochemicals and wastewater treatments. Methods: They hydrolyse a wide variety of amides (short-chain aliphatic amides, mid-chain amides, arylamides, α-aminoamides and α-hydroxyamides) and can be grouped on the basis of their catalytic site and preferred substrate. Despite their economic importance, we lack knowledge as to how these amidases withstand elevated pH and temperature whereas others cannot. Results: The present study focuses on the statistical comparison between the acid-tolerant, alkali tolerant and neutrophilic organisms. In silico analysis of amidases of acid-tolerant, alkali tolerant and neutrophilic organisms revealed some striking trends as to how amino acid composition varies significantly. Statistical analysis of primary and secondary structure revealed amino acid trends in amidases of these three groups of bacteria. The abundance of isoleucine (Ile, I) in acid-tolerant and leucine (Leu, L) in alkali tolerant showed the aliphatic amino acid dominance in extreme conditions of pH in acidtolerant and alkali tolerant amidases. Conclusion: The present investigation insights physiochemical properties and dominance of some crucial amino acid residues in the primary and secondary structure of some amidases from acid-tolerant, alkali tolerant and neutrophilic microorganisms.

Introduction: Human Herpesvirus 8 (HHV-8) causes classical, endemic (African), and Acquired Immunodeficiency Syndrome (AIDS)-related Kaposi’s Sarcoma (KS), Body Cavity-Based Primary Effusion Lymphomas (BCBL), HHV-8-associated peritoneal Primary Effusion Lymphoma (PEL), and Multicentric Castleman’s Disease (MCD). HHV8 genome encodes several structural and non-structural proteins, among which vIL6 is a functional homologue of Interleukin-6 (IL-6). It has been established that vIL6 plays a vital role in HHV8 infections; also, it has been suggested that its function was mediated through gp130, rather than the gp80 (IL-6 receptor [IL-6R]). This study aimed to investigate the physicochemical and structural properties as well as the immunological features, and finally the interaction between vIL6 and IL6 receptor (IL6R) by using several bioinformatics tools which could provide both valuable insight into vIL6 protein and advantageous data for further studies on HHV8 inhibitors and new vaccines. Material and Methods: vIL6, human IL6 (hIL6), and IL6R were obtained from NCBI GenBank and Uniport, which were aligned by The CLC Genomics Workbench. "Signal-BLAST" and “predisi" were employed to define signal peptide; also, “Expasy’sProtParam” was used to predict physicochemical properties as well as "DiANNA", and "SCRATCH" predicted the disulfide bonds. “NetPhosK”, “DISPHOS”, “NetPhos”, ”NetNGlyc”, and ”GlycoEP” were involved to determine post-modification sites. To define immunoinformatics analysis, “BcePred”, “ABCpred”, “Bepipred”, “AlgPred”, and "VaxiJen" were used. “SOPMA”, “I-TASSER”, “GalaxyRefine”, and “3D-Refine” predicted and refined the secondary and tertiary structures. TM-align server was used to align 3D structures. In addition, docking analysis was done by “Hex 5.0.”, and finally the results were illustrated by “Discovery Studio”. Results: A signal peptide (1-22) was defined in the vIL6 sequences and analysis has shown that vIL6 is an acidic protein which is significantly stable in all organisms. Three Disulfide bonds were predicted and immunoinformatics analysis showed 5 distinct B-cell epitopes. vIL6 is predicted as a non-allergen protein and the majority of its structure consists of Alpha helix. TM-align pointed the significant similarity between vIL6 and hIL6 in protein folding. The high energy value between vIL6 protein and IL6R was calculated and further analysis illustrated 5 conserved regions as well as 4 conserved amino acids which had a significant role in vIL6 and IL6R interaction. Discussion: An in silico study by numerous software determined the possible interaction between vIL6 and IL6R and the possible role of this interaction in HHV8 pathogenesis and the progress of infection. These have been overlooked by previous studies and will be beneficial to gain a more comprehensive understanding of vIL6 function during HHV8 lifecycle and infections. Structural analysis showed the significant similarity between vIL6 and hIL6 folding which can describe the similarity of the functions or interactions of both proteins. Furthermore, several conserved regions in the interaction site which interestingly were highly conserved among all vIL6 sequences can be used as new target for vIL6 inhibitors. Moreover, our results could predict immunological properties of vIL6 which suggested the ability of this protein in induction of the humoral immune response. Such a protein may be used for further studies on therapeutic vaccine fields.

Background: Cell-Penetrating Peptides (CPPs), a family of short peptides, are broadly used as the carrier in the delivery of drugs and different therapeutic agents. Thanks to the existence of valuable databases, computational screening of the experimentally validated CPPs can help the researchers to select more effective CPPs for the intercellular delivery of therapeutic proteins. Arginine deiminase of Mycoplasma hominis, an arginine-degrading enzyme, is currently in the clinical trial for treating several arginine auxotrophic cancers. However, some tumor cells have developed resistance to ADI treatment. The ADI resistance arises from the over-expression of argininosuccinate synthetase 1 enzyme, which is involved in arginine synthesis. Intracellular delivery of ADI into tumor cells is suggested as an efficient approach to overcome the aforesaid drawback. Objective: In this study, in-silico tools were used for evaluating the experimentally validated CPPs to select the best CPP candidates for the intracellular delivery of ADI. Results: In this regard, 150 CPPs of protein cargo available at CPPsite were retrieved and evaluated by the CellPPD server. The best CPP candidates for the intracellular delivery of ADI were selected based on stability and antigenicity of the ADI-CPP fusion form. The conjugated forms of ADI with each of the three CPPs including EGFP-hcT (9-32), EGFP-ppTG20, and F(SG)4TP10 were stable and nonantigenic; thus, these sequences were introduced as the best CPP candidates for the intracellular delivery of ADI. In addition, the proposed CPPs had appropriate positive charge and lengths for an efficient cellular uptake. Conclusion: These three introduced CPPs not only are appropriate for the intracellular delivery of ADI, but also can overcome the limitation of its therapeutic application, including short half-life and antigenicity.

Background: Arginine deiminase of Mycoplasma hominis, an arginine catabolizing enzyme, is currently in clinical trial for the treatment of arginine auxotrophic cancers. However, some drawbacks such as instability and antigenicity have limited its application as a protein drug. Arginine Deiminase (ADI) belongs to the guanidino-group modifying enzyme superfamily. Despite differences in the primary amino acid sequences of various members of this superfamily, the folding and secondary structures are conserved in all members. Despite structural similarities, ADIs in various species have different levels of catalytic activity and physicochemical properties due to the differences in their primary amino acid sequences. Therefore, investigating and comparing sequences between different ADI producing bacterial strains could be helpful in the rational engineering of ADI. Objective: In the current research, we used an in-silico approach to characterize and classify the available reviewed protein sequences of ADI. Results: 102 ADI sequences from SwissProt database were extracted. Subsequently, based on clustering analyses, the sequence sets were divided into five distinct groups. Different physicochemical properties, solubility, and antigenicity of the enzymes were determined. Some ADI sequences were introduced as well-suited candidates for protein engineering; Lactobacillus fermentum ADI for low pI value, Mycobacterium avium ADI for high aliphatic index, Bacillus licheniformis ADI for low GRAVY index, Bradyrhizobium diazoefficiens ADI for low antigenicity and high stability index, and among Mycoplasma ADIs, Mycoplasma arthritidis ADI for high stability and aliphatic index, and Mycoplasma capricolum for low antigenicity.

Background: The role of Apolipoprotein-E (APO-E) in lipid metabolism and cholesterol transport is a key component of lipid metabolism which plays a role in diseases like hypercholesterolemia, diabetes, and cardiovascular disease. The aim of this study was to determine the genotypes, allelic frequencies, gene expression and methylation related to apolipoprotein E polymorphism in Coronary Artery Disease (CAD) patients and compare with non-CAD healthy subjects of South Indian population. Methods: The APO-E alleles and genotypes were determined by PCR-RFLP. Gene expression profiles for E3/E3 genotypes were determined using RT-PCR and methylation status was determined using Methyl Specific PCR assay in one hundred patients and an equal number of controls. Results: Four APO-E genotypes (E4/E4, E3/E3, E3/E4, and E2/E3) were identified with different allele frequency. Among these, E3/E3 genotype and E3 allele were found to be significantly higher in cases than controls. The present study showed that the mRNA expression of APO-E was up-regulated in CAD patients with E3/E3 genotype in comparison with controls. Methylation status indicated a significant association of E3/E3 genotypes with the disease. Conclusion: Different populations studied worldwide showed inherent variable frequencies of the APO-E alleles and genotypes, with the most frequent allele being E3. In this study, the APO-E genotypes E2/E3/E4 showed variable response to CAD, further, there was a significant association of E3/E3 genotypes to CAD risk; this genotype can be suggested for the diagnosis of CAD.

Background: HCV Alternate Reading Frame Protein (ARFP) is a frameshift product of HCV-core encoding. Here, we characterized specific anti-ARFP antibodies in Liver Transplant Candidate (LTC) and chronic HCV-infected patients. Methods: The ARFP gene was cloned and the recombinant protein was purified using Nickel chromatography and confirmed by western blotting. ELISA was developed using recombinant core-1a, core- 1b, ARFP-1a protein, and 99-residue synthetic ARFP 1b peptide. By several Bioinformatics tools, general properties, immunogenic epitopes, and structures of these proteins were obtained. Results: The seroprevalence of anti-core and anti-ARFP antibodies was 100% in LTC patients, but only 75.2% and 94.3% of chronic patients had evidence of anti-ARFP and anti-core antibodies, respectively. In-silico results demonstrated physicochemical features, antigen properties and potential interactors that could describe progression toward advanced liver disease. Conclusion: As the first report, the prevalence of anti-ARFP antibodies in LTC patients is of the order of 100% and titer of anti-ARFP antibody was significantly higher in LTC patients compared to chronic individuals, suggesting the possible role of ARFP in the progression toward advanced liver disease. In addition, docking analysis determined several interactor proteins such as prefoldin 2, cathepsin B, vitronectin, and angiotensinogen that have an important role in progression to chronic infection and liver disease development.