Current Proteomics - Volume 17, Issue 1, 2020

Background: Staphylococcus aureus (S. aureus), an important pathogen, causes a wide range of infections in human starting from food poisoning to septicemia. It affects the host cells with various exotoxins, known as virulence factors, which are synthesized in growth phase-dependent manner of the bacteria. S. aureus has been reported to become resistant to antibiotics rapidly. Among two common clinical isolates, Methicillin-sensitive S. aureus (MSSA) and Methicillin-resistant S. aureus (MRSA), MRSA pose major problems across hospitals around the world. Objective: The objective of the present study was to profile the exoproteins of Methicillin-sensitive S. aureus (ATCC 25293) and subsequently to establish a proteomics-based method of characterization of S. aureus that is crucial in treating hospital-acquired infections. Methods: We used two-dimensional nanoLC/ESI-MS based proteomic platform to characterize and quantify the exoproteins isolated from Methicillin-sensitive S. aureus (ATCC 25293) strain. Results: A total of 69 proteins were identified from extracellular proteome pool of ATCC 25293 strain that includes 18 extracellular proteins, 40 cytoplasmic proteins, 2 membrane proteins, 3 cell wall proteins and 6 uncharacterized proteins. Conclusion: We propose that this mass spectrometry-based proteomics method of characterization of exoproteins might be useful to identify S. aureus strains that are resistant to antibiotics.

Background: The Cytochromes P450 (CYPs) have an essential role in the oxidation of endogenous and exogenous molecules. The CYPs are identified in all domains of life, but the CYP152A1 from Bacillus subtilis is specially considered for clinical and industrial applications. The molecular cloning of a new type of CYP from Bacillus subtilis was reported, previously. Here, we describe the hidden layer of biological information of the CYP152A1 enzyme, which can help researchers for better understanding of enzyme application. In this study, four rare codons of enzyme, including Arg63, Arg187, Arg276, and Arg338 were identified and evaluated using the bioinformatics web servers. Methods: Through in silico modeling of CYP152A1 via the I-TASSER server, the above-mentioned rare codons were studied in the structure of enzyme that may have an important role in the proper folding of CYP152A1. In the following, the substrate binding site of CYP152A1 was studied by AutoDock Vina, and the heme and palmitic acid were considered as the substrates. Results: The results of docking study elucidated the Arg242 in the active site is closely related to the substrate binding site of CYP152A1, which help us to further clarify the mechanism of the enzyme reaction. Conclusion: Studies of these hidden information’s can enhance our understanding of CYP152A1 folding and protein expression challenges. Moreover, identification of rare codons can help in the rational design of new and effective drugs.

Background: Cervical cancer arises from the cervix and it is the 3rd most diagnosed malignancy and a foremost cause of cancer-related death in females. On the other hand, the expressions of EGFR and p53 are two important proteins observed in various studies on cervical cancer. Objective: The study aims to evaluate the beneficial effect of radiotherapy based on the regulation of p53 and EGFR gene in patients with cervical cancer. Methods: In this investigation, the regulation of important molecules responsible for cancer cell proliferation and DNA repair in the cervical cancer cell line was evaluated. The study comprises of an evaluation based on clinical study design from the malignant biopsies of 15 cervical cancer patients. The patterns of expression for the p53 gene and Epidermal Growth Factor Receptor (EGFR) were evaluated in DoTc2 and SiHa cervical cancer cell lines using clonogenic assay, western blotting and immunohistochemistry techniques from the malignant biopsies of the 15 patients. Results: The study observed that the regulation of p53 and EGFR was very weak after the exposure of the radiation. In addition, the expression of p53 and EGFR was observed in malevolent biopsy samples after radiation with a dosage of 1.8 Gy radiations. Additionally, the expression of p53 and EGFR was able to induce by a single dose of radiotherapy in the malignant biopsies whereas it was unable to induce in DoTc2 and SiHa cervical cancer cells. Conclusion: The study observed that radiation exposed cancer cell lines modulates the expression of p53 and EGFR gene. The study also highlights the gap between in vitro experimental models and clinical study design.

Background: The firefly luciferase enzyme is widely used in protein engineering and diverse areas of biotechnology, but the main problem with this enzyme is low-temperature stability. Previous reports indicated that surface areas of thermostable proteins are rich in arginine, which increased their thermal stability. In this study, this aspect of thermophilic proteins evaluated by mutations of surface residues to Arg. Here, we report the construction, purification, and studying of these mutated luciferases. Methods: For mutagenesis, the QuikChange site-directed mutagenesis was used and the I108R, T156R, and N177R mutant luciferases were created. In the following, the expression and purification of wild-type and mutant luciferases were conducted and their kinetic and structural properties were analyzed. To analyze the role of these Arg in these loops, the 3D models of these mutants’ enzymes were constructed in the I-TASSER server and the exact situation of these mutants was studied by the SPDBV and PyMOL software. Results: Overall, the optimum temperature of these mutated enzymes was not changed. However, after 30 min incubation of these mutated enzymes at 30°C, the I108R, T156R, N177R, and wild-type kept the 80%, 50%, 20%, and 20% of their original activity, respectively. It should be noted that substitution of these residues by Arg preserved the specific activity of firefly luciferase. Conclusion: Based on these results, it can be concluded that T156R and N177R mutants by compacting local protein structure, increased the thermostability of luciferase. However, insertion of positively charged residues in these positions create the new hydrogen bonds that associated with a series of structural changes and confirmed by intrinsic and extrinsic fluorescence spectroscopy and homology modeling studies.

Background: The 1,4-alpha-glucan branching protein (GlgB) plays an important role in the glycogen biosynthesis and the deficiency in this enzyme has resulted in Glycogen storage disease and accumulation of an amylopectin-like polysaccharide. Consequently, this enzyme was considered a special topic in clinical and biotechnological research. One of the newly introduced GlgB belongs to the Neisseria sp. HMSC071A01 (Ref.Seq. WP_049335546). For in silico analysis, the 3D molecular modeling of this enzyme was conducted in the I-TASSER web server. Methods: For a better evaluation, the important characteristics of this enzyme such as functional properties, metabolic pathway and activity were investigated in the TargetP software. Additionally, the phylogenetic tree and secondary structure of this enzyme were studied by Mafft and Prabi software, respectively. Finally, the binding site properties (the maltoheptaose as substrate) were studied using the AutoDock Vina. Results: By drawing the phylogenetic tree, the closest species were the taxonomic group of Betaproteobacteria. The results showed that the structure of this enzyme had 34.45% of the alpha helix and 45.45% of the random coil. Our analysis predicted that this enzyme has a potential signal peptide in the protein sequence. Conclusion: By these analyses, a new understanding was developed related to the sequence and structure of this enzyme. Our findings can further be used in some fields of clinical and industrial biotechnology.

Background: Oral Squamous Cell Carcinoma (OSCC) is a major sub-type of oral cancer that shares 90% proportion of oral cavity cancers. It is declared as the sixth most frequent cancer among all cancer types throughout the world. Higher morbidity in Asian countries is reported due to frequent use of Smokeless Tobacco (SLT) products besides exposure to other risk factors. Hyperactivation of epidermal growth factor receptors is a molecular event in many solid tumors including oral cancer making them potential therapeutic targets. Objective: Current study was designed to explore the effect of varlitinib, a pan-HER inhibitor, on oral cancer cell line. We investigated key regulatory genes in downstream pathway in response to drug treatment. Furthermore, we also examined expression profile of these genes in malignant and healthy oral tissue. Methods: Gene expression pattern in drug treated and untreated cancer cell line along with OSCC tumor samples (n=45) and adjacent normal tissues was studied using real time PCR. Results: In response to varlitinib treatment, significant suppression of oncogenes (IGF1R, MAPK1, SFN and CDK2) was observed. Interestingly, mRNA expression level of CDKN1A and Akt1 was found to be the opposite of what was expected. In case of malignant tissue, over expression of oncogenes (IGF1R, Akt1, MAPK1, SFN and CDK2) with simultaneous down expression of tumor suppressor genes (Tp53 and CDKN1A) was noted. STRING analysis indicated a strong association among differentially expressed genes suggesting their combined role in carcinogenesis. Conclusion: In summary, our results indicate that varlitinib can be considered as a potential therapeutic agent in oral cancer due to its antitumor potential.

Background: Hyperuricemia and gout are the conditions, which is a response of accumulation of uric acid in the blood and urine. Uric acid is the product of purine metabolic pathway in humans. Uricase is a therapeutic enzyme that can enzymatically reduces the concentration of uric acid in serum and urine into more a soluble allantoin. Uricases are widely available in several sources like bacteria, fungi, yeast, plants and animals. Objective: The present study is aimed at elucidating the structure and physiochemical properties of uricase by insilico analysis. Methods: A total number of sixty amino acid sequences of uricase belongs to different sources were obtained from NCBI and different analysis like Multiple Sequence Alignment (MSA), homology search, phylogenetic relation, motif search, domain architecture and physiochemical properties including pI, EC, Ai, Ii, and were performed. Results: Multiple sequence alignment of all the selected protein sequences has exhibited distinct difference between bacterial, fungal, plant and animal sources based on the position-specific existence of conserved amino acid residues. The maximum homology of all the selected protein sequences is between 51-388. In singular category, homology is between 16-337 for bacterial uricase, 14-339 for fungal uricase, 12-317 for plants uricase, and 37-361 for animals uricase. The phylogenetic tree constructed based on the amino acid sequences disclosed clusters indicating that uricase is from different source. The physiochemical features revealed that the uricase amino acid residues are in between 300- 338 with a molecular weight as 33-39kDa and theoretical pI ranging from 4.95-8.88. The amino acid composition results showed that valine amino acid has a high average frequency of 8.79 percentage compared to different amino acids in all analyzed species. Conclusion: In the area of bioinformatics field, this work might be informative and a stepping-stone to other researchers to get an idea about the physicochemical features, evolutionary history and structural motifs of uricase that can be widely used in biotechnological and pharmaceutical industries. Therefore, the proposed in silico analysis can be considered for protein engineering work, as well as for gout therapy.

Background: Familial Mediterranean Fever (FMF) is a prototypical hereditary autoinflammatory disease affecting principally Mediterranean populations and characterized by recurrent frequent fever and inflammation. The disease is essentially caused by inherited mutations in the MEFV gene which encodes pyrin protein. The reported mutations are mostly located on the B30.2 domain in the C-terminal end of the protein. Objective: The present study reports a structural comparison of the five most common mutated structures including M694V, V726A, M694I, R761H, and M680I. The aim of this study was to determine the structural and functional disorders caused by the mutations in the human pyrin protein. Results: The comparison revealed that all mutations make overall changes in the structure of the domain. Further, the effects of these mutations on structural and molecular behavior of the B30.2 domain were compared with the native structure using MD simulation by GROMACS software. The results revealed that all the studied mutants have a destabilizing effect on the protein structure. Additionally, analyzing the projection of the motions of the proteins in phase space demonstrates high rigidity of the mutated structures in comparison with the native protein. Conclusion: The results of simulations elucidate how the mutations affect the physiological functioning of the pyrin B30.2 domain and cause the occurrence of the FMF disease.