Evaluation and biosynthetic incorporation of chlorotyrosine into recombinant proteins

Recently, non-canonical amino acids (NCAA) incorporation was developed to enhance the functional properties of proteins. Incorporation of NCAA containing chlorine atom is conceptually an attractive approach to prepare pharmacologically active substances, which is a difficult task since chlorine is bulky atom. In this study, we evaluated the efficiency and extent of in vivo incorporation of tyrosine analogue 3-chlorotyrosine [(3-Cl)Tyr] into the recombinant proteins GFP and GFPHS (highly stable GFP). The incorporation of (3-Cl)Tyr into GFP leads to dramatic reduction in the expression level of protein. On the other hand, the incorporation of (3-Cl)Tyr into GFPHS was expressed well as a soluble form. In addition we used bioinformatics tools for the analysis to explore the possible constraints in micro-environment of each natural amino acid residue to be replaced with chlorine atom accommodation into GFPHS. In conclusion, our approaches are reliable and straightforward way to enhance the translation of chlorinated amino acids into proteins.     

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV “O” 100 GPID₅₀ challenge virus.

     

Role of plant phytochemicals and microbial enzymes in biosynthesis of metallic nanoparticles

Applied Microbiology and Biotechnology - Metal-based nanoparticles have gained tremendous popularity because of their interesting physical, biological, optical, and magnetic properties. These...     

Multifunctional theranostic applications of biocompatible green-synthesized colloidal nanoparticles

Applied Microbiology and Biotechnology - Phytochemicals offer immense promise for sustainable development and production of nanotechnology-enabled products. In the present study, Olax nana Wall. ex...     

Copper oxide nanoparticles induce anticancer activity in A549 lung cancer cells by inhibition of histone deacetylase

Objectives

Copper oxide nanoparticles (CuO NPs) promoting anticancer activity may be due to the regulation of various classes of histone deacetylases (HDACs).

Results

Green-synthesized CuO NPs significantly arrested total HDAC level and also suppressed class I, II and IV HDACs mRNA expression in A549 cells. A549 cells treated with CuO NPs downregulated oncogenes and upregulated tumor suppressor protein expression. CuO NPs positively regulated both mitochondrial and death receptor-mediated apoptosis caspase cascade pathway in A549 cells.

Conclusion

Green-synthesized CuO NPs inhibited HDAC and therefore shown apoptosis mediated anticancer activity in A549 lung cancer cell line.

     

Analysis of secondary structural and physicochemical changes in protein–protein complexes

Conformation switching in protein–protein complexes is considered important for the molecular recognition process. Overall analysis of 123 protein–protein complexes in a benchmark data-set showed that 6.8% of residues switched over their secondary structure conformation upon complex formation. Amino acid residue-wise preference for conformation change has been analyzed in binding and non-binding site residues separately. In this analysis, residues such as Ser, Leu, Glu, and Lys had higher frequency of secondary structural conformation change. The change of helix to coil and sheet to coil conformation and vice versa has been observed frequently, whereas the conformation change of helix to extended sheet occurred rarely in the studied complexes. Influence of conformation change toward the N and C terminal on either side of the binding site residues has been analyzed. Further, analysis on φ and ψ angle variation, conservation, stability, and solvent accessibility have been performed on binding site residues. Knowledge obtained from the present study could be effectively employed in the protein–protein modeling and docking studies.     

Burden of Liver Disease among Community-Based People Who Inject Drugs (PWID) in Chennai,India

Background and Objective

We characterize the burden of liver disease in a cohort of PWID in Chennai, India, with a high prevalence of HCV.

Materials and Methods

1,042 PWID were sampled through community outreach in Chennai. Participants underwent fasting blood draw, questionnaire and an examination that included liver stiffness assessment using transient elastography (Fibroscan) and assessment of steatosis via ultrasound.

Results

The median age was 39 years, all were male, 14.8% were HIV infected and 35.6% were HCV antibody positive, of whom 78.9% were chronically infected (HCV RNA positive). Median liver stiffness was 6.2 kPA; 72.9% had no evidence of or mild stiffness, 14.5% had moderate stiffness, and 12.6% had evidence of severe stiffness/cirrhosis. Prevalence of severe stiffness/cirrhosis was significantly higher among persons who were older, had a longer duration of injecting drugs, higher body mass index, higher prevalence of insulin resistance, higher prevalence of steatosis, higher HCV RNA levels and evidence of alcohol dependence. An estimated 42.1% of severe stiffness/cirrhosis in this sample was attributable to HCV. 529 (53.0%) had some evidence of steatosis. Prevalence of steatosis was higher among those who had larger waist circumference, insulin resistance, higher HDL cholesterol and a history of antiretroviral therapy.

Conclusions

We observed a high burden of liver disease in this relatively young cohort that was primarily driven by chronic HCV infection, metabolic factors (insulin resistance and steatosis) and heavy alcohol use. Interventions to improve access to HCV treatment and reduce alcohol use are needed to prevent further progression of liver disease.     

Probing the “fingers” domain binding pocket of Hepatitis C virus NS5B RdRp and D559G resistance mutation via molecular docking,molecular dynamics simulation and binding free energy calculations

The NS5B RdRp polymerase is a prominent enzyme for the replication of Hepatitis C virus (HCV). During the HCV replication, the template RNA binding takes place in the “fingers” sub-domain of NS5B. The “fingers” domain is a new emerging allosteric site for the HCV drug development. The inhibitors of the “fingers” sub-domain adopt a new antiviral mechanism called RNA intervention. The details of essential amino acid residues, binding mode of the ligand, and the active site intermolecular interactions of RNA intervention reflect that this mechanism is ambiguous in the experimental study. To elucidate these details, we performed molecular docking analysis of the fingers domain inhibitor quercetagetin (QGN) with NS5B polymerase. The detailed analysis of QGN-NS5B intermolecular interactions was carried out and found that QGN interacts with the binding pocket amino acid residues Ala97, Ala140, Ile160, Phe162, Gly283, Gly557, and Asp559; and also forms π?π stacking interaction with Phe162 and hydrogen bonding interaction with Gly283. These are found to be the essential interactions for the RNA intervention mechanism. Among the strong hydrogen bonding interactions, the QGN?Ala140 is a newly identified important hydrogen bonding interaction by the present work and this interaction was not resolved by the previously reported crystal structure. Since D559G mutation at the fingers domain was reported for reducing the inhibition percentage of QGN to sevenfold, we carried out molecular dynamics (MD) simulation for wild and D559G mutated complexes to study the stability of protein conformation and intermolecular interactions. At the end of 50?ns MD simulation, the π?π stacking interaction of Phe162 with QGN found in the wild-type complex is altered into T-shaped π stacking interaction, which reduces the inhibition strength. The origin of the D559G resistance mutation was studied using combined MD simulation, binding free energy calculations and principal component analysis. The results were compared with the wild-type complex. The mutation D559G reduces the binding affinity of the QGN molecule to the fingers domain. The free energy decomposition analysis of each residue of wild-type and mutated complexes revealed that the loss of non-polar energy contribution is the origin of the resistance.

Communicated by Ramaswamy H. Sarma