A comprehensive proteomic profiling of urinary exosomes and the identification of early non-invasive biomarker in patients with coronary artery disease.

Urinary small extracellular vesicles or exosomes (uEVs) source could be an emerging trove of biomarkers in coronary artery disease (CAD). It is a chronic inflammatory disease having a long asymptomatic phase of fatty-fibrous development in arteries leading to angina, myocardial infarction, and death. Our study was aimed at identifying differential protein expression profiling of uEVs in CAD. We collected urine samples of CAD patients (n = 41) age 18-65 years and gender matched healthy controls (n = 41). We isolated uEVs using differential ultracentrifugation. Further, uEV samples were characterized by western blotting exosome markers (Flotillin, TSG, CD63, and CD9), nano tracking analysis, and transmission and scanning electron microscopy. A total of 508 proteins were identified by iTRAQ-based mass spectrometry. We observed protein expression levels of AZGP1, SEMG1/2, ORM1, IGL, SERPINA5, HSPG2, prosaposin, gelsolin, and CD59 were upregulated, and UMOD, KNG1, AMBP, prothrombin, and TF were downregulated. Protein-protein interactions, gene ontology and pathway analysis were performed to functionally annotate identified uEVs proteins. A novel uEVs differential protein signature is shown. On validating UMOD protein by ELISA in two clinically different CAD, stable-CAD patients had lower levels than healthy controls whereas recent myocardial infarction patients had lowest. Our findings suggest UMOD importance as early diagnostic biomarker. SIGNIFICANCE: Coronary artery disease is a chronic inflammatory disease caused by gradual deposition of cholesterol and fat along with other proteins to develop plaque inside arteries. This further leads to blockage of artery, heart attack and death. There are no identifiable early biomarkers to diagnose this. For the first time, we have identified the differentially expressed proteins isolated from non-invasive uEV of CAD patients compared to healthy controls by using MS Orbitrap and iTRAQ labelling of peptides. We have identified decreased levels of UMOD protein in CAD. These findings have been confirmed by ELISA. Furthermore, the levels of UMOD were observed as more highly decreased in recent myocardial infarction CAD patients, indicating the importance of this protein as an early diagnostic biomarker. Conclusively, our study represents a non-invasive urinary EVs trove of differentially expressed proteins in CAD. This will form a groundwork for understanding the pathophysiology of CAD and will help in future translational research utilizing uEVs.

New biochemical insights of CCA enzyme role in tRNA maturation and an efficient method to synthesize the 3'-amino-tailed tRNA.

The maturation of tRNA and its quality control is crucial for aminoacylation and protein synthesis. The CCA enzyme, also known as tRNA nucleotidyltransferase, catalyzes the addition or repair of CCA at the 3'-terminus of tRNAs to facilitate aminoacylation. Structural studies of CCA enzyme in complex with ATP and CTP suggested that adding CCA at the 3'-terminus of tRNAs is a sequential process [1-4]. However, there are many inconsistent results of CCA addition from the biochemical studies, which raise the ambiguity about the CCA enzyme specificity in vitro [5-7]. On the other hand, there are no effective methods for preparing the 3'-amino-tailed tRNA to provide a stable amide linkage, which is vital to make homogeneous samples for structural studies of stalling peptides to understand ribosome mediated gene regulation [7-11]. In this study, we examined the functional specificity of the Class II CCA enzyme from E. coli, and optimized the benchmark experimental conditions to prepare the 3'-NH2-tRNA using the CCA enzyme. Our results suggest that the CCA enzyme has a specific ability to catalyze the CCA addition/repair activity within the stoichiometric range of the reactants, and excess amounts of nucleotides lead to non-specific polymerization of the tRNA. Further, we developed an efficient method for synthesizing 3'-amino tRNA, which can facilitate stable aminoacyl/peptidyl-tRNA preparation.

Repurposing of FDA Approved Drugs Against SARS-CoV-2 Papain-Like Protease: Computational, Biochemical, and in vitro Studies.

The pandemic caused by SARS-CoV-2 (SCoV-2) has impacted the world in many ways and the virus continues to evolve and produce novel variants with the ability to cause frequent global outbreaks. Although the advent of the vaccines abated the global burden, they were not effective against all the variants of SCoV-2. This trend warrants shifting the focus on the development of small molecules targeting the crucial proteins of the viral replication machinery as effective therapeutic solutions. The PLpro is a crucial enzyme having multiple roles during the viral life cycle and is a well-established drug target. In this study, we identified 12 potential inhibitors of PLpro through virtual screening of the FDA-approved drug library. Docking and molecular dynamics simulation studies suggested that these molecules bind to the PLpro through multiple interactions. Further, IC50 values obtained from enzyme-inhibition assays affirm the stronger affinities of the identified molecules for the PLpro. Also, we demonstrated high structural conservation in the catalytic site of PLpro between SCoV-2 and Human Coronavirus 229E (HCoV-229E) through molecular modelling studies. Based on these similarities in PLpro structures and the resemblance in various signalling pathways for the two viruses, we propose that HCoV-229E is a suitable surrogate for SCoV-2 in drug-discovery studies. Validating our hypothesis, Mefloquine, which was effective against HCoV-229E, was found to be effective against SCoV-2 as well in cell-based assays. Overall, the present study demonstrated Mefloquine as a potential inhibitor of SCoV-2 PLpro and its antiviral activity against SCoV-2. Corroborating our findings, based on the in vitro virus inhibition assays, a recent study reported a prophylactic role for Mefloquine against SCoV-2. Accordingly, Mefloquine may further be investigated for its potential as a drug candidate for the treatment of COVID.

Biochemical and Biophysical Characterisation of the Hepatitis E Virus Guanine-7-Methyltransferase.

Hepatitis E virus (HEV) is an understudied pathogen that causes infection through fecal contaminated drinking water and is prominently found in South Asian countries. The virus affects ~20 million people annually, leading to ~60,000 infections per year. The positive-stranded RNA genome of the HEV genotype 1 has four conserved open reading frames (ORFs), of which ORF1 encodes a polyprotein of 180 kDa in size, which is processed into four non-structural enzymes: methyltransferase (MTase), papain-like cysteine protease, RNA-dependent RNA polymerase, and RNA helicase. MTase is known to methylate guanosine triphosphate at the 5'-end of viral RNA, thereby preventing its degradation by host nucleases. In the present study, we cloned, expressed, and purified MTase spanning 33-353 amino acids of HEV genotype 1. The activity of the purified enzyme and the conformational changes were established through biochemical and biophysical studies. The binding affinity of MTase with magnesium ions (Mg2+) was studied by isothermal calorimetry (ITC), microscale thermophoresis (MST), far-UV CD analysis and, fluorescence quenching. In summary, a short stretch of nucleotides has been cloned, coding for the HEV MTase of 37 kDa, which binds Mg2+ and modulate its activity. The chelation of magnesium reversed the changes, confirming its role in enzyme activity.

Characterization of active/binding site residues of peptidyl-tRNA hydrolase using biophysical and computational studies.

All mRNAs cannot be translated into full-length proteins due to ribosome-stalling that leads to release of peptidyl-tRNA which can be lethal for bacterial survival. The enzyme peptidyl-tRNA hydrolase (PtH) hydrolyses the ester bond between nascent peptide and tRNA of peptidyl-tRNA and rescues the cells from toxicity. PtH is an essential enzyme in bacteria and inhibiting this crucial enzyme can serve to combat bacterial diseases. But due to lack of understanding about the catalytic mechanism of PtH, its inhibitors have not been developed. In this work, we have carried out the binding studies of M. tuberculosis and E. coli PtH with the peptidyl-tRNA analogue (puromycin) using ITC, FTIR, CD experiments followed by docking and MD simulations to identify the potential active site residues that would help to design PtH inhibitors. Binding studies of puromycin with both PtH by ITC experiments demonstrate similar thermodynamic parameters and three fold difference in their KD. CD and FTIR studies detected changes in secondary structure composition of PtH in the presence of puromycin with different degree of perturbation. Though interactions with puromycin are conserved in both proteins, modelling studies revealed that water mediated interactions in M. tb-PtH resulting in higher affinity to puromycin.