Metal-binding proteins and proteases in RNA viruses: unravelling functional diversity and expanding therapeutic horizons.

IMPORTANCE: Metal-binding proteins are pivotal components with diverse functions in organisms, including viruses. Despite their significance, many metalloproteins in viruses remain uncharacterized, posing challenges to understanding viral systems. This study addresses this knowledge gap by identifying and analyzing metal-binding proteins and proteases in RNA viruses. The findings emphasize the prevalence of these proteins as essential functional classes within viruses and shed light on the role of metal ions and metalloproteins in viral replication and pathogenesis. Moreover, this research serves as a crucial foundation for further investigations in this field, offering the potential for developing innovative antiviral strategies. Additionally, the study enhances our understanding of the distribution and evolutionary patterns of metal-binding proteases in major human viruses. Continually exploring metal-binding proteomes across diverse viruses will deepen our knowledge of metal-dependent biological processes and provide valuable insights for combating viral infections, including respiratory viruses and other life-threatening diseases.

Metalloproteome of human-infective RNA viruses: a study towards understanding the role of metal ions in virology.

Metalloproteins and metal-based inhibitors have been shown to effectively combat infectious diseases, particularly those caused by RNA viruses. In this study, a diverse set of bioinformatics methods was employed to identify metal-binding proteins of human RNA viruses. Seventy-three viral proteins with a high probability of being metal-binding proteins were identified. These proteins included 40 zinc-, 47 magnesium- and 14 manganese-binding proteins belonging to 29 viral species and eight significant viral families, including Coronaviridae, Flaviviridae and Retroviridae. Further functional characterization has revealed that these proteins play a critical role in several viral processes, including viral replication, fusion and host viral entry. They fall under the essential categories of viral proteins, including polymerase and protease enzymes. Magnesium ion is abundantly predicted to interact with these viral enzymes, followed by zinc. In addition, this study also examined the evolutionary aspects of predicted viral metalloproteins, offering essential insights into the metal utilization patterns among different viral species. The analysis indicates that the metal utilization patterns are conserved within the functional classes of the proteins. In conclusion, the findings of this study provide significant knowledge on viral metalloproteins that can serve as a valuable foundation for future research in this area.

Genomic Variation Affecting MPV and PLT Count in Association with Development of Ischemic Stroke and Its Subtypes.

Platelets play a significant role in the pathophysiology of ischemic stroke since they are involved in the formation of intravascular thrombus after erosion or rupture of the atherosclerotic plaques. Platelet (PLT) count and mean platelet volume (MPV) are the two significant parameters that affect the functions of platelets. In the current study, MPV and PLT count was evaluated using flow cytometry and a cell counter. SonoClot analysis was carried out to evaluate activated clot timing (ACT), clot rate (CR), and platelet function (PF). Genotyping was carried out using GSA and Sanger sequencing, and expression analysis was performed using RT-PCR. In silico analysis was carried out using the GROMACS tool and UNAFold. The interaction of significant proteins with other proteins was predicted using the STRING database. Ninety-six genes were analyzed, and a significant association of THPO (rs6141) and ARHGEF3 (rs1354034) was observed with the disease and its subtypes. Altered genotypes were associated significantly with increased MPV, decreased PLT count, and CR. Expression analysis revealed a higher expression in patients bearing the variant genotypes of both genes. In silico analysis revealed that mutation in the THPO gene leads to the reduced compactness of protein structure. mRNA encoded by mutated ARHGEF3 gene increases the half-life of mRNA. The two significant proteins interact with many other proteins, especially the ones involved in platelet activation, aggregation, erythropoiesis, megakaryocyte maturation, and cytoskeleton rearrangements, suggesting that they could be important players in the determination of MPV values. In conclusion, the current study demonstrated the role of higher MPV affected by genetic variation in the development of IS and its subtypes. The results of the current study also indicate that higher MPV can be used as a biomarker for the disease and altered genotypes, and higher MPV can be targeted for better therapeutic outcomes.

Structural Distortion of ß-Cyclodextrin Plays a Key Role in the pH-dependent Host-Guest Chemistry with Doxorubicin, Evident by the Electrochemical and Molecular Dynamics Approach.

ß-Cyclodextrin (ß-CD) is the potential drug carrier to deliver antitumor drugs like doxorubicin (DOX). However, the mechanism for the inclusion complex formation is still unclear and needs to be explored. This study investigated the effect of pH on the inclusion of DOX into thiolated ß-CD (ß-CD-SH) by electrochemical and molecular dynamics (MD) simulation. The electrochemical study shows a clear difference at different pH values. The redox peak due to the DOX is strongly influenced by pH. At neutral pH, the peak intensity decreases with time, while slight variation is observed at acidic and basic pH, depicting the association of DOX to the ß-CD-SH cavity at neutral pH. Also, due to the association, the charge transfer resistance variation increased with time at neutral pH and decreased at basic and acidic pH. The electrochemical study was further supported by MD simulation, suggesting that the cyclodextrin (CD) ring gets slightly elongated due to the flipping of glucose units, specifically at neutral pH leading to a strong association. Also, another significant result observed that the DOX forms an inclusion complex with ß-CD-SH in quinol conformation, not in quinone. Briefly, the study provides the necessary molecular binding information for designing an effective ß-CD-based targeted drug delivery system.

The putative metal-binding proteome of the Coronaviridae family.

Metalloproteins are well-known for playing various physicochemical processes in all life forms, including viruses. Some life-threatening viruses (such as some members of the Coronaviridae family of viruses) are emerged and remerged frequently and are rapidly transmitted throughout the globe. This study aims to identify and characterize the metal-binding proteins (MBPs) of the Coronaviridae family of viruses and further provides insight into the MBP's role in sustaining and propagating viruses inside a host cell and in the outer environment. In this study, the available proteome of the Coronaviridae family was exploited. Identified potential MBPs were analyzed for their functional domains, structural aspects, and subcellular localization. We also demonstrate phylogenetic aspects of all predicted MBPs among other Coronaviridae family members to understand the evolutionary trend among their respective hosts. A total of 256 proteins from 51 different species of coronaviruses are predicted as MBPs. These MBPs perform various key roles in the replication and survival of viruses within the host cell. Cysteine, aspartic acid, threonine, and glutamine are key amino acid residues interacting with respective metal ions. Our observations also indicate that the metalloproteins of this family of viruses circulated and evolved in different hosts, which supports the zoonotic nature of coronaviruses. The comprehensive information on MBPs of the Coronaviridae family may be further helpful in designing novel therapeutic metalloprotein targets. Moreover, the study of viral MBPs can also help to understand the roles of MBPs in virus pathogenesis and virus-host interactions.

Functional assignment to hypothetical proteins in Orientia tsutsugamushistrain Ikeda.

Orientia tsutsugamushi(O. tsutsugamushi) is an intracellular bacterial pathogen which causes zoonosis scrub typhus in humans. Genome of O. tsutsugamushi strain Ikeda contains 214 hypothetical proteins (HPs) which is nearly 20% of the total proteins. Domain and family based functional analysis of HPs results in the annotation of 44 hypothetical proteins. The annotated HPs were classified in to five main classes namely, gene expression and regulation, transport, metabolism, cell signaling and proteolysis. Thus, computational analysis of HPs helps to understand their putative roles in various biological and cellular processes, including pathogenesis for further consideration as potential therapeutic targets.

YAPPIS-Finder: A novel method for protein-protein interaction site predictions.

We describe a multi parametric-approach, YAPPIS-Finder, for predicting the PPI sites on protein surface. A non-redundant database of comprised of 2,265 protein-protein interaction interfaces (PPIIs) involving 4,530 protein-protein interacting partners (PPIPs) and depicting the interaction between protein-chains of experimentally determined PPCs was used in designing the YAPPIS-Finder. Parametric score obtained on analyzing these 4,530 PPIPs with respect to their residue interface propensity, their hydrophobic content, and amount of solvation free energy associated with them provided the basis of YAPPIS-Finder. By applying YAPPIS-Finder on another dataset 4,290 PPIPs from 2,145 PPIIs, the optimal range of the parametric scores and protein-probe van der Waals energy of interaction was determined. Subsequently, taking the optimal range of PPIP parametric scores and threshold for protein-probe van der Waals energy of interaction into the consideration, the YAPPIS-Finder was tested on a blind dataset of 554 protein-chains and it was found predicting 69.67% sites correctly. On predicting only one PPI site on each protein-chain, the YAPPIS-Finder found covering 22.91% of actually sites in the predicted site. Contrary to this, the sites predicted by SPPIDER covered 22.7% of actual sites. However, on predicting two PPI sites for each protein-chain, the percentage coverage of actual sites in the predicted sites by YAPPIS-Finder exceeded two-fold (i.e. 41.81%), thus making the YAPPIS-Finder a better method.

Geometrical and electro-static determinants of protein-protein interactions.

Protein-protein interactions (PPI) are pivotal to the numerous processes in the cell. Therefore, it is of interest to document the analysis of these interactions in terms of binding sites, topology of the interacting structures and physiochemical properties of interacting interfaces and the of forces interactions. The interaction interface of obligatory protein-protein complexes differs from that of the transient interactions. We have created a large database of protein-protein interactions containing over100 thousand interfaces. The structural redundancy was eliminated to obtain a non-redundant database of over 2,265 interaction interfaces. Therefore, it is of interest to document the analysis of these interactions in terms of binding sites, topology of the interacting structures and physiochemical properties of interacting interfaces and the offorces interactions. The residue interaction propensity and all of the rest of the parametric scores converged to a statistical indistinguishable common sub-range and followed the similar distribution trends for all three classes of sequence-based classifications PPInS. This indicates that the principles of molecular recognition are dependent on the preciseness of the fit in the interaction interfaces. Thus, it reinforces the importance of geometrical and electrostatic complementarity as the main determinants for PPIs.

Evaluation of in silico designed inhibitors targeting MelF (Rv1936) against Mycobacterium marinum within macrophages.

We recently identified inhibitors targeting Mycobacterium marinum MelF (Rv1936) by in silico analysis, which exhibited bacteriostatic/bactericidal activity against M. marinum and M. tuberculosis in vitro. Herein, we evaluated the effect of best four inhibitors (# 5175552, # 6513745, # 5255829, # 9125618) obtained from the ChemBridge compound libraries, on intracellular replication and persistence of bacteria within IFN-γ activated murine RAW264.7 and human THP-1 macrophages infected with M. marinum. Inhibitors # 5175552 and # 6513745 significantly reduced (p < 0.05) the intracellular replication of bacilli during day 7 post-infection (p.i.) within RAW264.7 and THP-1 macrophages infected at multiplicity of infection (MOI) of ~1.0. These observations were substantiated by electron microscopy, which revealed the protective effect of # 5175552 in clearing the bacilli inside murine macrophages. Strikingly, # 6513745 displayed synergism with isoniazid against M. marinum in murine macrophages, whereas # 5175552 significantly suppressed (p < 0.05) the persistent bacilli during day 10-14 p.i. in infected RAW264.7 and THP-1 macrophages (MOI of ~ 0.1). Moreover, # 5175552 and # 6513745 were non-cytotoxic to host macrophages at both 1X and 5X MIC. Further validation of these inhibitors against M. tuberculosis-infected macrophages and animal models has potential for development as novel anti-tubercular agents.

A study of mechanistic mapping of novel SNPs to male breast cancer.

Alterations in BRCA2, PALB2, CHEK2, and p53 genes have been identified for their association with male breast cancer in various studies. The incidence of male breast cancer in India is consistent with its global rate. The present study was carried out with an aim to evaluate the genetic alterations in male breast cancer patients from Malwa region of Punjab, India. Four male breast cancer patients belonging to different families were recruited from Guru Gobind Singh Medical College and Hospital, Faridkot, India. A total of 51 genes reported with implications in the pathogenesis of breast cancer were screened using next generation sequencing. Germline variations were found in BRCA1, BRCA2, PMS2, p53, and PALB2 genes, previously reported to be associated with MBC as well as FBC. In addition to these, 13 novel missense alterations were detected in eight genes including STK11, FZR1, PALB2, BRCA2, NF2, BAP1, BARD1, and CHEK2. Impact of these missense alterations on structure and function of protein was also analyzed through molecular dynamics simulation. Structural analysis of these single nucleotide polymorphisms (SNPs) revealed significant impact on the encoded protein functioning.

PPInS: a repository of protein-protein interaction sitesbase.

Protein-Protein Interaction Sitesbase (PPInS), a high-performance database of protein-protein interacting interfaces, is presented. The atomic level information of the molecular interaction happening amongst various protein chains in protein-protein complexes (as reported in the Protein Data Bank [PDB]) together with their evolutionary information in Structural Classification of Proteins (SCOPe release 2.06), is made available in PPInS. Total 32468 PDB files representing X-ray crystallized multimeric protein-protein complexes with structural resolution better than 2.5 Å had been shortlisted to demarcate the protein-protein interaction interfaces (PPIIs). A total of 111857 PPIIs with ~32.24 million atomic contact pairs (ACPs) were generated and made available on a web server for on-site analysis and downloading purpose. All these PPIIs and protein-protein interacting patches (PPIPs) involved in them, were also analyzed in terms of a number of residues contributing in patch formation, their hydrophobic nature, amount of surface area they contributed in binding, and their homo and heterodimeric nature, to describe the diversity of information covered in PPInS. It was observed that 42.37% of total PPIPs were made up of 6-20 interacting residues, 53.08% PPIPs had interface area ≤1000 Å2 in PPII formation, 82.64% PPIPs were reported with hydrophobicity score of ≤10, and 73.26% PPIPs were homologous to each other with the sequence similarity score ranging from 75-100%. A subset "Non-Redundant Database (NRDB)" of the PPInS containing 2265 PPIIs, with over 1.8 million ACPs corresponding to the 1931 protein-protein complexes (PDBs), was also designed by removing structural redundancies at the level of SCOP superfamily (SCOP release 1.75). The web interface of the PPInS ( http://www.cup.edu.in:99/ppins/home.php ) offers an easy-to-navigate, intuitive and user-friendly environment, and can be accessed by providing PDB ID, SCOP superfamily ID, and protein sequence.

Insights from the Molecular dynamics simulation of BcsD Subunit from K. xylinus.

Biofilms are bacteria living in micro-colonies with a protective coating in sessile form. The biofilm protects bacteria from harsh surroundings as well as help in antibiotics resistance using a semi-fluid substance. Cellulose is the major component of biofilm, which provides the sticky appearance to bacteria for attaching to the substratum. The bacteria communicate in biofilm with the help of quorum sensing hormones Acylated Homoserine Lactones (AHL's). In Komagataeibacter xylinus the four genes Bcs A, Bcs B, Bcs C, Bcs D are associated with cellulose biosynthesis. The Bcs D subunits have a hypothetical octamer pore-like structure through which glucan molecule pass to form the cellulose. Therefore, it is of interest to document a structural understanding of Bcs D. Hence a homology model of Bcs D was simulated and analyzed further to gain functional insight towards biofilm formation.

Insights from the analysis of alginate lyase protein model from Pseudomonas fluorescens towards the understanding of mucoid biofilm disruption.

Bacterial biofilm is a protective, slippery and slimy coat secreted by bacterial cells. It helps in attaching to moisturized surfaces during colonization. Alginate is an important component as it is essential for retention of water and nutrients in biofilms. It is a polysaccharide consisting of ß-D-mannuronic acid (M) and α-L-guluronic acid (G) monomers with 1-4 linkage. The alginate lyase (AlgL) secreted by certain bacteria is capable of degrading alginate into oligo-uronides by ß-elimination of the glycosidic bond. Therefore, it is of interest to analyze the simulated (GROMACS force filed) structure protein model (homology based on template 4OZV) of AlgL from Pseudomonas fluorescens to gain functional insight mucoid biofilm disruption. We report root mean square deviation (RMSD) and radius of gyration (Rg) profiles of the simulated (molecular dynamics) AlgL protein homology model in this context towards biofilm discruption.

Rational design of drug-like compounds targeting Mycobacterium marinum MelF protein.

The mycobacterial mel2 locus (mycobacterial enhanced infection locus, Rv1936-1941) is Mycobacterium marinum and M. tuberculosis specific, which can withstand reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced stress. A library of over a million compounds was screened using in silico virtual ligand screening (VLS) to identify inhibitors against the modeled structure of MelF protein expressed by melF of mel2 locus so that M. marinum's ability to withstand ROS/RNS stress could be reduced. The top ranked 1000 compounds were further screened to identify 178 compounds to maximize the scaffold diversity by manually evaluating the interaction of each compound with the target site. M. marinum melF was cloned, expressed and purified as maltose binding protein (MBP)-tagged recombinant protein in Escherichia coli. After establishing the flavin dependent oxidoreductase activity of MelF (~ 84 kDa), the inhibitors were screened for the inhibition of enzyme activity of whole cell lysate (WCL) and the purified MelF. Amongst these, 16 compounds could significantly inhibit the enzyme activity of purified MelF. For the six best inhibitory compounds, the minimal inhibitory concentration (MIC) was determined to be 3.4-19.4 µM and 13.5-38.8 µM for M. marinum and M. tuberculosis, respectively. Similarly, the minimal bactericidal concentration (MBC) was determined to be 6.8-38.8 µM and 27-38.8 µM against M. marinum and M. tuberculosis, respectively. One compound each in combination with isoniazid (INH) also showed synergistic inhibitory effect against M. marinum and M. tuberculosis with no cytotoxicity in HeLa cells. Interestingly, these inhibitors did not display any non-specific protein-structure destabilizing effect. Such inhibitors targeting the anti-ROS/RNS machinery may facilitate the efficient killing of replicating and nonreplicating mycobacteria inside the host cells.

Understanding human thiol dioxygenase enzymes: structure to function, and biology to pathology.

Amino acid metabolism is a significant metabolic activity in humans, especially of sulphur-containing amino acids, methionine and cysteine (Cys). Cys is cytotoxic and neurotoxic in nature; hence, mammalian cells maintain a constant intracellular level of Cys. Metabolism of Cys is mainly regulated by two thiol dioxygenases: cysteine dioxygenase (CDO) and 2-aminoethanethiol dioxygenase (ADO). CDO and ADO are the only human thiol dioxygenases reported with a role in Cys metabolism and localized to mitochondria. This metabolic pathway is important in various human disorders, as it is responsible for the synthesis of antioxidant glutathione and is also for the synthesis of hypotaurine and taurine. CDO is the most extensively studied protein, whose high-resolution crystallographic structures have been solved. As compared to CDO, ADO is less studied, even though it has a key role in cysteamine metabolism. To further understand ADO's structure and function, the three-dimensional structures have been predicted from I-TASSER and SWISS-MODEL servers and validated with PROCHECK software. Structural superimposition approach using iPBA web server further confirmed near-identical structures (including active sites) for the predicted protein models of ADO as compared to CDO. In addition, protein-protein interaction and their association in patho-physiology are crucial in understanding protein functions. Both ADO and CDO interacting partner profiles have been presented using STRING database. In this study, we have predicted a 3D model structure for ADO and summarized the biological roles and the pathological consequences which are associated with the altered expression and functioning of ADO and CDO in case of cancer, neurodegenerative disorders and other human diseases.

Rational design of small molecules targeting the C2 domain of coagulation factor VIII.

The C domains of coagulation factors V (FV) and VIII (FVIII) are structurally conserved domains and share a common and essential function in membrane binding. In vivo regulation of thrombin formation strongly depends on the expression and regulation of the cofactor activities of FVIII and FV. With this study, we explored the possibility of inhibition of thrombin formation in full blood with small druglike molecules. Such compounds may serve as lead molecules for the development of a new type of orally available coagulation inhibitors that act by blocking the interaction between the C domains of FVIII and the membrane surface. We identified 9 novel molecules that are able to inhibit binding of the FVIII C2 domain to a model membrane by application of a combined ligand-based and target structure-based virtual screening approach that took into account the knowledge of a set of previously identified low-molecular-weight FVIII binders that were, however, not active in full blood. The half-maximal inhibitory concentration values of our newly identified compounds varied from 2.1 to 19.9 µM, of which 7 of 9 molecules did not appreciably inhibit FV membrane binding and were thus specific for FVIII. The most active bioactive compound showed activity in both plasma and in full blood.

Homology modeling and structural validation of tissue factor pathway inhibitor.

Blood coagulation is a cascade of complex enzymatic reactions which involves specific proteins and cellular components to interact and prevent blood loss. The coagulation process begins by either "Tissue Dependent Pathway" (also known as extrinsic pathway) or by "contact activation pathway" (also known as intrinsic pathway). TFPI is an endogenous multivalent Kunitz type protease inhibitor which inhibits Tissue factor dependent pathway by inhibiting Tissue Factor:Factor VIIa (TF:FVIIa) complex and Factor Xa. TFPI is one of the most studied coagulation pathway inhibitor which has various clinical and potential therapeutic applications, however, its exact mechanism of inhibition is still unknown. Structure based mechanism elucidation is commonly employed technique in such cases. Therefore, in the current study the generated a complete TFPI structural model so as to understand the mechanistic details of it's functioning. The model was checked for stereochemical quality by PROCHECK-NMR, WHATIF, ProSA, and QMEAN servers. The model was selected, energy minimized and simulated for 1.5ns. The result of the study may be a guiding point for further investigations on TFPI and its role in coagulation mechanism.

Goodpasture antigen-binding protein/ceramide transporter binds to human serum amyloid P-component and is present in brain amyloid plaques.

Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease.

InCa-SiteFinder: a method for structure-based prediction of inositol and carbohydrate binding sites on proteins.

Carbohydrate binding sites are considered important for cellular recognition and adhesion and are important targets for drug design. In this paper we present a new method called InCa-SiteFinder for predicting non-covalent inositol and carbohydrate binding sites on the surface of protein structures. It uses the van der Waals energy of a protein-probe interaction and amino acid propensities to locate and predict carbohydrate binding sites. The protein surface is searched for continuous volume envelopes that correspond to a favorable protein-probe interaction. These volumes are subsequently analyzed to demarcate regions of high cumulative propensity for binding a carbohydrate moiety based on calculated amino acid propensity scores. InCa-SiteFinder(1) was tested on an independent test set of 80 protein-ligand complexes. It efficiently identifies carbohydrate binding sites with high specificity and sensitivity. It was also tested on a second test set of 80 protein-ligand complexes containing 40 known carbohydrate binders (having 40 carbohydrate binding sites) and 40 known drug-like compound binders (having 58 known drug-like compound binding sites) for the prediction of the location of the carbohydrate binding sites and to distinguish these from the drug-like compound binding sites. At 73% sensitivity the method showed 98% specificity. Almost all of the carbohydrate and drug-like compound binding sites were correctly identified with an overall error rate of 12%.