Delineating virulence of Vibrio campbellii: a predominant luminescent bacterial pathogen in Indian shrimp hatcheries.

Luminescent vibriosis is a major bacterial disease in shrimp hatcheries and causes up to 100% mortality in larval stages of penaeid shrimps. We investigated the virulence factors and genetic identity of 29 luminescent Vibrio isolates from Indian shrimp hatcheries and farms, which were earlier presumed as Vibrio harveyi. Haemolysin gene-based species-specific multiplex PCR and phylogenetic analysis of rpoD and toxR identified all the isolates as V. campbellii. The gene-specific PCR revealed the presence of virulence markers involved in quorum sensing (luxM, luxS, cqsA), motility (flaA, lafA), toxin (hly, chiA, serine protease, metalloprotease), and virulence regulators (toxR, luxR) in all the isolates. The deduced amino acid sequence analysis of virulence regulator ToxR suggested four variants, namely A123Q150 (AQ; 18.9%), P123Q150 (PQ; 54.1%), A123P150 (AP; 21.6%), and P123P150 (PP; 5.4% isolates) based on amino acid at 123rd (proline or alanine) and 150th (glutamine or proline) positions. A significantly higher level of the quorum-sensing signal, autoinducer-2 (AI-2, p = 2.2e-12), and significantly reduced protease activity (p = 1.6e-07) were recorded in AP variant, whereas an inverse trend was noticed in the Q150 variants AQ and PQ. The pathogenicity study in Penaeus (Litopenaeus) vannamei juveniles revealed that all the isolates of AQ were highly pathogenic with Cox proportional hazard ratio 15.1 to 32.4 compared to P150 variants; PP (5.4 to 6.3) or AP (7.3 to 14). The correlation matrix suggested that protease, a metalloprotease, was positively correlated with pathogenicity (p > 0.05) and negatively correlated (p < 0.05) with AI-2 and AI-1. The syntenic organization of toxS-toxR-htpG operon in V. campbellii was found to be similar to pathogenic V. cholerae suggesting a similar regulatory role. The present study emphasizes that V. campbellii is a predominant pathogen in Indian shrimp hatcheries, and ToxR plays a significant role as a virulence regulator in the quorum sensing-protease pathway. Further, the study suggests that the presence of glutamine at 150th position (Q150) in ToxR is crucial for the pathogenicity of V. campbellii.

Bioactive Molecules of Tea as Potential Inhibitors for RNA-Dependent RNA Polymerase of SARS-CoV-2.

The coronavirus disease (COVID-19), a worldwide pandemic, is caused by the severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2). At this moment in time, there are no specific therapeutics available to combat COVID-19. Drug repurposing and identification of naturally available bioactive molecules to target SARS-CoV-2 are among the key strategies to tackle the notorious virus. The enzyme RNA-dependent RNA polymerase (RdRp) performs a pivotal role in replicating the virus. RdRp is a prime target for Remdesivir and other nucleotides analog-based antiviral drugs. In this study, we showed three bioactive molecules from tea (epicatechin-3,5-di-O-gallate, epigallocatechin-3,5-di-O-gallate, and epigallocatechin-3,4-di-O-gallate) that showed better interaction with critical residues present at the catalytic center and the NTP entry channel of RdRp than antiviral drugs Remdesivir and Favipiravir. Our computational approach to identify these molecules included molecular docking studies, followed by robust molecular dynamics simulations. All the three molecules are readily available in tea and could be made accessible along with other medications to treat COVID-19 patients. However, these results require validation by further in vitro and in vivo studies.

An in-silico evaluation of different bioactive molecules of tea for their inhibition potency against non structural protein-15 of SARS-CoV-2.

Immensely aggravated situation of COVID-19 has pushed the scientific community towards developing novel therapeutics to fight the pandemic. Small molecules can possibly prevent the spreading infection by targeting specific vital components of the viral genome. Non-structural protein 15 (Nsp15) has emerged as a promising target for such inhibitor molecules. In this investigation, we docked bioactive molecules of tea onto the active site of Nsp15. Based on their docking scores, top three molecules (Barrigenol, Kaempferol, and Myricetin) were selected and their conformational behavior was analyzed via molecular dynamics simulations and MMPBSA calculations. The results indicated that the protein had well adapted the ligands in the binding pocket thereby forming stable complexes. These molecules displayed low binding energy during MMPBSA calculations, substantiating their strong association with Nsp15. The inhibitory potential of these molecules could further be examined by in-vivo and in-vitro investigations to validate their use as inhibitors against Nsp15 of SARS-CoV2.

Identification of bioactive molecules from tea plant as SARS-CoV-2 main protease inhibitors.

The SARS-CoV-2 is the causative agent of COVID-19 pandemic that is causing a global health emergency. The lack of targeted therapeutics and limited treatment options have triggered the scientific community to develop new vaccines or small molecule therapeutics against various targets of SARS-CoV-2. The main protease (Mpro) is a well characterized and attractive drug target because of its crucial role in processing of the polyproteins which are required for viral replication. In order to provide potential lead molecules against the Mpro for clinical use, we docked a set of 65 bioactive molecules of Tea plant followed by exploration of the vast conformational space of protein-ligand complexes by long term molecular dynamics (MD) simulations (1.50 µs). Top three bioactive molecules (Oolonghomobisflavan-A, Theasinensin-D, and Theaflavin-3-O-gallate) were selected by comparing their docking scores with repurposed drugs (Atazanavir, Darunavir, and Lopinavir) against SARS-CoV-2. Oolonghomobisflavan-A molecule showed a good number of hydrogen bonds with Mpro and higher MM-PBSA binding energy when compared to all three repurposed drug molecules. during the time of simulation. This study showed Oolonghomobisflavan-A as a potential bioactive molecule to act as an inhibitor for the Mpro of SARS-CoV-2.

Pathological role of a point mutation (T315I) in BCR-ABL1 protein-A computational insight.

BCR-ABL protein is one of the most potent target to treat chronic myeloid leukemia (CML). Apart from other mutations, T315I is especially challenging as it confers resistance to all first- and second-generation tyrosine kinase inhibitors. So, a thorough study of altered behavior upon mutation is crucially needed. To understand the resistance mechanism of mutant BCR-ABL protein, we organized a long-term molecular dynamics simulation (500 ns) and performed the detailed comparative conformational analysis. We found that due to mutation at 315th position (threonine to isoleucine), original structures deviated from normal, and attained a flexible conformation. Our observations pave a clear path toward designing new inhibitors against resistant BCR-ABL1 protein and suggest a strategy where additional flexibility governed by mutation could be given an appropriate consideration.

Draft Genome Sequence of the Luminescent Strain Vibrio campbellii LB102, Isolated from a Black Tiger Shrimp (Penaeus monodon) Broodstock Rearing System.

We report here the genome sequence of Vibrio campbellii LB102, isolated from the broodstock rearing system of a shrimp hatchery in India. Sequence analysis revealed the presence of effector toxins of the type III (YopT, sharing 39% identity with Yersinia pestis) and type VI (VgrG-3 and hemolysin coregulated protein of V. cholerae) secretion systems.

Impact of point mutation P29S in RAC1 on tumorigenesis.

A point mutation (P29S) in the RAS-related C3 botulinum toxin substrate 1 (RAC1) was considered to be a trigger for melanoma, a form of skin cancer with highest mortality rate. In this study, we have investigated the pathogenic role of P29S based on the conformational behavior of RAC1 protein toward guanosine triphosphate (GTP). Molecular interaction, molecular dynamics trajectory analysis (RMSD, RMSF, Rg, SASA, DSSP, and PCA), and shape analysis of binding pocket were performed to analyze the interaction energy and the dynamic behavior of native and mutant RAC1 at the atomic level. Due to this mutation, the RAC1 switch I region acquired more flexibility and, to compensate it, the switch II region becomes rigid in their conformational space, as a result of which the interaction energy of the protein for GTP increased. The overall results strongly implied that the changes in atomic conformation of the switch I and II regions in mutant RAC1 protein were a significant reason for its malignant transformation and tumorigenesis. We raised the opportunity for researchers to design possible therapeutic molecule by considering our findings.

Structural analysis of oncogenic mutation of isocitrate dehydrogenase 1.

Arginine to histidine mutation at position 132 (R132H) in isocitrate dehydrogenase 1 (IDH1) led to reduced affinity of the respective enzymes for isocitrate and increased affinity for α-ketoglutarate (AKG) and NADPH. This phenomenon retarded oxidative decarboxylation of isocitrate to AKG and conferred a novel enzymatic activity that facilitated the reduction of AKG to d-2-hydroxyglutarate (d-2HG). The loss of isocitrate utilization and gain of 2HG production from IDH1 R132H had been taken up as a fundamental problem and to solve this, structural biology approaches were adopted. Interaction analysis was carried out to investigate the IDH1 substrate binding environment. The altered behaviour of mutant and native IDH1 in interaction analysis was explored by performing long-term molecular dynamics simulations (∼300 ns). This study reports a comprehensive atomic behaviour of the gain-of-function mutation (R132H) in the IDH1 enzyme which in turn provides a direction towards new therapeutics.

Biophysical aspect of phosphatidylinositol 3-kinase and role of oncogenic mutants (E542K & E545K).

Genetic variations in oncogenes can often promote uncontrolled cell proliferation by altering the structure of the encoded protein, thereby altering its function. The PI3KCA oncogene that encodes for p110α, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is one the most frequently mutated oncogenes in humans. PI3K plays a pivotal role in cell division. PI3K consists of two subunits: the catalytic (p110α) and regulatory (p85α). The regulatory subunit usually controls the catalytic subunit and switches off the enzyme when not required. It is believed that mutations in PI3KCA gene can alter the control of p85α over p110α and can sustain p110α in a prolonged active state. This in turn results in uncontrolled cell division. In this study, we investigate the pathogenic role of two point mutations: E542K and E545K on p110α subunit and how they alter its binding with the regulatory subunit. Molecular interaction and molecular dynamic simulation analysis are performed to study the dynamic behaviour of native and mutant structures at atomic level. Mutant p110α showed less interaction with its regulatory partner p85α than the native did, due to its expanded and rigid structure. Our analysis clearly points out that the structural and functional consequences of the mutations could promote tumour proliferation.

Mutational analysis of FUS gene and its structural and functional role in amyotrophic lateral sclerosis 6.

Amyotrophic lateral sclerosis 6 (ALS6) is an autosomal recessive disorder caused by heterozygous mutation in the Fused in Sarcoma (FUS) gene. ALS6 is a neurodegenerative disorder, which affects the upper and lower motor neurons in the brain and spinal cord, resulting in fatal paralysis. ALS6 is caused by the genetic mutation in the proline/tyrosine-nuclear localization signals of the Fused in sarcoma Protein (FUS). FUS gene also known as TLS (Translocated in liposarcoma), which encodes a protein called RNA-binding protein-Fus (FUS), has a molecular weight of 75 kDa. In this analysis, we applied computational approach to filter the most deleterious and neurodegenerative disease of ALS6-associated mutation on FUS protein. We found H517Q as most deleterious and disease associated using PolyPhen 2.0, I-Mutant 3.0, SIFT, SNPs&GO, PhD-SNP, Pmut, and Mutpred tools. Molecular dynamics simulation (MDS) approach was conducted to investigate conformational changes in the mutant protein structure with respect to its native conformation. MDS results showed the flexibility loss in mutant (H517Q) FUS protein. Due to mutation, FUS protein became more rigid in nature and might alter the structural and functional behavior of protein and play a major role in inducing ALS6. The results obtained from this investigation would help in the field of pharmacogenomics to develop a potent drug target against FUS-associated neurodegenerative diseases.

Role of centrosome in regulating immune response.

Centrosomes are the vital component of cell cycle progression pathway. Recent investigations have suggested their role in regulating the immune response system. Centrosome polarization delivers secretory granules to the immunological synapse (IS). The Cytotoxic T lymphocytes use a specific mechanism, controlled by centrosome delivery to the plasma membrane for delivering the secretory granules to the immunological synapse. Moreover, the polarization of centrioles to the immunological synapse directs secretion from cytolytic cells of innate as well as adaptive immune systems. Although the recent investigations have suggested their strong role in mediating the crucial events of immunological response, there are few discrepancies that are yet to be resolved. Furthermore, a clear picture of their molecular mechanism along with their cellular functions has not been reported. In this manuscript we have reviewed some important points that explain the importance of centrosomes in mediating the immunological signals and the delivery of lytic discharge from the cytotoxic and killer cells.

Drug resistance mechanism of PncA in Mycobacterium tuberculosis.

Tuberculosis continues to be a global health threat. Pyrazinamide (PZA) is an important first-line drug in multidrug-resistant tuberculosis treatment. The emergence of strains resistant to PZA represents an important public health problem, as both first- and second-line treatment regimens include PZA. It becomes toxic to Mycobacterium tuberculosis when converted to pyrazinoic acid by the bacterial pyrazinamidase (PncA) enzyme. Resistance to PZA is caused mainly by the loss of enzyme activity by mutation, the mechanism of resistance is not completely understood. In our studies, we analysed three mutations (D8G, S104R and C138Y) of PncA which are involved in resistance towards PZA. Binding pocket analysis solvent accessibility analysis, molecular docking and interaction analysis were performed to understand the interaction behaviour of mutant enzymes with PZA. Molecular dynamics simulations were conducted to understand the three-dimensional (3D) conformational behaviour of native and mutants PncA. Our analysis clearly indicates that the mutation (D8G, S104R and C138Y) in PncA is responsible for rigid binding cavity which in turn abolishes conversion of PZA to its active form and is the sole reason for PZA resistance. Excessive hydrogen bonding between PZA binding cavity residues and their neighbouring residues are the reason of rigid binding cavity during simulation. We present an exhaustive analysis of the binding site flexibility and its 3D conformations that may serve as new starting points for structure-based drug design and helps the researchers to design new inhibitors with consideration of rigid criterion of binding residues due to mutation of this essential target. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:11.

Relationship between a point mutation S97C in CK1δ protein and its affect on ATP-binding affinity.

CK1δ (Casein kinase I isoform delta) is a member of CK1 kinase family protein that mediates neurite outgrowth and the function as brain-specific microtubule-associated protein. ATP binding kinase domain of CK1δ is essential for regulating several key cell cycle signal transduction pathways. Mutation in CK1δ protein is reported to cause cancers and affects normal brain development. S97C mutation in kinase domain of CK1δ protein has been involved to induce breast cancer and ductal carcinoma. We performed molecular docking studies to examine the effect of this mutation on its ATP-binding affinity. Further, we conducted molecular dynamics simulations to understand the structural consequences of S97C mutation over the kinase domain of CK1δ protein. Docking results indicated the loss of ATP-binding affinity of mutant structure, which were further rationalized by molecular dynamics simulations, where a notable loss in 3-D conformation of CK1δ kinase domain was observed in mutant as compared to native. Our results explained the underlying molecular mechanism behind the observed cancer associated phenotype caused by S97C mutation in CK1δ protein.

Computational SNP analysis: current approaches and future prospects.

The computational approaches in determining disease-associated Non-synonymous single nucleotide polymorphisms (nsSNPs) have evolved very rapidly. Large number of deleterious and disease-associated nsSNP detection tools have been developed in last decade showing high prediction reliability. Despite of all these highly efficient tools, we still lack the accuracy level in determining the genotype-phenotype association of predicted nsSNPs. Furthermore, there are enormous questions that are yet to be computationally compiled before we might talk about the prediction accuracy. Earlier we have incorporated molecular dynamics simulation approaches to foster the accuracy level of computational nsSNP analysis roadmap, which further helped us to determine the changes in the protein phenotype associated with the computationally predicted disease-associated mutation. Here we have discussed on the present scenario of computational nsSNP characterization technique and some of the questions that are crucial for the proper understanding of pathogenicity level for any disease associated mutations.

AKT kinase pathway: a leading target in cancer research.

AKT1, a serine/threonine-protein kinase also known as AKT kinase, is involved in the regulation of various signalling downstream pathways including metabolism, cell proliferation, survival, growth, and angiogenesis. The AKT kinases pathway stands among the most important components of cell proliferation mechanism. Several approaches have been implemented to design an efficient drug molecule to target AKT kinases, although the promising results have not been confirmed. In this paper we have documented the detailed molecular insight of AKT kinase protein and proposed a probable doxorubicin based approach in inhibiting miR-21 based cancer cell proliferation. Moreover, the inhibition of miR-21 activation by raising the FOXO3A concentration seems promising in reducing miR-21 mediated cancer activation in cell. Furthermore, the use of next generation sequencing and computational drug design approaches will greatly assist in designing a potent drug molecule against the associated cancer cases.

In-silico screening of cancer associated mutation on PLK1 protein and its structural consequences.

The Polo-like kinases (Plks) are a conserved subfamily of serine-threonine protein kinases that have significant roles in cell proliferation. The serine/threonine protein kinases or polo-like kinase 1 (PLK1) exist in centrosome during interphase and is an important regulatory enzyme in cell cycle progression during M phase. Mutations in mammalian PLK1 were found to be over expressed in various human cancers and it is disrupting the binding ability of polo box domain with target peptide. In this analysis we implemented a computational approach to filter the most deleterious and cancer associated mutation on PLK1 protein. We found W414F as the most deleterious and cancer associated by Polyphen 2.0, SIFT, I-mutant 3.0, PANTHER, PhD-SNP, SNP&GO, Mutpred and Dr Cancer tools. Molecular docking and molecular dynamics simulation (MDS) approach was used to investigate the structural and functional behavior of PLK1 protein upon mutation. MDS and docking results showed stability loss in mutant PLK1 protein. Due to mutation, PLK1 protein became more flexible and alters the dynamic property of protein which might affect the interaction with target peptide and leads to cell proliferation. Our study provided a well designed computational methodology to examine the cancer associated nsSNPs and their molecular mechanism. It further helps scientists to develop a drug therapy against PLK1 cancer-associated diseases.

Molecular dynamic simulation reveals damaging impact of RAC1 F28L mutation in the switch I region.

Ras-related C3 botulinum toxin substrate 1 (RAC1) is a plasma membrane-associated small GTPase which cycles between the active GTP-bound and inactive GDP-bound states. There is wide range of evidences indicating its active participation in inducing cancer-associated phenotypes. RAC1 F28L mutation (RAC(F28L)) is a fast recycling mutation which has been implicated in several cancer associated cases. In this work we have performed molecular docking and molecular dynamics simulation (~0.3 µs) to investigate the conformational changes occurring in the mutant protein. The RMSD, RMSF and NHbonds results strongly suggested that the loss of native conformation in the Switch I region in RAC1 mutant protein could be the reason behind its oncogenic transformation. The overall results suggested that the mutant protein attained compact conformation as compared to the native. The major impact of mutation was observed in the Switch I region which might be the crucial reason behind the loss of interaction between the guanine ring and F28 residue.

Evidence of colorectal cancer-associated mutation in MCAK: a computational report.

Computational prediction of disease-associated non-synonymous polymorphism (nsSNP) has provided a significant platform to filter out the pathological mutations from large pool of SNP datasets at a very low cost input. Several methodologies and complementary protocols have been previously implemented and has provided significant prediction results. Although the previously implicated prediction methods were capable of investigating the most likely deleterious nsSNPs, but due to the lack of genotype-phenotype association analysis, the prediction results lacked in accuracy level. In this work we implemented the computational compilation of protein conformational changes as well as the probable disease-associated phenotypic outcomes. Our result suggested E403K mutation in mitotic centromere-associated kinesin protein as highly damaging and showed strong concordance to the previously observed colorectal cancer mutations aggregation tendency and energy value changes. Moreover, the molecular dynamics simulation results showed major loss in conformation and stability of mutant N-terminal kinesin-like domain structure. The result obtained in this study will provide future prospect of computational approaches in determining the SNPs that may affect the native conformation of protein structure and lead to cancer-associated disorders.

CEP proteins: the knights of centrosome dynasty.

Centrosome forms the backbone of cell cycle progression mechanism. Recent debates have occurred regarding the essentiality of centrosome in cell cycle regulation. CEP family protein is the active component of centrosome and plays a vital role in centriole biogenesis and cell cycle progression control. A total of 31 proteins have been categorized into CEP family protein category and many more are under candidate evaluation. Furthermore, by the recent advancements in genomics and proteomics researches, several new CEP proteins have also been characterized. Here we have summarized the importance of CEP family proteins and their regulation mechanism involved in proper cell cycle progression. Further, we have reviewed the detailed molecular mechanism behind the associated pathological phenotypes and the possible therapeutic approaches. Proteins such as CEP57, CEP63, CEP152, CEP164, and CEP215 have been extensively studied with a detailed description of their molecular mechanisms, which are among the primary targets for drug discovery. Moreover, CEP27, CEP55, CEP70, CEP110, CEP120, CEP135, CEP192, CEP250, CEP290, and CEP350 also seem promising for future drug discovery approaches. Since the overview implicates that the overall researches on CEP proteins are not yet able to present significant details required for effective therapeutics development, thus, it is timely to discuss the importance of future investigations in this field.

Investigation of binding phenomenon of NSP3 and p130Cas mutants and their effect on cell signalling.

Members of the novel SH2-containing protein (NSP3) and Crk-associated substrate (p130Cas) protein families form a multi-domain signalling platforms that mediate cell signalling process. We analysed the damaging consequences of three mutations, each from NSP3 (NSP3(L469R), NSP3(L623E), NSP3(R627E)) and p130Cas (p130Cas(F794R), p130Cas(L787E), p130Cas(D797R)) protein with respect to their native biological partners. Mutations depicted notable loss in interaction affinity towards their corresponding biological partners. NSP3(L469R) and p130Cas(D797R) mutations were predicted as most prominent in docking analysis. Molecular dynamics (MD) studies were conducted to evaluate structural consequences of most prominent mutation in NSP3 and p130Cas obtained from the docking analysis. MD analysis confirmed that mutation in NSP3(L469R) and p130Cas(D797R) showed significant structural deviation, changes in conformations and increased flexibility, which in turn affected the binding affinity with their biological partners. Moreover, the root mean square fluctuation has indicated a rise in fluctuation of residues involved in moderate interaction acquired between the NSP3 and p130Cas. It has significantly affected the binding interaction in mutant complexes. The results obtained in this work present a detailed overview of molecular mechanisms involved in the loss of cell signalling associated with NSP3 and p130Cas protein.