Ponnusamy Kalaiarasan

miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2 : a potential for therapeutic intervention

IntroductionNew levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored.MethodsChanges in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2.ResultsIt was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2.Conclusionsmir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests that miR-24-2 is more effective in controlling H2AX gene expression, regardless of the change in gene copy number. Further, the study indicates that combination therapy with miR-24-2 along with an anticancer drug such as cisplatin could provide a new avenue in cancer therapy for patients with tumors otherwise resistant to drugs.

Biophysical insight into the anti-amyloidogenic behavior of taurine

In this work, we investigated the inhibitory ability of taurine on the aggregation of Human serum albumin (HSA) and also examined how it controls the kinetic parameters of the aggregation process. We demonstrated the structural alterations in the HSA after binding to the taurine at 65 °C by exploiting various biophysical techniques. UV-vis spectroscopy was used to check the turbidometric changes in the protein. Thioflavin T fluorescence kinetics was subjected to explore kinetic parameters comparing the amyloid formation in the presence of varying concentration of taurine. Further, Congo red binding and ANS binding assays were performed to determine the inhibitory effect of taurine on HSA fibrillation process and surface hydrophobicity modifications occurring before and after the addition of taurine with protein, respectively. Far UV CD and Dynamic Light Scattering (DLS) confirmed that taurine stabilized the protein α-helical structure and formed complex with HSA which is further supported by differential scanning calorimetry (DSC). Moreover, microscopic imaging techniques were also done to analyze the morphology of aggregation formed. Taurine is also capable of altering the cytotoxicity of the proteinaceous aggregates. Molecular docking study also deciphered the possible residues involved in protein and drug interaction.

Genetic variations and interactions in anti-inflammatory cytokine pathway genes in the outcome of leprosy: a study conducted on a MassARRAY platform.

BACKGROUND Mycobacterium leprae is the etiologic pathogen that causes leprosy. The outcome of disease is dependent on the host genetic background. METHODS We investigated the association of 51 single-nucelotide polymorphisms (SNPs) in anti-inflammatory cytokines (IL-10, TGFB1, IL-6, IL-4, and IL-13) and receptors (IL-10RA, IL-10RB, TGFBR1, TGFBR2, IL-6R, IL-4R, IL-5RA, IL-5RB, and IL-13RA1) with susceptibility to leprosy in a case-control study from New Delhi in northern India. This was followed by replication testing of associated SNPs in a geographically distinct and unrelated population from Orissa in eastern India. The functional potential of SNPs was established with in vitro reporter assays. RESULTS Significant associations (P < .05) were observed for 8 polymorphisms (rs1800871, rs1800872, and rs1554286 of IL-10; rs3171425 and rs7281762 of IL-10RB; rs2228048 and rs744751 of TGFBR2; and rs1800797 of IL-6) with leprosy. This association was replicated for 4 SNPs (rs1554286 of IL-10, rs7281762 of IL-10RB, rs2228048 of TGFBR2, and rs1800797 of IL-6). The interaction study revealed a significantly greater association with leprosy risk than was obtained for any SNP individually. CONCLUSIONS This study provides an interesting insight on the cumulative polygenic host component that regulates leprosy pathogenesis.

Dominant negative mutations affect oligomerization of human pyruvate kinase M2 isozyme and promote cellular growth and polyploidy.

This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM2) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM2 in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM2 by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM2 showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM2 by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.

mtDNA germ line variation mediated ROS generates retrograde signaling and induces pro-cancerous metabolic features

mtDNA non-synonymous germ line variation (G10398A; p.A114T) has remained equivocal with least mechanistic understanding in showing an association with cancer. This has necessitated showing in-vitro how an over-expression within mitochondria of either of the variants produces higher intracellular ROS, resulting in differential anchorage dependent and independent growth. Both these features were observed to be relatively higher in ND3:114T variant. An elevated amount of intracellular carbonylated proteins and a reduced activity of a key glycolytic enzyme, Pyruvate kinase M2, along with high glucose uptake and lactate production were other pro-cancerous features observed. The retrograde signaling through surplus ROS was generated by post-ND3 over-expression regulated nuclear gene expression epigenetically, involving selectively the apoptotic-DDR-pathways. The feature of ND3 over-expression, inducing ROS mediated pro-cancerous features in the cells in in vitro, was replicated in a pilot study in a limited number of sporadic breast tumors, suggesting the importance of mitochondrial germ-line variant(s) in enabling the cells to acquire pro-cancerous features.

Mapping of PARK2 and PACRG overlapping regulatory region reveals LD structure and functional variants in association with leprosy in unrelated indian population groups.

Leprosy is a chronic infectious disease caused by Mycobacterium Leprae, where the host genetic background plays an important role toward the disease pathogenesis. Various studies have identified a number of human genes in association with leprosy or its clinical forms. However, non-replication of results has hinted at the heterogeneity among associations between different population groups, which could be due to differently evolved LD structures and differential frequencies of SNPs within the studied regions of the genome. A need for systematic and saturated mapping of the associated regions with the disease is warranted to unravel the observed heterogeneity in different populations. Mapping of the PARK2 and PACRG gene regulatory region with 96 SNPs, with a resolution of 1 SNP per 1 Kb for PARK2 gene regulatory region in a North Indian population, showed an involvement of 11 SNPs in determining the susceptibility towards leprosy. The association was replicated in a geographically distinct and unrelated population from Orissa in eastern India. In vitro reporter assays revealed that the two significantly associated SNPs, located 63.8 kb upstream of PARK2 gene and represented in a single BIN of 8 SNPs, influenced the gene expression. A comparison of BINs between Indian and Vietnamese populations revealed differences in the BIN structures, explaining the heterogeneity and also the reason for non-replication of the associated genomic region in different populations.

Structure-based screening of inhibitors against KPC-2: designing potential drug candidates against multidrug-resistant bacteria.

KPC-2 β-lactamase demonstrates a wide substrate spectrum that includes carbapenamases, oxyimino-cephalosporins, and cephamycins. In addition, strains harboring KPC-type β-lactamases are often identified as resistant to standard β-lactamase inhibitors. Thus, KPC-2 carbapenems present a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain mysterious. Inhibiting the function of these resistance enzymes could control the hydrolysis of antibiotics. In the present study, we have reported two novel (non-β-lactatam) compounds that inhibit the activity of the KPC-2 enzyme. These compounds were identified by structure-based virtual screening using computational docking programs and molecular dynamics simulations with the solved crystal structure. Two compounds (ZINC01807204 and ZINC02318494) were selected on the basis of fitness scores from docking program and 5 ns molecular dynamics simulations. These commercially available compounds have been procured and their biological activity was experimentally evaluated on the E. coli strain carrying recombinant KPC-2. These new compounds in combination with ceftazidime and cefoxitin exhibited the Minimum Inhibitory Concentration (MIC) values of 2 and 8 μg/ml respectively, which were found to be lower as compared to known β-lactamase inhibitors. Moreover, these compounds were also found to have comparable MICs values being 64 μg/ml in combination with ceftriaxone. This study explored novel inhibitors against KPC-2, a class A β-lactamase, which may be putative drug candidates against KPC-2 producing bacterial infection.

Insight into the Effect of Inhibitor Resistant S130G Mutant on Physico-Chemical Properties of SHV Type Beta-Lactamase: A Molecular Dynamics Study

Bacterial resistance is a serious threat to human health. The production of β-lactamase, which inactivates β-lactams is most common cause of resistance to the β-lactam antibiotics. The Class A enzymes are most frequently encountered among the four β-lactamases in the clinic isolates. Mutations in class A β-lactamases play a crucial role in substrate and inhibitor specificity. SHV and TEM type are known to be most common class A β-lactamases. In the present study, we have analyzed the effect of inhibitor resistant S130G point mutation of SHV type Class-A β-lactamase using molecular dynamics and other in silico approaches. Our study involved the use of different in silico methods to investigate the affect of S130G point mutation on the major physico-chemical properties of SHV type class A β-lactamase. We have used molecular dynamics approach to compare the dynamic behaviour of native and S130G mutant form of SHV β-lactamase by analyzing different properties like root mean square deviation (RMSD), H-bond, Radius of gyration (Rg) and RMS fluctuation of mutation. The results clearly suggest notable loss in the stability of S130G mutant that may further lead to decrease in substrate specificity of SHV. Molecular docking further indicates that S130G mutation decreases the binding affinity of all the three inhibitors in clinical practice.

PARK2 and proinflammatory/anti-inflammatory cytokine gene interactions contribute to the susceptibility to leprosy: a case–control study of North Indian population

Objectives Cytokines and related molecules in immune-response pathways seem important in deciding the outcome of the host–pathogen interactions towards different polar forms in leprosy. We studied the role of significant and functionally important single-nucleotide polymorphisms (SNPs) in these genes, published independently from our research group, through combined interaction with an additional analysis of the in silico network outcome, to understand how these impact the susceptibility towards the disease, leprosy. Design The study was designed to assess an overall combined contribution of significantly associated individual SNPs to reflect on epistatic interactions and their outcome in the form of the disease, leprosy. Furthermore, in silico approach was adopted to carry out protein–protein interaction study between PARK2 and proinflammatory/anti-inflammatory cytokines. Setting Population-based case–control study involved the data of North India. Protein–protein interaction networks were constructed using cytoscape. Participants Study included the data available from 2305 Northern Indians samples (829 patients with leprosy; 1476 healthy controls), generated by our research group. Primary and secondary outcome measures For genotype interaction analysis, all possible genotype combinations between selected SNPs were used as an independent variable, using binary logistic regression with the forward likelihood ratio method, keeping the gender as a covariate. Results Interaction analysis between PARK2 and significant SNPs of anti-inflammatory/proinflammatory cytokine genes, including BAT1 to BTNL2-DR spanning the HLA (6p21.3) region in a case–control comparison, showed that the combined analysis of: (1) PARK2, tumour necrosis factor (TNF), BTNL2-DR, interleukin (IL)-10, IL-6 and TGFBR2 increased the risk towards leprosy (OR=2.54); (2) PARK2, BAT1, NFKBIL1, LTA, TNF-LTB, IL12B and IL10RB provided increased protection (OR=0.26) in comparison with their individual contribution. Conclusions Epistatic SNP–SNP interactions involving PARK2 and cytokine genes provide an additive risk towards leprosy susceptibility. Furthermore, in silico protein–protein interaction of PARK2 and important proinflammatory/anti-inflammatory molecules indicate that PARK2 is central to immune regulation, regulating the production of different cytokines on infection.

In Silico Screening, Genotyping, Molecular Dynamics Simulation and Activity Studies of SNPs in Pyruvate Kinase M2

Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs) and 2 mutations of Human Pyruvate Kinase (PK) M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP) was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD) simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.