Department of Life Sciences

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Browsing Department of Life Sciences by Author "Dey, Sanghamitra"

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    Comparative Analysis of Eukaryotic Homo sapiens and Oryza sativa indica Class IV Sirtuins
    Dey, Sanghamitra
    Sirtuins are a class of proteins biologically acting as epigenetic modulators. They are generally found to be involved in regulating the essential cellular processes like metabolism, aging and genome stability. In our studies we have investigated the class IV sirtuins to explore the mechanistic and regulatory aspect of this protein family. To understand the role of class IV sirtuins better we took representatives from both human and plant. Human class IV sirtuins HsSIRT6 and HsSIRT7 are nuclear and nucleolar proteins, respectively whereas OsSRT1 is a nuclear protein. Using HsSIRT6 as a control/model, we investigated the biochemical and epigenetic activities of HsSIRT7 and OsSRT1. From our studies, we found that N and C terminal region of these proteins have important role in their enzymatic activities and substrate recognition. N-terminal region in HsSIRT7 is associated with protein-protein interactions and C-terminus is often associated with subcellular localization. In case of OsSRT1, the extended C-terminal region plays important role in substrate recognition and catalysis. Sirtuins in general have a unique substrate specificity. There were available reports of H3K9Ac deacetylation by OsSRT1 and H3K18Ac deacetylation by HsSIRT7. Our studies shed light on its dual enzyme capability with preference for mono ADP ribosylation (mono ADPr) over deacetylation. Just like its plant counterpart, a comparison of these dual activities suggests HsSIRT7's preference for the mono ADPr transfer over its deacetylation of H3K18Ac. Several new targets for both the deacetylation and ADP ribosylation were detected. Class IV sirtuins can specifically transfer the single ADP ribose group on its substrates in an enzymatic manner. So, this study is the first one to report the ADP ribosylation activity in HsSIRT7 and OsSRT1, which were previously reported as only deacetylases. This mono ADPr effect is not well known in plants, more so for deacetylases. The products of this reaction (NAM and ADP ribose) have immense negative effect on these enzymes suggesting a tighter regulation. Under various ranges of abiotic stress conditions in rice plants, we could link this ADPr activity to the DNA repair pathway by activating the enzyme PARP1. The histone specific deacetylation is also related with OsSRT1 upregulation under these stress situations. Metal toxicity in plants also influences these enzyme activities. Mono ADP ribosylation in cells is often linked to different metabolic disease conditions. This kind of modification of transcription factors, p53 and ELK4 by HsSIRT7 may play a key role in maintaining the tumor phenotype. Thus, HsSIRT7 becomes an important therapeutic hotspot for drug designing to counter several disease conditions. Screening of several chemical compounds with HsSIRT7 active site has been carried out in this study.
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    Structural and Biochemical characterization of a class III sirtuin, OsCobB in Oryza sativa indica and its association to stress response in plants
    Dey, Sanghamitra
    Sirtuins have attained recognition in the sphere of epigenetic regulation, owing to its affiliation with numerous biological processes including metabolism and genomic stability. Many post translational modifications (PTMs) are controlled by these sirtuins, playing critical roles in escaping abiotic and biotic stresses, although their pattern in plants have been poorly understood. In our investigation, a novel class III sirtuin has been discovered in Oryza sativa var indica with a high sequence identity with bacterial CobB. No class III sirtuins have been reported in plants till this study. Correlating molecular docking analysis with in-vitro and in-vivo deacylation assays helped in the understanding of various aspects of enzymatic mechanisms of OsCobB. We identified the important motifs for substrate and NAD+ binding like GAGISA, FGE, YXXR and TQNID, which were unique to class III sirtuin family. Although all modifications could be accommodated at the catalytic site, its selectivity also depended on the orientation of the peptide backbone and its interaction (bonded and non-bonded) with the sirtuin. OsCobB had a preference for the negatively charged lysine modifications like malonyl, succinyl and glutaryl through H-bonds with conserved YXXR motif. OsCobB can also accommodate the longer acyl modifications like myristoyl by a possible shift of its α4 helix. It was also established that an initial attachment of its co-substrate, NAD+ was mandatory to facilitate a conformational change for the correct binding of the peptide. In contrast to some class III members, OsCobB didnot exhibit any ADP-ribosyl transferase activity. We have identified this sirtuin to be majorly localized in the mitochondria with trace amounts in the nucleus. However, the preferential localization of this protein in the nucleus in response to low temperature and dehydration conditions could be related to its functioning. Histones (H3 and H4) as well as ACS were identified as the nuclear targets for this enzyme. Though OsCobB was incapable of efficiently deacetylating histone H4, it could deacetylate H3 at K9 and K18 residues. To cope with dehydration, OsCobB was capable of regulating the metabolism by removing different acyl groups like succinyl in H4 and butyryl, HIB and BHB in H3. Arsenic toxicity in the soil was also linked to OsCobB catalyzed H3 demalonylation and H4 desuccinylation. This enzyme was also capable of modulating mitochondrial ACS and IDH2 activity, favouring its deglutarylation and desuccinylation. This is the first mitochondrial CobB targeting important plant machinery under pathogen attack. NAD+ could be replaced by NADP+ as a cosubstrate in deacetylation reaction. OsCobB activity is insensitive to its product, NAM while showing sensitivity towards Fe2+ and Mn2+ ions. All-inclusive, this study discovered the catalytic abilities of rice class III sirtuin in conjunction with their substrates. A correlation between OsCobB overexpression and its preferential removal of lysine modifications on target substrates may hint at its regulation under stress.