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Inhibition studies of SIRT1

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Silent Information Regulator 2 Sir2 of Saccharomyces cerevisiae is the progenitor of an ancient family of proteins known as sirtuins. Sirtuins act on substrates in various regulatory pathways critical to normal cell function. Human SIRT1 is the closest known homologue of yeast Sir2. This study focuses on the function of human SIRT1 and the influence of this protein on the host inflammatory response.To better understand the role of SIRT1 and its role in disease, a working clone that expresses the SIRT1 protein is necessary. In this study, the human SIRT1 gene was amplified from commercially available template DNA using the Polymerase Chain Reaction PCR. The SIRT1 gene was ligated into pET28b+, a bacterial expression vector that works in Escherichia coli. The SIRT1 gene is expressed under control of the T7 promoter, which is not recognized by the transcriptional machinery in E.coli. Protein expression does not occur until a source of T7 RNA polymerase is provided. Once obtained, the SIRT1-pET28b+ plasmid was transferred into TOP10, a bacterial maintenance strain.Once established, the SIRT1-pET28b+ construct is transformed into an expression host containing a chromosomal copy of the T7 RNA polymerase gene. Induced by the addition of Isopropylthio-β-Galactoside IPTG, the expression cell diverts almost all of its resources to express the target SIRT1 gene. Overexpression of the SIRT1 protein allows for characterization of enzyme activity. Characterization of the mechanism where SIRT1 is inhibited by the native inhibitor nicotinamide NAM and the clinical antimycobacterial agent pyrazinamide PZA is needed.The focus of this thesis is elucidation of the mechanism of inhibition of SIRT1. PZA, a clinical agent used to treat tuberculosis TB, has been shown to allosterically inhibit SIRT1. It is of particular interest to determine where PZA, a NAM derivative, binds SIRT1 and if this binding can be included in natural regulatory network mechanisms within the cell. At the heart of this study, are attempts to understand the complex binding interactions of both PZA and pyrazinoic acid POA, the chemotherapeutically active metabolite of PZA, in the presence of NAM. Competition experiments with these agents may prove useful to determine if these molecules interact at the same site, if the molecules can be displaced competitively, or if certain residues within parts of the enzyme are essential for characteristic binding.

Boston University Theses and Dissertations -

Autor: Bonetti, Kelly - -


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