SIRT1, human ortholog of yeast SIR2 protein, deacetylates histones and several other transcription factors. Recently, SIRT1 has emerged as a drug target for treating age related diseases, type II diabetes, neurodegeneration, inflammation and cancer. Here, we have optimized production of functionally active wild type full-length SIRT1 protein and its N-terminal deleted mutants. In a comparative study, we found that the region containing 192–208 amino acids towards the N-terminus is critical for right conformational folding of the protein to retain its deacetylase activity. The EC 50 and IC 50 values obtained with standard modulators showed that the SRT 748 & SRT 556 can deacetylate substrate and are activated by resveratrol, whereas, deacetylase activity of all the other deletion mutants (SRT 540 , SRT 532 , SRT 507 and SRT 503 ) was lost. We further report that the peptide substrate K m for SRT 748 (70 ± 5.2 μM) was comparable to SRT 556 (93 ± 5.4 μM). The K m for NAD + substrate was 176 & 274 μM for SRT 748 and SRT 556 , respectively. Similar substrate affinity studies demonstrate that either of the protein (SRT 748 or SRT 556 ) can be utilized for screening SIRT1 modulators. We have also examined critical regions in SIRT1 required for deacetylase activity as well as kinetic analyses of SIRT1 proteins.
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Comparative deacetylase activity of wild type and mutants of SIRT1
Malik, Renu; Kashyap, Akriti; Bansal, Karuna; Sharma, Pratibha; Rayasam, Geeta Vani; Davis, Joseph Alex; Bora, Roop Singh; Ray, Abhijit; Saini, Kulvinder Singh
Follow Biochemical and Biophysical Research Communications
, Volume 391 (1)
Elsevier – Jan 1, 2010
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