Department of Biochemistry, Bose Institute, Kolkata, India.
Cell Microbiol. 2010 Jul;12(7):1002-14. doi: 10.1111/j.1462-5822.2010.01449.x. Epub 2010 Feb 9.
We have discovered four sirtuin genes in Entamoeba histolytica, two of which are similar to eukaryotic sirtuins and two to bacterial and archaeal sirtuins. The eukaryotic sirtuin homologue, EhSir2a, showed NAD(+)-dependent deacetylase activity and was sensitive to class III HDAC inhibitors. Localization of EhSir2a at different cellular sites suggested that this deacetylase could have multiple targets. Using an E. histolytica cDNA library in the yeast two-hybrid genetic screen, we identified several proteins that bound to EhSir2a. These proteins included Eh alpha-tubulin, whose interaction with EhSir2a was validated in E. histolytica. We have shown that EhSir2a deacetylated tubulin and localized with microtubules in E. histolytica. Increased expression levels of EhSir2a in stable transformants led to reduced number of microtubular assemblies in serum synchronized cells. This effect was abrogated by mutations in the deacetylase domain of EhSir2a, showing that EhSir2a deacetylase activity affected the stability and number of microtubular assemblies during the cell cycle of E. histolytica. Our results suggest that epigenetic modification of tubulin by EhSir2a is one of the mechanisms that regulates microtubular assembly in E. histolytica.
我们在溶组织内阿米巴中发现了 4 个组蛋白脱乙酰酶基因，其中 2 个与真核生物的组蛋白脱乙酰酶相似，2 个与细菌和古菌的组蛋白脱乙酰酶相似。真核生物组蛋白脱乙酰酶同源物 EhSir2a 表现出 NAD(+)依赖的脱乙酰酶活性，并对 III 类组蛋白去乙酰化酶抑制剂敏感。EhSir2a 在不同细胞部位的定位表明，这种脱乙酰酶可能有多个靶标。我们利用酵母双杂交遗传筛选中的溶组织内阿米巴 cDNA 文库，鉴定出几种与 EhSir2a 结合的蛋白质。这些蛋白质包括 Eh alpha-微管蛋白，其与 EhSir2a 的相互作用在溶组织内阿米巴中得到了验证。我们已经表明 EhSir2a 使微管蛋白脱乙酰化，并在溶组织内阿米巴中与微管共存。稳定转化体中 EhSir2a 的表达水平增加导致血清同步化细胞中微管组装数量减少。EhSir2a 的脱乙酰酶结构域中的突变消除了这种效应，表明 EhSir2a 脱乙酰酶活性影响溶组织内阿米巴细胞周期中微管组装的稳定性和数量。我们的结果表明，EhSir2a 通过组蛋白修饰对微管蛋白进行表观遗传修饰，是调节溶组织内阿米巴微管组装的机制之一。
