特定位点乙酰化及小分子对锰超氧化物歧化酶活性调控机理的研究

Superoxide anion is an important molecule which mediates cellular signal transduction and aging, and manganese superoxide dismutase (MnSOD) is its crucial scavenger in mitochondria. Preliminary research indicates that lysine acetylation can regulate MnSOD activities dynamically, however, the regulatory mechanisms underlying which have not yet been elucidated..In the study, acetyllysines will be incorporated at the specific sites of MnSOD by the genetic code expansion technology, in order to investigate the regulatory mechanisms of acetylation modification for MnSOD activities employing enzymology and structural biology. Based on that, the activators of SIRT3, the upstream deacetylase of MnSOD, will be developed to realize the regulation of MnSOD activities and superoxide anion levels in vivo..We have examined one of lysine acetylation sites and investigated its effects on the structure and activities of MnSOD. Based on that, we will screen for small molecules which can activate SIRT3, reduce MnSOD acetylation level and increase MnSOD activities. And their antioxidative activities will be investigated in vivo as well. We will devote ourselves to solving the crystal structures of the two complexes of activator-SIRT3 and MnSOD-SIRT3. The strategy of controlling superoxide anion levels in vivo by posttranslational modification regulation will provide fundamental basis for developing the new anticancer and antiaging drugs targeting SIRT3.

超氧阴离子是介导细胞信号转导及老化的重要分子，而锰超氧化物歧化酶（MnSOD）则是线粒体中重要的超氧阴离子清除剂。初步研究表明赖氨酸乙酰化能动态调控MnSOD的活性，但其调控机理尚不清楚。.本研究将利用基因密码子扩展技术在MnSOD特定位点插入乙酰赖氨酸，利用酶学及结构生物学手段研究乙酰化修饰对MnSOD活性的调控机理，并在此基础上开发MnSOD去乙酰化酶SIRT3的激活剂，实现活体水平MnSOD酶活及超氧阴离子水平的调控。 .我们已经检测了一个位点乙酰化修饰对MnSOD结构和酶活的调控作用。在此基础上，我们将筛选激活SIRT3，降低MnSOD乙酰化并提高其酶活的小分子，同时在体内验证其抗氧化活性。我们将致力于解析激活剂-SIRT3和MnSOD-SIRT3两个复合物的晶体结构。这种通过翻译后修饰调控来控制超氧水平的策略将为靶向SIRT3新型抗癌抗衰老药物的研发奠定理论基础。

锰超氧化物歧化酶（MnSOD）是线粒体中重要的超氧阴离子清除剂，其活性受SIRT3介导的去乙酰化作用调控，特别是它的68位赖氨酸。为了探究68位赖氨酸乙酰化作用对MnSOD活性的影响，我们通过基因遗传密码子扩展技术将68位赖氨酸替换为乙酰赖氨酸，从而获得MnSOD突变体蛋白—MnSODK68AcK。我们解析了该乙酰化MnSOD的晶体结构，从而展示了68位赖氨酸乙酰化后MnSOD活性显著下降的结构基础和静电学基础。此外，基于SIRT3介导的MnSODK68AcK去乙酰化反应体系，我们筛选识别到了一个新的SIRT3激活剂—7-羟基-3-（4‘-甲氧苯基）香豆素（C12），它以高亲和力与SIRT3结合，能够在体外和胞内促进SIRT3去乙酰化从而提高MnSOD的活性，它将通过调控细胞乙酰化作用对众多疾病的治疗具有重要的潜在价值。