钙网蛋白酰基转移酶整合调控血管内皮稳态的机制研究

最近发现具酰基转移酶活性的钙网蛋白(CRT)呈多亚细胞定位，课题组证实胞浆CRT减轻而核CRT加重细胞损伤。本项目提出“钙网蛋白酰基转移酶是整合调控内皮稳态的关键分子：通过乙酰化胞浆肌动蛋白促进其功能成熟而维持内皮稳态；在核内通过乙酰化激活ATF4致血管内皮损伤“的假说。在证实过表达胞浆CRT保护而过表达核CRT损伤内皮的基础上，分别制备有酰基转移酶活性的重组胞浆或核CRT，研究胞浆CRT乙酰化肌动蛋白和核CRT乙酰化ATF4的作用及其修饰位点；研究肌动蛋白乙酰化位点突变对过表达胞浆CRT内皮保护的影响，证实胞浆CRT通过乙酰化肌动蛋白维持内皮稳态；研究ATF4乙酰化位点突变对过表达胞核CRT活化ATF4致内皮损伤的影响，证实核CRT乙酰化激活ATF4致内皮损伤。从CRT整合调控内皮稳态的角度，为血管损伤及心血管病防治提供新靶点。

Recently, it is found that calreticulin (CRT) possessed transacetylase and located in nucleus, cytoplasm, and other sub-cellular organelles, besides endoplasmic reticulum. We found that CRT in cytoplasm attenuated, while in nucleus aggravated cell damage. We hypothesize that “Calreticulin transacetylase is a master regulator for integratively regulating endothelial homeostasis. CRT in cytoplasm maintains endothelial homeostasis by acetylation of actin to promote actin’s maturation and to maintain actin’s structural/functional properties, while in nucleus CRT damages endothelial cells (ECs) through activation of ATF4 by acetylation modification”. In the present project, we shall verify that CRT in cytoplasm is cytoprotective, while CRT in nucleus damages ECs. The recombinant CRT with transacetylase specifically localized in cytoplasm or nucleus will be prepared. We shall identified the acetylation modification and its sites of actin acetylated by cytosol CRT and the acetylation modification and its sites of activating transcription factor 4 (ATF4) acetylated by nuclear CRT. To demonstrate cytosol CRT regulating endothelial homeostasis by acetylating actin, the effects of cytosol CRT over-expression on structure and function of actin, and ECs protection will be investigated, after actin is mutated at putative acetylation sites. The effects of nuclear CRT over-expression on ATF4 and ECs damage will be investigated following ATF4’s mutation at putative acetylation sites, to demonstrate that nuclear CRT damages ECs by acetylating ATF4. The results of the project will provide a new target for mechanisms underlying vascular damage and treatment of cardio-cerebrovascular diseases from CRT’s integrative regulation of endothelial homeostasis.