钙网蛋白酰基转移酶整合调控血管内皮稳态的机制研究

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.

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。

血管内皮功能障碍是心脑血管疾病的共同发病环节，改善内皮功能已成为心血管疾病治疗的新策略。本课题以钙网蛋白酰基转移酶调控血管内皮功能为切入点，研究同型半胱氨酸和微波致微血管内皮损伤特点，证实同型半胱氨酸呈剂量和时间依赖性损伤微血管内皮细胞；研究外源性钙网蛋白对于内皮细胞微波辐射损伤的影响，证实钙网蛋白减轻微波辐射造成的细胞死亡、凋亡和功能异常；研究外源性钙网蛋白对于微血管内皮细胞骨架和actin蛋白乙酰化及聚合的影响，证实钙网蛋白通过乙酰化actin减轻受损伤内皮的actin解聚而减轻微血管内皮细胞损伤；进一步研究钙网蛋白对于缺氧/复氧后心肌细胞的保护作用及其机制，证实钙网蛋白通过改善自噬流减轻缺氧/复氧造成的H9c2细胞损伤。上述研究结果从钙网蛋白调控细胞骨架和自噬的角度为内皮损伤相关心脑血管疾病的防治提供了新思路和新药研发靶点。本项目部分研究结果获得国家发明专利一项，国家科技进步二等奖一项。