Alcohol effect on Golgi morphology and function

描述（由申请人提供）：慢性酒精滥用和酒精中毒与高发病率和死亡率有关，并已知会导致严重的健康问题，如酒精性肝病。据报道，乙醇暴露的肝细胞中蛋白质转运和糖基化发生改变，细胞凋亡增加。但其机制仍未得到解决。最近，我们发现非肌肉肌球蛋白IIA （NMIIA）是一种运动蛋白，可与高尔基糖基转移酶（GT）的细胞质尾部相互作用，诱导应激细胞中的高尔基断裂。在体外和体内暴露于酒精的肝细胞中检测到高尔基体碎片。酒精代谢物是造成这种效果的原因。酒精处理也增加了Rab6A GTPase、NMIIA、caspase-3活性、NMIIA-GT复合物，但降低了高尔基基质蛋白、巨肽肽和GT。在对照细胞中，巨肽肽的敲除保留了内质网中的GT。NMIIA或Rab6A的下调可防止酒精治疗引起的高尔基体碎裂。结果表明NMIIA和Rab6A与酒精诱导的高尔基体断裂密切相关。此外，酒精治疗诱导的GT减少可以解释为：(a) NMIIA-GT复合物增加导致高尔基体向内质网逆行运输升高，以及(b) caspase-3活性激活导致巨肽肽降解升高导致高尔基体靶向性受损。我们提出验证酒精治疗诱导的高尔基体碎片是导致糖基化和asialal糖蛋白受体功能降低以及诱导细胞凋亡的原因。拟议研究的四个具体目标是：1。研究在酒精特异性高尔基断裂过程中，Rab6A如何调节NMIIA与GT的相互作用，随后增加NMIIA-GT复合物；2. 研究酒精处理诱导的caspase -3活性升高如何损害GT内质网到高尔基体的转运；3. 确定酒精处理诱导的高尔基体碎裂如何影响糖结构和asialal糖蛋白受体的功能，包括细胞凋亡；和4。验证在酒精处理小鼠肝细胞VA-13细胞中获得的特异性aims 1-3的结果，无论有无功能性asialal糖蛋白受体。完成这项研究的目标将扩大我们对酒精滥用引起的细胞死亡调控的理解，并有助于确定开发治疗酒精性肝病的潜在靶点。

DESCRIPTION (provided by applicant): Chronic alcohol abuse and alcoholism are associated with high morbidity and mortality and known to cause major health problems such as alcoholic liver disease. Altered protein trafficking and glycosylation, and increased apoptosis have been reported in ethanol-exposed liver cells. But the mechanism remains unresolved. Recently, we found that non-muscle myosin IIA (NMIIA), a motor protein, interacts with the cytoplasmic tail of Golgi glycosyltransferases (GT) to induce Golgi fragmentation in cells under stress. The Golgi fragmentation was detected in hepatocytes exposed to alcohol in vitro and in vivo. Alcohol metabolites are responsible for this effect. Alcohol treatment also increases Rab6A GTPase, NMIIA, caspase-3 activity, NMIIA-GT complexes but decreases Golgi matrix protein, Giantin, and GT. In control cells, knockdown of Giantin retains GT in the endoplasmic reticulum. Knockdown of NMIIA or Rab6A prevents alcohol treatment- induced Golgi fragmentation. The results suggest that NMIIA and Rab6A are intimately involved in Golgi fragmentation induced by alcohol treatment. Further, the reduction of GT induced by alcohol treatment could be explained by (a) its elevated Golgi-to-endoplasmic reticulum retrograde transport forced by increased NMIIA-GT complexes coupled with (b) its impaired Golgi targeting resulted from elevated degradation of Giantin caused by activated caspase-3 activity. We propose to test the hypothesis that alcohol treatment- induced Golgi fragmentation is responsible for reduced glycosylation and function of asialoglycoprotein receptors as well as induction of apoptosis. The four specific aims of the proposed study are to: 1. Examine how during alcohol-specific Golgi fragmentation Rab6A regulates the interaction of NMIIA with GT followed by increased NMIIA-GT complexes; 2. Examine how elevated caspases-3 activity induced by alcohol treatment impairs ER-to-Golgi transport of GT; 3. Determine how the alcohol treatment-induced Golgi fragmentation affects glycan structure and function of asialoglycoprotein receptors including apoptosis; and 4. Validate the results of specific aims 1-3 obtained in VA-13 cells in the hepatocytes of alcohol-treated mice with and without functional asialoglycoprotein receptors. Accomplishment of the goal of the proposed study would expand our understanding of the regulation of cell death caused by ethanol abuse and help identify potential targets for developing therapy to treat alcoholic liver disease.