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相关，并已知会导致重大健康问题，如酒精性肝病。在乙醇暴露的肝细胞中，蛋白质运输和糖基化发生改变，细胞凋亡增加。但机制仍然没有解决。最近，我们发现，非肌肉肌球蛋白IIA（NMIIA），一个运动蛋白，与高尔基体糖基转移酶（GT）的胞质尾相互作用，诱导高尔基体破碎在细胞应激。在体外和体内乙醇暴露的肝细胞中检测到高尔基体断裂。酒精代谢物是造成这种效应的原因。酒精处理还增加Rab 6A GT3，NMIIA，半胱天冬酶-3活性，NMIIA-GT复合物，但降低高尔基体基质蛋白，Giantin，和GT。在对照细胞中，Giantin的敲低保留内质网中的GT。NMIIA或Rab 6A的敲低防止酒精处理诱导的高尔基体片段化。结果表明，NMIIA和Rab 6A密切参与了由酒精处理引起的高尔基体片段化。此外，由酒精处理诱导的GT的减少可以通过以下来解释：（a）其由增加的NMIIA-GT复合物强制的高尔基体至内质网的逆行转运增加，以及（B）其受损的高尔基体靶向由活化的半胱天冬酶-3活性引起的Giantin的降解增加引起。我们建议检验这一假设，即酒精处理诱导的高尔基体断裂是负责降低糖基化和功能的去唾液酸糖蛋白受体，以及诱导细胞凋亡。建议研究的四个具体目标是：1。研究在酒精特异性高尔基体片段化过程中Rab 6A如何调节NMIIA与GT的相互作用，然后增加NMIIA-GT复合物; 2.研究如何升高半胱天冬酶-3活性诱导酒精治疗损害ER到高尔基体运输GT; 3.确定酒精处理诱导的高尔基体断裂如何影响聚糖结构和去唾液酸糖蛋白受体的功能，包括细胞凋亡;和4.在有和无功能性去唾液酸糖蛋白受体的酒精处理小鼠的肝细胞中，验证在VA-13细胞中获得的特定目标1-3的结果。实现拟议研究的目标将扩大我们对酒精滥用引起的细胞死亡调节的理解，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.