Targeting and Function of Golgi Membrane Proteins

描述(申请人提供)：高尔基复合体是一种普遍存在的真核细胞器，在新合成的蛋白质和脂类的翻译后处理和分类中发挥核心作用。高尔基复合体研究得最多的功能之一是寡糖加工，这是由糖基转移酶和糖苷酶的表达和定位精确控制的。糖基化模式在发育过程中发生变化，异常的糖基化模式可能有助于肿瘤细胞的转移。高尔基复合体的分选功能也是至关重要的，因为蛋白质和脂肪的错误定位可能会导致疾病。在哺乳动物细胞中，高尔基复合体具有一种不同寻常的结构，由堆叠在一起的池膜组成，在细胞核附近形成一条带状结构。这种精心设计的结构的功能尚不清楚。具有大卷曲结构域的外周高尔基体膜蛋白被称为高尔基体，与高尔基体的结构和功能有关。以前的工作表明，Golgin-160是有效分选某些货物分子所必需的，包括β-1肾上腺素能受体和葡萄糖转运体GLUT4。Golgin-160也是促凋亡刺激后半胱氨酸酶切割的早期靶点，这表明在细胞应激过程中运输功能可能迅速失活。以Golgin-160为模型，探讨了货物运输、应力传感和高尔基结构的耦合问题。该项目的具体目标是：(1)通过确定Golgin-160在货物运输中发挥作用的机制，验证Golgin-160与特定货物分子相互作用是其有效的高尔基后分选所必需的假说；(2)确定Caspase抗性版本的Golgin-160扰乱细胞对应激反应所需的膜运输步骤的机制；以及(3)通过使用靶向高尔基复合体的药物，测量高尔基体膜上caspase-2的局部激活，并确定阻止高尔基体裂解片段的核积累的后果，来检验高尔基体定位的caspase-2对高尔基体蛋白的切割是响应特定压力所必需的假设。这些研究将加深对高尔基体结构及其与哺乳动物细胞高尔基体功能关系的理解，并有可能发现一条从高尔基体到细胞核的新的信号通路。与公众健康相关高尔基复合体是一种无处不在的细胞器，有助于将货物分子运送到细胞表面。Golgin-160是一种驻留在高尔基体中的蛋白质，与影响心脏病和糖尿病的分子的适当表面递送有关，它也在受到导致细胞死亡的侮辱后早期被蛋白质分解。在Golgin-160上拟议的实验将增加我们对向细胞表面运送货物的理解，以及高尔基复合体在将应力信号传递到细胞其余部分中的作用。

DESCRIPTION (provided by applicant): The Golgi complex is a ubiquitous eukaryotic organelle that plays a central role in post-translational processing and sorting of newly synthesized proteins and lipids. One of the best-studied functions of the Golgi complex is oligosaccharide processing, which is precisely controlled by expression and localization of glycosyltransferases and glycosidases. Glycosylation patterns change during development, and aberrant glycosylation patterns may contribute to metastasis of tumor cells. The sorting function of the Golgi complex is also critical, since protein and lipid mistargeting can lead to disease. In mammalian cells, the Golgi complex has an unusual structure consisting of sets of stacked cisternal membranes gathered into a ribbon near the cell nucleus. The function of this elaborate structure is not known. Peripheral Golgi membrane proteins with large coiled-coil domains called golgins have been implicated in Golgi structure and function. Previous work has shown that golgin-160 is required for efficient sorting of certain cargo molecules, including the beta-1 adrenergic receptor and the glucose transporter GLUT4. Golgin-160 is also an early target for cleavage by caspases after pro-apoptotic stimuli, suggesting that the trafficking function may be rapidly inactivated during cellular stress. Using golgin-160 as a model, the coupling of cargo traffic, stress sensing and Golgi structure will be explored. The specific aims of the project are to: (1) Test the hypothesis that interaction of golgin-160 with specific cargo molecules is required for their efficient post-Golgi sorting by determining the mechanism by which golgin-160 functions in cargo trafficking; (2) Determine the mechanism by which a caspase-resistant version of golgin-160 disrupts membrane trafficking steps required for cellular response to stress; and (3) Test the hypothesis that cleavage of golgins by Golgi- localized caspase-2 is required for response to specific stresses by using drugs that target the Golgi complex, measuring local activation of caspase-2 at Golgi membranes, and determining the consequences of blocking nuclear accumulation of golgin cleavage fragments. These studies will enhance the understanding of Golgi structure and how it relates to Golgi function in mammalian cells, and potentially uncover a novel signaling pathway from the Golgi to the nucleus. PUBLIC HEALTH RELEVANCE The Golgi complex is a ubiquitous cellular organelle that is instrumental for delivering cargo molecules to the cell surface. Golgin-160 is a Golgi resident protein that is implicated in proper surface delivery of molecules that impact heart disease and diabetes, and it is also proteolytically cleaved early after insults that lead to cell death. The proposed experiments on golgin-160 will add to our understanding of cargo delivery to the cell surface and to the role of the Golgi complex in transducing stress signals to the rest of the cell.