Targeting and Function of Golgi Membrane Proteins

描述（由申请人提供）：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质的翻译后加工和分选中发挥核心作用。高尔基复合体的最佳研究功能之一是寡糖加工，其通过糖基转移酶和糖苷酶的表达和定位精确控制。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。高尔基复合体的分类功能也很重要，因为蛋白质和脂质的错误定位会导致疾病。在哺乳动物细胞中，高尔基复合体有一种不寻常的结构，由一组堆叠的池膜组成，在细胞核附近聚集成一条带状。这个复杂结构的功能尚不清楚。具有大卷曲螺旋结构域的外周高尔基体膜蛋白称为golgins，其与高尔基体的结构和功能有关。先前的研究表明，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蛋白GLUT 4。Golgin-160也是促凋亡刺激后半胱天冬酶裂解的早期靶点，表明在细胞应激期间运输功能可能迅速失活。以Golgin-160为模型，探讨货物运输、压力感知和高尔基体结构的耦合。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，检验Golgin-160与特定货物分子的相互作用是其有效的高尔基后分选所必需的假设;（2）确定抗半胱天冬酶形式的Golgin-160破坏细胞对应激反应所需的膜运输步骤的机制;和（3）通过使用靶向高尔基体复合体的药物、测量高尔基体膜上半胱天冬酶-2的局部活化、并确定阻断高尔基体切割片段的核积累的结果，来检验高尔基体定位的半胱天冬酶-2切割高尔基体是响应特异性应激所必需的假设。这些研究将增强对高尔基体结构及其与哺乳动物细胞中高尔基体功能的关系的理解，并可能揭示从高尔基体到细胞核的新信号通路。高尔基复合体是一种普遍存在的细胞器，有助于将货物分子运送到细胞表面。Golgin-160是一种高尔基体常驻蛋白，涉及影响心脏病和糖尿病的分子的适当表面递送，并且在导致细胞死亡的损伤后早期也被蛋白水解切割。对高尔基体-160的拟议实验将增加我们对货物递送到细胞表面的理解，以及高尔基体复合体在将应激信号转导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.