Control of COPII vesicle trafficking by intracellular protein glycosylation

项目摘要/摘要 三分之一的真核蛋白质通过分泌途径分选到特定的位置，包括 内质网、高尔基体、质膜或细胞外环境。因为错误定向的蛋白质 不能发挥作用，分泌途径对建立和维持正常的细胞和组织至关重要 生理学。COPII外壳蛋白复合体是一个关键的控制因素，它介导了内质网的顺行运输 以蛋白质为靶点。事实上，COPII基因的突变会导致一系列人类疾病，包括颅脑- 晶状体缝合不良(CLSD)，成骨不全(OI)的一个亚型，血液系统疾病和无数 神经缺陷。需要对COPII转运的详细知识才能理解它在细胞生理学中的作用 并治疗它被扰乱的障碍。然而，尽管核心COPII机制定义得很好，但几乎没有定义 了解脊椎动物细胞如何调节COPII活性，以响应正常或病理信号或压力。 我们和其他人发现，几种COPII蛋白可以被O-连接的b-N-乙酰氨基葡萄糖修饰 (O-GlcNAc)，一种细胞内蛋白质糖基化的动态形式。在前一个项目期开始时， O-GlcN酰化对COPII的影响几乎完全未知。从那时起，我们定义了范围 在核心COPII系统中O-GlcN酰化的研究，确定了O-GlcNAc在囊泡中循环的功能效应 走私，设计了新的定量糖蛋白组学方法来描述O-GlcNAc的变化 分泌途径应激和其他刺激，并证明特定的Sec23A O-GlcNAc位点是 培养的人细胞和发育中的软骨细胞内源性胶原运输所需 斑马鱼。综上所述，这些结果表明，COPII蛋白的位点特异性O-GlcN酰化控制 脊椎动物细胞和组织的货物贩运。然而，主要的悬而未决的问题仍然存在，包括 O-GlcNAc对COPII蛋白施加的机制作用，上游刺激调节COPII O- GlcN酰化与O-GlcNAc信号在早期分泌途径中的全球格局。在这里，我们建议 在下一次颁奖期间解决这些重要问题。 在目标1中，我们将剖析O-GlcNAc循环影响COPII囊泡的分子机制 贩卖人口。在目标2中，我们将识别控制COPII蛋白O-GlcN酰化的上游刺激因素，并确定 这种信号在蛋白质分泌和细胞周期进程中的下游作用。在目标3中，我们将使用我们的 糖蛋白质组学方法研究响应COPII货物贩运的蛋白质组范围的O-GlcNAc信号， 并提供了蛋白质分泌中O-GlcN酰化和磷酸化之间的串扰的完整图像。 我们的工作将为O-GlcNAc如何调控细胞和组织的贩运提供新的线索，并可能揭示新的 通过操纵蛋白质糖基化来治疗COPII功能障碍疾病的机会。

Project Summary/Abstract One third of eukaryotic proteins transit the secretory pathway for sorting to specific locations, including the endoplasmic reticulum (ER), Golgi, plasma membrane or extracellular milieu. Since misdirected proteins cannot function, the secretory pathway is critical for establishing and maintaining normal cell and tissue physiology. The COPII coat protein complex, which mediates anterograde trafficking from the ER, is a key control point for protein targeting. Indeed, mutations in COPII genes cause a range of human diseases, including cranio- lenticulo-sutural dysplasia (CLSD), a subtype of osteogenesis imperfecta (OI), hematologic disorders and myriad neurological defects. Detailed knowledge of COPII trafficking is required to understand its role in cell physiology and to treat disorders in which it is disrupted. However, while the core COPII machinery is well defined, little is known about how vertebrate cells regulate COPII activity in response to normal or pathological signals or stress. We and others have found that several COPII proteins are modified by O-linked b-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation. At the start of the prior project period, the effects of O-GlcNAcylation on COPII remained almost entirely unknown. Since then, we have defined the scope of O-GlcNAcylation in the core COPII system, identified functional effects of O-GlcNAc cycling in vesicle trafficking, devised new quantitative glycoproteomics methods to profile O-GlcNAc changes in response to secretory pathway stress and other stimuli, and demonstrated that particular Sec23A O-GlcNAc sites are required for endogenous collagen trafficking in cultured human cells and in the chondrocytes of developing zebrafish. Together, these results demonstrate that site-specific O-GlcNAcylation of COPII proteins governs cargo trafficking in vertebrate cells and tissues. However, major unanswered questions remain, including the mechanistic effects that O-GlcNAc exerts on COPII proteins, the upstream stimuli that modulate COPII O- GlcNAcylation and the global landscape of O-GlcNAc signaling in the early secretory pathway. Here, we propose to address these important questions in the next award period. In Aim 1, we will dissect the molecular mechanisms by which O-GlcNAc cycling influences COPII vesicle trafficking. In Aim 2, we will identify upstream stimuli that control COPII protein O-GlcNAcylation and determine the downstream effects of this signaling in protein secretion and cell cycle progression. In Aim 3, we will use our glycoproteomics methods to canvass proteome-wide O-GlcNAc signaling in response to COPII cargo trafficking, and provide an integrated picture of crosstalk between O-GlcNAcylation and phosphorylation in protein secretion. Our work will shed new light on how O-GlcNAc regulates trafficking in cells and tissues, and may reveal new opportunities to treat diseases of COPII dysfunction by manipulating protein glycosylation.