Control of COPII vesicle trafficking by intracellular protein glycosylation

三分之一的真核蛋白通过分泌途径靶向特定位置， 包括内质网（ER）、高尔基体、质膜或细胞外环境。由于误导 蛋白质不能发挥功能，分泌途径对于建立和维持正常细胞和组织至关重要 physiology.特别地，COPII蛋白复合物，其介导囊泡从ER运输到高尔基体， 是蛋白质靶向的关键控制点此外，COPII基因的突变导致一系列人类疾病。 疾病，包括颅-颅-缝发育不良（CLSD）和成骨不全（OI）。详细 需要了解COPII囊泡运输的知识以了解其在细胞生理学中的作用并治疗疾病 在其中它被破坏。然而，尽管COPII的核心机制已经得到了很好的定义， 哺乳动物细胞响应于发育、代谢或病理学线索而调节COPII活性。 最近，我们和其他人发现几种COPII蛋白被O-连接的b-N-修饰， 乙酰葡糖胺（O-GlcNAc），细胞内蛋白质糖基化的动态形式。有趣的是，糖基化 COPII组分包括Sec 23 A和Sec 24 D，其分别在CLSD和OI中突变，表现出 在胶原蛋白的错构和骨骼的畸形方面。然而，O-GlcNAc酰化对COPII的影响 路径仍不清楚。在初步工作中，我们使用化学生物学方法表明，至少有四个 COPII组分，包括Sec 23和Sec 24，参与O-GlcNAc介导的蛋白质-蛋白质相互作用， 人体细胞此外，我们发现，药理学抑制O-GlcNAc循环阻碍COPII 贩卖人口最后，我们发现Sec 23 A的非糖基化突变体不能拯救胶原运输， 和骨骼发育缺陷的Sec 23 A-突变破碎机斑马鱼。总之，这些结果表明，网站- 单个COPII蛋白的特异性O-GlcNAc酰化控制脊椎动物细胞和组织中的囊泡运输。 本项目的目的是确定O-GlcNAc酰化作用对 COPII途径。我们将通过三个具体目标来实现我们的目标。在目标1中，我们将剖析 O-GlcNAc循环对COPII囊泡运输的功能影响。在目标2中，我们将定义特定地点的 人细胞中Sec 23 A和Sec 24 D的O-GlcNAc酰化。在目标3中，我们将确定COPII的贡献 CLSD和OI脊椎动物模型中的O-GlcNAc化。我们的工作将为O-GlcNAcylation 调节细胞和组织中的蛋白质运输，并可能揭示治疗COPII疾病的新机会 通过靶向蛋白质糖基化来治疗功能障碍，如CLSD和OI。

One third of all eukaryotic proteins pass through the secretory pathway for targeting 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. In particular, the COPII protein complex, which mediates vesicle trafficking from the ER to the Golgi, is a key control point for protein targeting. Moreover, mutations in COPII genes cause a range of human diseases, including cranio-lenticulo-sutural dysplasia (CLSD) and osteogenesis imperfecta (OI). Detailed knowledge of COPII vesicle 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 mammalian cells regulate COPII activity in response to developmental, metabolic or pathological cues. Recently, we and others found that several COPII proteins are modified by O-linked b-N- acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation. Interestingly, glycosylated COPII components include Sec23A and Sec24D, which are mutated in CLSD and OI, respectively, manifesting in collagen mistrafficking and skeletal dysmorphology. However, the effects of O-GlcNAcylation on the COPII pathway remain unclear. In preliminary work, we used a chemical biology approach to show that at least four COPII components, including Sec23 and Sec24, engage in O-GlcNAc-mediated protein-protein interactions in human cells. In addition, we showed that pharmacological inhibition of O-GlcNAc cycling hinders COPII trafficking. Finally, we found that an unglycosylatable mutant of Sec23A failed to rescue the collagen trafficking and skeletogenesis defects of Sec23A-mutant crusher zebrafish. Together, these results suggest that site- specific O-GlcNAcylation of individual COPII proteins governs vesicle trafficking in vertebrate cells and tissues. The objective of this project is to define the mechanistic and functional effects of O-GlcNAcylation on the COPII pathway. We will accomplish our objective through three Specific Aims. In Aim 1, we will dissect the functional impact of O-GlcNAc cycling on COPII vesicle trafficking. In Aim 2, we will define the role of site-specific O-GlcNAcylation of Sec23A and Sec24D in human cells. In Aim 3, we will determine the contribution of COPII O-GlcNAcylation in vertebrate models of CLSD and OI. Our work will shed new light on how O-GlcNAcylation tunes protein trafficking in cells and tissues, and may reveal new opportunities to treat diseases of COPII dysfunction, such as CLSD and OI, by targeting protein glycosylation.