Control of COPII vesicle trafficking by intracellular protein glycosylation

所有真核蛋白中的三分之一通过分泌途径靶向特定位置， 包括内质网、高尔基体、质膜或细胞外环境。因为被误导了 蛋白质不能发挥作用，分泌途径是建立和维持正常细胞和组织的关键 生理学。特别是，COPII蛋白复合体，它介导囊泡从内质网到高尔基体的运输， 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，包括颅豆状结构发育不良(CLSD)和成骨不全(OI)。详细 需要了解COPII囊泡运输的知识才能了解它在细胞生理学中的作用并治疗疾病 在那里它被打乱了。然而，尽管核心COPII机制被很好地定义，但人们对它是如何定义的知之甚少 哺乳动物细胞调节COPII活性以响应发育、代谢或病理信号。 最近，我们和其他人发现一些COPII蛋白被O-连接的b-N-修饰。 乙酰氨基葡萄糖(O-GlcNAc)，一种细胞内蛋白质糖基化的动态形式。有趣的是，糖基化 COPII组分包括分别在CLSD和OI中突变的Sec23A和Sec24D，表现为 胶原蛋白断裂和骨骼畸形。然而，O-GlcN酰化对COPII的影响 途径仍不清楚。在前期工作中，我们使用化学生物学方法证明了至少有四种 COPII组分，包括Sec23和SEC24，参与O-GlcNAc介导的蛋白质-蛋白质相互作用 人类细胞。此外，我们还发现，O-GlcNAc循环的药理抑制会阻碍COPII 贩卖人口。最后，我们发现Sec23A的一个不能糖基化的突变体未能挽救胶原的运输 以及Sec23A突变粉碎斑马鱼的骨骼发育缺陷。总而言之，这些结果表明，网站- 单个COPII蛋白的特异性O-GlcN酰化调控脊椎动物细胞和组织中的囊泡运输。 本项目的目标是确定O-GlcN酰化对细胞的机制和功能的影响 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，我们将定义特定于站点的角色 人细胞中Sec23A和Sec24D的O-GlcN酰化。在目标3中，我们将确定COPII的贡献 O-GlcN酰化在CLSD和OI脊椎动物模型中的作用我们的工作将为O-GlcN酰化反应提供新的线索 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 功能障碍，如慢性阻塞性肺疾病和OI，通过靶向蛋白质糖基化。

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.