Cellular protein maturation and degradation

总结 通过哺乳动物分泌系统运输的约7，000种蛋白质中的绝大多数被一个或多个修饰。 更多的聚糖这些改变包括Asn（N-连接）和Ser/Thr（O-连接）残基的修饰。 附加在蛋白质上的碳水化合物可以帮助蛋白质折叠、质量控制和运输，或控制蛋白质的质量。 它们的活动和功能。在这项建议中，我们会研究加入 内质网（ER）中葡萄糖和甘露糖的己糖差向异构体。 通过质量控制过程监测蛋白质成熟，该质量控制过程评估蛋白质的结构完整性。 成熟的新生链，并允许正确折叠的蛋白质通过。或者，非本地 蛋白质被标记为ER保留，以便可以修复缺陷，或者如果无法修复，则靶向 降解钙连接蛋白和钙网蛋白是ER碳水化合物结合分子伴侣，其指导ER糖链的形成。 通过选择性地结合到糖基化侧链上的分泌途径货物的折叠和运输 成熟蛋白因此，糖蛋白的糖基化状态在很大程度上控制其流动 分泌途径中的蛋白质。糖基化状态由UGGT家族成员控制 UGGT 1和UGGT 2似乎有选择地修改不成熟或非本机客户端，以支持持久性 伴侣结合这些可溶性UGGT将葡萄糖从UDP-葡萄糖转移到早期成熟的货物中， 分泌途径在本提案的前两个目标中，我们将检验UGGT是 中央质量控制守门人，通过检查它们的蛋白质， 特异性和再葡萄糖基化在细胞中的作用。 己糖差向异构体甘露糖也被添加到ER中的蛋白质中。与再葡萄糖基化相反， 甘露糖基化涉及甘露糖通过膜包埋的转移酶从多萜醇-β-甘露糖基转移到甘露糖基。 前体直接连接到Ser/Thr残基以形成O-糖苷键。有两个家庭的假定O- 甘露糖基转移酶，其存在于哺乳动物细胞的ER膜中，POMT（POMT 1和2）和 最近发现的TMTCs（TMTC 1 -4）。对它们的作用机制知之甚少， 将甘露糖添加到ER中的成熟蛋白质的功能。我们最近发现TMTC 3是 参与E-钙粘蛋白的O-甘露糖基化，E-钙粘蛋白的O-甘露糖基化有助于细胞粘附 和神经发育。TMTC 3突变与神经发育疾病有关， 强调了这种翻译后修饰的生物学意义。具体目标三是 了解ER中O-甘露糖基化的机制和意义。长期目标是 本计画旨在了解己糖加成反应的过程，包括底物的选择与修饰 步骤，以及这些翻译后修饰如何控制蛋白质的运输和功能。

Summary The vast majority of the ~7,000 proteins that traffic through the mammalian secretory are modified by one or more glycans. These alterations include the modifications of Asn (N-linked) and Ser/Thr (O-linked) residues. Carbohydrates appended to proteins can assist with protein folding, quality control and trafficking, or control their activity and function. In this proposal, we will study the mechanism and role of the addition of the hexose epimers of glucose and mannose in the endoplasmic reticulum (ER). Protein maturation is monitored by a quality control process that evaluates the structural integrity of maturing nascent chains, and permits the passage of properly folded proteins. Alternatively, non-native proteins are marked for ER retention so that the defect can be repaired, or if irreparable, targeted for degradation. Calnexin and calreticulin are ER carbohydrate binding molecular chaperones that direct the folding and trafficking of secretory pathway cargo by selectively binding to monoglucosylated side chains on maturing proteins. Therefore, to a large extent the glucosylation state of a glycoprotein controls its flow proteins in the secretory pathway. The glucosylation state is controlled by the UGGT family members UGGT1 and UGGT2 that appear to selectively modify immature or non-native clients to support persistent chaperone binding. These soluble UGGTs transfer glucose from UDP-glucose to maturing cargo in the early secretory pathway. In the first two aims of this proposal, we will test the hypothesis that the UGGTs are central quality control gatekeepers that control the flux of proteins through the ER by examining their specificity and the role of reglucosylation in the cell. The hexose epimer mannose is also added to proteins in the ER. In contrast to reglucosylation, mannosylation involves the transfer of mannose by a membrane embedded transferase from a dolichol-P- precursor directly to Ser/Thr residues to form an O-glycosidic bond. There are two families of putative O- mannosyltransferases that reside in the ER membrane in mammalian cells, the POMTs (POMT1 and 2) and the recently discovered TMTCs (TMTC1-4). Little is known about their mechanisms of action and the function of adding a mannose to maturing proteins in the ER. We have recently found that TMTC3 is involved in the O-mannosylation of E-cadherin and that E-cadherin’s O-mannosylation aids in cell adhesion and neurodevelopment. Mutations in TMTC3 are associated with the neurodevelopment diseases, underscoring the biological significance of this post-translational modification. Specific aim 3 is to understand the mechanism and significance of O-mannosylation in the ER. The long-term goal of this project is to understand the process of hexose addition including the substrate selection and modification steps, and how these post-translational modifications control the trafficking and functions of proteins.