Cellular protein maturation and degradation

摘要 在哺乳动物分泌物中运输的~7000种蛋白质中，绝大多数是由一种或多种蛋白质修饰的 更多的甘露糖。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 它们的活动和功能。在这项建议中，我们会研究加入 内质网中葡萄糖和甘露糖的己糖同分异构体。 蛋白质的成熟度通过质量控制过程进行监测，该过程评估 成熟的新生链，并允许适当折叠的蛋白质通过。或者，非本机的 蛋白质被标记为内质网保留，以便可以修复缺陷，或者如果无法修复，则针对 退化。钙粘蛋白和钙网织蛋白是内质网碳水化合物结合的分子伴侣，指导 通过选择性地与单糖化侧链结合来折叠和运输分泌途径的货物 成熟的蛋白质。因此，糖蛋白的糖基化状态在很大程度上控制着它的流动 分泌途径中的蛋白质。糖基化状态由UGGT家族成员控制 UGGT1和UGGT2似乎选择性地修改不成熟或非本机客户端以支持持久性 伴侣结合。这些可溶的UGGT在早期将葡萄糖从UDP-葡萄糖转移到成熟的货物 分泌途径。在本提案的前两个目标中，我们将测试UGGT是 中央质量控制把关人员，通过检查通过ER的 糖基化在细胞中的特异性和作用。 己糖同分异构体甘露糖也被添加到内质网中的蛋白质中。与糖基化不同， 甘露糖化反应涉及通过膜包埋的转移酶将甘露糖从多酚-P-甘露糖转移到 前体直接与Ser/Thr残基形成O-糖苷键。有两个推测的O-家族 存在于哺乳动物细胞内质网膜上的甘露糖基转移酶，POMTs(POMT1和POMT2)和 最近发现的TMTCs(TMTC1-4)。人们对它们的作用机制和 向内质网中成熟的蛋白质中添加甘露糖的功能。我们最近发现TMTC3是 参与E-钙粘蛋白的O-甘露糖化以及E-钙粘附素的O-甘露糖化有助于细胞黏附 和神经发育。TMTC3的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3是 了解O-甘露糖化在内质网中的作用机制和意义。这样做的长期目标是 项目是了解己糖加成的过程，包括底物的选择和修饰 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.