Cellular protein maturation and degradation

描述（由申请方提供）：本提案的总体目标是阐明N-连接聚糖在穿过分泌途径的蛋白质成熟和质量控制中的作用。碳水化合物在细胞中蛋白质的成熟和降解中起着关键作用。在多肽骨架上添加大的亲水性和柔性结构改变了蛋白质的溶解性和稳定性。近年来，N-连接的聚糖已成为分泌途径中的内腔标签，其通过为具有碳水化合物结合特性的常驻ER蛋白提供附着位点来指导新生糖蛋白的折叠、质量控制和降解。 我们假设，蛋白质上的N-连接聚糖的位置在细胞中的糖蛋白的有效成熟中起着重要的作用，通过介导的时间和位置之间的相互作用的新生链和居民ER蛋白，协助成熟过程。驻留ER蛋白可以保护成熟蛋白的有问题的区域免于过早折叠或聚集，帮助折叠催化剂的募集或远端结构域的连接。因此，通过控制凝集素伴侣钙连接蛋白和钙网蛋白的结合以及随后募集其相关的氧化还原酶ERp 57，聚糖可以指导糖蛋白的共翻译和翻译后成熟。此外，N-连接的聚糖还充当ER内的质量控制分选信号-一种靶向ER相关蛋白降解（ERAD）的异常蛋白的“内腔泛素”。因此，调节聚糖结构的酶和识别特定聚糖结构的碳水化合物结合蛋白在指导分泌途径中的蛋白质质量控制运输中起关键作用。 本研究的主要目的是：（1）研究糖链与折叠区域之间的相互作用，重点是糖链与折叠过程中易受损伤的Cys残基之间的关系;（2）阐明凝集素ER伴侣钙连接蛋白和钙网蛋白及其相关的折叠酶ERp 57在糖蛋白成熟过程中的作用机制;（3）用新的细胞生物学方法研究酪氨酸酶的结构和内质网成熟途径，（4）揭示N-连接聚糖引导蛋白质进入降解途径的机制;（5）确定ER常驻和推定的质量控制受体EDEM在异常糖蛋白降解中的作用。这些研究将深入了解蛋白质如何能够在细胞中高效折叠，以及细胞如何降解与疾病状态相关的异常蛋白质。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to elucidate the role of N-linked glycans in the maturation and quality control of proteins that traverse the secretory pathway. Carbohydrates play a pivotal role in maturation and degradation of proteins in the cell. The addition of the large hydrophilic and flexible structures to the polypeptide backbone modifies the solubility and stability of proteins. In recent years, N-linked glycans have emerged as the lumenal tags within the secretory pathway that direct the folding, quality control and degradation of nascent glycoproteins by providing attachment sites for resident ER proteins possessing carbohydrate-binding properties. We hypothesize that the position of N-linked glycans on a protein plays an important role in the efficient maturation of glycoproteins in the cell by mediating the timing and location of interactions between the nascent chain and resident ER proteins that assist in the maturation process. Resident ER proteins can protect problematic regions of a maturing protein from premature folding or aggregation, aid in the recruitment of folding catalysts, or the joining of distal domains. Therefore, by controlling the binding of the lectin chaperones calnexin and calreticulin, and the subsequent recruitment of their associated oxidoreductase ERp57, glycans can direct the co- and post-translational maturation of glycoproteins. In addition, N-linked glycans also act as quality control sorting signals within the ER- a type of 'lumenal ubiquitin' that targets aberrant proteins for ER associated-protein degradation (ERAD). Thus, enzymes that regulate the structures of glycans and carbohydrate-binding proteins that recognize specific glycan structures play key roles in directing the protein quality control traffic in the secretory pathway. The specific aims of this proposal are: (l) to investigate the interplay between glycans and problematic folding regions focusing on the relationship between glycans and vulnerable Cys residues during the folding process; (2) to elucidate the mechanisms by which the lectin ER chaperones calnexin and calreticulin, and their associated foldase ERp57 aid in the maturation of glycoproteins; (3) to characterize the structure and ER maturation pathway of tyrosinase, a model ERAD substrate whose misfolding is associated with albinism and melanoma, by using novel cell biological approaches; (4) to uncover the mechanism by which N-linked glycans direct proteins to the degradative pathway; and (5) to determine the role of he ER resident and putative quality control receptor EDEM in the degradation of aberrant glycoproteins. These studies will provide insights into how proteins are able to fold with high efficiencies in the cell and how the cell degrades aberrant proteins associated with disease states.