Interplay of Intrinsic and Extrinsic Effects of N-glycans on Glycoproteostasis

N-聚糖对糖蛋白稳态的内在和外在影响的相互作用

• 批准号: 9520024
• 负责人: JEFFERY W KELLY
• 金额: $ 43.31万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-27 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9520024
• 关键词: Affect; Area; Binding; Catalytic Domain; Cellular biology; Congenital Disorders; Cystic Fibrosis; Defect; Disease; Ensure; Enzymes; Eukaryota; Event; Failure; Glycoproteins; Lysosomal Storage Diseases; Maintenance; Mammals; Mannosidase; Mediating; Molecular Chaperones; Molecular Conformation; Multienzyme Complexes; Mutation; N-Glycosylation Site; Neighborhoods; Pathway interactions; Polysaccharides; Property; Protein Isoforms; Protein Region; Proteins; Proteome; Quality Control; Role; Structure; Substrate Specificity; Testing; Transferase; Variant; biophysical techniques; experience; glycosylation; paralogous gene; protein aggregate; protein folding; public health relevance; recruit; trafficking

 DESCRIPTION (provided by applicant): About one-third of the proteome of eukaryotes traverses the cellular secretory pathway, and the majority of these proteins are N-glycosylated. Within the secretory pathway there is an elaborate network of chaperones, folding enzymes, and degradation machinery dedicated to maintaining glycoprotein homoeostasis, or "glycoproteostasis". Failures of glycoproteostasis, either because of mutations in N-glycoproteins themselves or defects in the glycoproteostasis network, are responsible for many diseases, including cystic fibrosis. N-glycans affect glycoproteostasis through intrinsic mechanisms, by directly stabilizing glycoproteins and/or inhibiting their aggregation, and through extrinsic mechanisms, by mediating their interactions with the glycoproteostasis network. We have considerable experience studying the extrinsic role of N-glycans in glycoproteostasis maintenance through our studies of the folding and trafficking of glycoproteins associated with lysosomal storage diseases. We have also investigated in depth the intrinsic effects of N-glycans on protein folding and have carefully studied the effects of local sequence on the efficiency of protein N-glycosylation, and their influence on the N-glycan structures produced. In this proposal, we will fuse these areas of expertise to study how N- glycans intrinsically and extrinsically affect folding and trafficking vs. degradation decisions by the glycoproteostasis network. In Specific Aim 1, we will examine how the initial N-glycosylation event by oligosaccharyl transferase (OST) influences downstream trafficking vs. degradation (i.e., quality control) decisions by the glycoproteostasis network. We will explore the effect of N- glycosylation by OSTSTT3A vs. OSTSTT3B (where STT3A and STT3B are the two paralogs of the catalytic subunit of OST) on folding and trafficking vs. degradation decisions, by determining the effect of co-translational folding on substrate selectivity by OSTSTT3A vs OSTSTT3B, and by characterizing the interactomes of nascent glycoproteins and the various isoforms of OST itself. In Specific Aim 2, we will determine how the conformational properties of the N-glycoprotein determine the processing of N-glycans by glycoproteostasis network components. Many components of the glycoproteostasis network bind to N-glycoproteins in a bidentate fashion, interacting with both the N-glycan and the protein. This binding mode enables them to sense both the folding status of the protein and the mode and extent of N-glycan trimming, but it is unclear to what extent this sensing is a function of the immediate protein neighborhood of the N- glycan (neighborhood-local), the entire domain to which the N-glycan is attached (domain-local), or the domains that are remote from the N-glycosylation site (non-local).

 描述（由申请人提供）：大约三分之一的真核生物蛋白质组穿过细胞分泌途径，并且这些蛋白质中的大多数是N-糖基化的，在分泌途径中存在一个复杂的分子伴侣、折叠酶和降解网络。致力于维持糖蛋白稳态的机制，或“糖蛋白稳态” 由于 N-糖蛋白本身的突变而导致的糖蛋白稳态失败。糖蛋白稳态网络中的缺陷或缺陷是许多疾病的原因，包括囊性纤维化，N-聚糖通过直接稳定糖蛋白和/或抑制其聚集的内在机制，以及通过介导其与糖蛋白稳态网络的相互作用的外在机制影响糖蛋白稳态。通过我们的研究，我们在研究 N-聚糖在糖蛋白稳态维持中的外在作用方面拥有丰富的经验。我们还深入研究了 N-聚糖对蛋白质折叠的内在影响，并仔细研究了局部序列对蛋白质 N-糖基化效率的影响及其影响。在本提案中，我们将融合这些领域的专业知识来研究 N-聚糖如何内在和外在地影响折叠和运输与糖蛋白稳态的降解决定。在具体目标 1 中，我们将研究寡糖转移酶 (OST) 的初始 N-糖基化事件如何影响糖蛋白稳态网络的下游运输与降解（即质量控制）决策。 OSTSTT3A 与 OSTSTT3B（其中 STT3A 和 STT3B 是 OST 催化亚基的两个旁系同源物）在折叠和运输方面的比较在特定目标 2 中，通过确定 OSTSTT3A 与 OSTSTT3B 的共翻译折叠对底物选择性的影响，并通过表征新生糖蛋白和 OST 本身的各种亚型的相互作用组，我们将确定构象特性。 N-糖蛋白的结构决定了糖蛋白稳态网络组件对 N-聚糖的加工。 糖蛋白稳态网络的许多组件结合到 N-聚糖上。 N-糖蛋白以双齿方式与 N-聚糖和蛋白质相互作用，这种结合模式使它们能够感知蛋白质的折叠状态以及 N-聚糖修剪的模式和程度，但目前尚不清楚。这种感应的程度是 N-聚糖的直接蛋白质邻域（邻域局部）、N-聚糖所附着的整个域（域局部）或远离该域的函数。 N-糖基化位点（非局部）。