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(申请人提供)：真核生物中约三分之一的蛋白质组穿过细胞分泌途径，其中大多数蛋白质是N-糖基化的。在分泌途径中，有一个由伴侣、折叠酶和降解机制组成的精心设计的网络，致力于维持糖蛋白同质平衡或“糖蛋白平衡”。由于N-糖蛋白本身的突变或糖蛋白平衡网络的缺陷，糖蛋白稳定的失败是许多疾病的原因，包括囊性纤维化。N-葡聚糖通过直接稳定糖蛋白和/或抑制糖蛋白聚集的内在机制，以及通过介导其与糖蛋白平衡网络的相互作用的外在机制来影响糖蛋白稳定。我们通过研究与溶酶体储存疾病相关的糖蛋白的折叠和运输，研究了N-葡聚糖在维持糖蛋白稳定中的外在作用，积累了相当多的经验。我们还深入研究了N-糖链对蛋白质折叠的内在效应，并仔细研究了局部序列对蛋白质N-糖基化效率的影响，以及它们对N-糖链结构的影响。在这项提案中，我们将融合这些专业领域的知识，以研究N-葡聚糖如何内在和外在地影响折叠和运输，而不是通过糖蛋白平衡网络做出降解决定。在特定的目标1中，我们将研究寡糖转移酶(OST)最初的N-糖基化事件如何影响糖蛋白平衡网络对下游运输和降解(即质量控制)的决定。我们将通过确定OSTT3A和OSTT3B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本身的各种异构体，来探索OSTT3A和OSTT3B(其中STT3A和STT3B是OST催化亚单位的两个平行对)的N-糖基化对折叠、运输和降解决策的影响。在特定的目标2中，我们将确定N-糖蛋白的构象特性如何通过糖蛋白稳定网络组件决定N-糖蛋白的加工。糖蛋白平衡网络的许多成分以双齿方式与N-糖蛋白结合，与N-糖蛋白和蛋白质相互作用。这种结合模式使它们能够感觉到蛋白质的折叠状态以及N-糖链的修剪方式和程度，但尚不清楚这种感觉在多大程度上是N-糖链的邻近蛋白质邻域(邻域-局部)、N-糖链连接到的整个区域(区域-局部)或远离N-糖基化位点的区域(非局部)的作用。