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: 8946941
• 负责人: JEFFERY W KELLY
• 金额: $ 43.14万
• 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/8946941
• 关键词: Affect; Area; Binding; Catalytic Domain; Cellular biology; Congenital Disorders; Cystic Fibrosis; Defect; Disease; Ensure; Enzymes; Eukaryota; Event; Failure; Glycoproteins; Homeostasis; Housing; Lysosomal Storage Diseases; Maintenance; Mammals; Mannosidase; Mediating; Molecular Chaperones; Multienzyme Complexes; Mutation; N-Glycosylation Site; Neighborhoods; Pathway interactions; Polysaccharides; Process; Property; Protein Isoforms; Protein Region; Proteins; Proteome; Quality Control; Recruitment Activity; Role; Structure; Substrate Specificity; Testing; Transferase; Variant; biophysical techniques; experience; glycosylation; paralogous gene; protein aggregate; protein folding; public health relevance; 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（由适用提供）：大约三分之一的真核生物蛋白质遍历细胞秘密途径，并且这些蛋白质中的大多数是N-糖基化的。在秘密途径中，有一个精致的伴侣网络，折叠酶和降解机制，该网络用于维持糖蛋白同种异体或“ Glycoproteotostasis”。糖蛋白抑制性的失败是由于N-糖蛋白本身的突变或糖蛋白溶裂网络中的缺陷是导致许多疾病（包括囊性纤维化）的原因。 N-聚糖通过固有机制，直接稳定糖蛋白和/或通过介导其与糖蛋白蛋白骨化网络的相互作用来影响糖蛋白溶血症。通过研究与溶酶体储存疾病相关的糖蛋白的折叠和运输，我们拥有研究N-聚糖在糖蛋白抑制性维持中的外在作用的丰富经验。我们还深入研究了N-聚糖对蛋白质折叠的内在作用，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对产生的N-聚糖结构的影响。在此提案中，我们将融合这些专业知识领域，以研究n-Glycans如何本质上和外在影响折叠和贩运与降解决策的折叠和贩运决策。在特定的目标1中，我们将研究寡糖胆碱转移酶（OST）最初的N-糖基化事件如何影响下游贩运与降解（即质量控制）的决策。我们将通过OSTST3A与OSTSTT3B（stt3a和stt3b是OST的催化亚基的两个旁系同源物）对N-糖基化对折叠和运输的两个旁系同源物的影响，并通过确定由共旋转式折叠式选择的效果和降级决策的影响。新生的糖蛋白和OST本身的各种同工型。在特定的目标2中，我们将确定N-糖蛋白的构象性能如何通过糖蛋白酶抑制性网络组件确定N-聚糖的加工。糖蛋白酶抑制网络的许多成分以边界方式与N-糖蛋白结合，与N-聚糖和蛋白质相互作用。 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 sensor 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).