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

 描述（由申请人提供）：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，存在一个由分子伴侣、折叠酶和降解机制组成的复杂网络，致力于维持糖蛋白稳态或"糖蛋白稳态"。由于N-糖蛋白本身的突变或糖蛋白抑制网络的缺陷，糖蛋白抑制的失败是许多疾病的原因，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和通过介导其与糖蛋白抑制网络的相互作用的外在机制影响糖蛋白抑制。通过研究与溶酶体贮积病相关的糖蛋白的折叠和运输，我们在研究N-聚糖在糖蛋白稳态维持中的外在作用方面具有相当丰富的经验。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，以及它们对所产生的N-聚糖结构的影响。在本提案中，我们将融合这些领域的专业知识，研究N-聚糖如何内在地和外在地影响糖蛋白抑制网络的折叠和运输与降解决定。在具体目标1中，我们将研究寡糖基转移酶（OST）的初始N-糖基化事件如何影响下游运输与降解（即，质量控制）的决定。我们将探索OSTSTT3A与OSTSTT3B（其中STT3A和STT3B是OST催化亚基的两个旁系同源物）的N-糖基化对折叠和运输与降解决定的影响，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的相互作用组和OST本身的各种亚型。在具体目标2中，我们将确定N-糖蛋白的构象特性如何通过糖蛋白抑制网络组件决定N-聚糖的加工。糖蛋白稳态网络的许多组分以双齿方式结合N-糖蛋白，与N-聚糖和蛋白质相互作用。这种结合模式使它们能够感测蛋白质的折叠状态以及N-聚糖修剪的模式和程度，但尚不清楚这种感测在多大程度上是N-聚糖的直接蛋白质邻域（邻域-局部）、N-聚糖所连接的整个结构域（结构域-局部）或远离N-糖基化位点的结构域（非局部）的函数。

 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).