N-Glycosylation And ER Stress

描述(申请人提供)：许多重要的蛋白质分泌(如免疫球蛋白和多肽激素)或存在于细胞表面(包括细胞黏附分子和信号受体)由内质网(ER)产生和折叠。如果内质网有折叠这些蛋白质的问题，一个代偿性的“内质网应激反应”(又名。未折叠蛋白反应)被触发以增强内质网相关的折叠过程。内质网产生的蛋白质经常与天冬酰胺结合的糖聚合物(多糖)进行N-糖基化。糖链与N-糖蛋白的折叠密切相关，也可以在分泌后或到达细胞表面后参与其功能。内质网相关的脂质连接寡糖(LLO)Glc3Man9GlcNAc2-P-P-dolicol提供用于制造N-糖蛋白的糖(Glc3Man9GlcNAc2)。LLO合成的许多关键特征早在20多年前就已为人所知，但对该途径的功能急性调节知之甚少。本研究的重点是P.I.S发现内质网应激反应调节LLO的合成，当LLO不足是内质网功能障碍的原始原因时，介导一个反馈回路进行补偿。这涉及到一个“LLO生物合成”手臂和一个反直觉的“LLO降解”手臂。因此，LLO的生产并不是硬性的，而是不断地进行监测和调整。目的阐明内质网应激反应控制LLO生物合成臂的一种成分，即作为糖链前体的核苷酸-糖水平升高的机制。这一目标将：确定应激诱导的己糖磷酸如何提高核苷酸-糖库；确定响应核苷酸-糖控制的LLO合成步骤；以及探索涉及的胁迫信号转导和效应器。目的II将提供有关甘露糖-6-磷酸(M6P)的一种意外活性及其在LLO降解臂中的关键作用的重要新信息。P.I.发现内质网应激导致M6P升高，并导致Glc3Man9GlcNAc2-P-P-Dolicol释放多糖。这一目标将：开发M6P的模拟物和拮抗剂以阐明其细胞作用；探索M6P释放的多糖在内质网稳态中的作用；并以单纯疱疹病毒1型为模型验证降解的臂代表针对病毒包膜N-糖蛋白合成(使用LLO的过程并可能诱导内质网应激)的新的宿主防御机制的假设。临床上的相关性是双重的。首先，人类有13种遗传性疾病，属于I型先天性糖基化紊乱(CDG)家族，LLO合成缺陷。CDG-I细胞由于N-糖基化能力差而存在内质网功能障碍，患者临床上存在许多困难。这项工作将从根本上对可能影响CDG-I的监管系统有新的见解。其次，这项工作将产生有关内质网应激反应的新信息，内质网应激反应调节内质网的生产力，对浆细胞(免疫球蛋白)和胰岛(胰岛素)的分泌功能至关重要，当控制异常时，可能会导致神经系统疾病、胆固醇失衡和肥胖。公共卫生相关性：这项研究的公共卫生相关性有三个方面。首先，将评估治疗患者碳水化合物与蛋白质异常附着的人类疾病的新策略。其次，这项工作将产生关于细胞如何应对压力的新信息，这是产生免疫球蛋白以对抗感染、产生胰岛素以预防糖尿病以及涉及神经系统、胆固醇和肥胖的疾病的重要因素。第三，我们将评估针对某些传染性人类病毒的潜在宿主防御机制。

DESCRIPTION (provided by applicant): Many vitally important proteins which are secreted (such as immunoglobulins and polypeptide hormones) or present at the cell surface (including cell adhesion molecules and signaling receptors) are produced and folded by the endoplasmic reticulum (ER). If the ER has problems folding these proteins, a compensatory "ER stress response" (a.k.a. Unfolded Protein Response) is triggered to enhance ER-related folding processes. ER-produced proteins are frequently N-glycosylated with asparagine-bound sugar polymers (glycans). The glycans are intimately involved in folding of N-glycoproteins, and can also participate in their functions after secretion or reaching the cell surface. The ER-associated lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol provides the glycan (Glc3Man9GlcNAc2) used to make N-glycoproteins. Many key features of LLO synthesis have been known for over 20 years, but functional acute regulation of the pathway has been poorly understood. This research proposal focuses on the P.I.'s discovery of LLO synthesis regulation by the ER stress response, mediating a feedback loop which can compensate when LLO insufficiency is the original cause of ER dysfunction. This involves both a "LLO biosynthetic" arm and a counter-intuitive "LLO degradative" arm. Thus LLO production is not hard-wired, but instead is constantly monitored and adjusted. Aim I will elucidate the mechanism by which the ER stress response controls one component of the LLO biosynthetic arm, elevation of levels of nucleotide-sugars which are the precursors of glycans. This Aim will: determine how stress-induced hexose phosphates elevate nucleotide-sugar pools; identify steps in LLO synthesis responsive to nucleotide-sugar control; and explore the stress signal transducer and effector involved. Aim II will provide important new information about an unexpected activity of mannose- 6-phosphate (M6P) and its key role in the LLO degradative arm. The P.I. discovered that M6P is elevated by ER stress, and causes release of glycan from Glc3Man9GlcNAc2-P-P-dolichol. This Aim will: develop mimics and antagonists of M6P to elucidate its cellular action; explore the role of M6P-released glycans in ER homeostasis; and test the hypothesis that the degradative arm represents a novel host defense mechanism against viral envelope N-glycoprotein synthesis (a process using LLO and likely to induce ER stress) with herpes simplex-1 as a model. The clinical relevance is two-fold. First, there are 13 human genetic diseases in the family "Congenital Disorders of Glycosylation" (CDG) Type I, with defective LLO synthesis. CDG-I cells have ER dysfunction due to poor N-glycosylation, and patients have many clinical difficulties. Fundamental new insights into the regulatory systems which may impact CDG-I will be gained from this work. Second, this work will generate new information about the ER stress response, which governs the productivity of the ER, is essential for the secretory functions of plasma cells (immunoglobulins) and pancreatic islets (insulin), and when aberrantly controlled can cause neurological diseases, cholesterol imbalance, and obesity. Public Health Relevance: The public health relevance of this research is three-fold. First, new strategies for treating human diseases in which patients have abnormal carbohydrate attachment to protein will be evaluated. Second, this work will generate new information about the how cells respond to stress, which is an important factor in the production of immunoglobulins to fight infection, in the production of insulin to prevent diabetes, and in diseases involving the nervous system, cholesterol, and obesity. Third, we will evaluate a potential host- defense mechanism against certain infectious human viruses.