N-Glycosylation And ER Stress

描述（由申请人提供）：许多分泌（如免疫球蛋白和多肽激素）或存在于细胞表面（包括细胞粘附分子和信号受体）的至关重要的蛋白质由内质网（ER）产生和折叠。如果ER有问题折叠这些蛋白质，一个补偿性的“ER应激反应”（a.k.a.未折叠蛋白质反应）被触发以增强ER相关的折叠过程。ER产生的蛋白质通常被天冬酰胺结合的糖聚合物（聚糖）N-糖基化。聚糖与N-糖蛋白的折叠密切相关，也可以在分泌或到达细胞表面后参与其功能。ER相关的脂质连接寡糖（LLO）Glc 3 Man 9 GlcNAc 2-P-P-dolichol提供用于制备N-糖蛋白的聚糖（Glc 3 Man 9 GlcNAc 2）。LLO合成的许多关键特征已经知道了20多年，但对该途径的功能性急性调节知之甚少。本研究的重点是P.I.的发现LLO合成调节的ER应激反应，介导的反馈回路，可以补偿时，LLO不足是原来的原因，ER功能障碍。这涉及“LLO生物合成”臂和反直觉的“LLO降解”臂。因此，LLO生产不是硬连线的，而是不断监测和调整。目的阐明内质网应激反应控制LLO生物合成臂的一个组成部分，即聚糖前体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升高核苷酸-糖库;确定响应核苷酸-糖控制的LLO合成步骤;并探索涉及的应激信号转导子和效应子。目的II将提供关于甘露糖-6-磷酸（M6 P）的意外活性及其在LLO降解臂中的关键作用的重要新信息。发现M6 P通过ER应激升高，并导致聚糖从Glc 3 Man 9 GlcNAc 2-P-P-多萜醇释放。这一目标将：开发M6 P的模拟物和拮抗剂，以阐明其细胞作用;探索M6 P释放的聚糖在ER稳态中的作用;并以单纯疱疹病毒-1为模型，检验降解臂代表针对病毒包膜N-糖蛋白合成（一种使用LLO并可能诱导ER应激的过程）的新型宿主防御机制的假设。临床相关性是双重的。首先，在家族“先天性糖基化障碍”（CDG）I型中有13种人类遗传疾病，具有LLO合成缺陷。CDG-I细胞由于N-糖基化差而具有ER功能障碍，患者具有许多临床困难。从这项工作中将获得对可能影响CDG-I的监管系统的基本新见解。其次，这项工作将产生关于ER应激反应的新信息，该反应控制ER的生产力，对于浆细胞（免疫球蛋白）和胰岛（胰岛素）的分泌功能至关重要，并且当异常控制时可能导致神经系统疾病，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是三方面的。首先，将评估治疗患者与蛋白质有异常碳水化合物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，我们将评估一个潜在的宿主防御机制，以对抗某些传染性的人类病毒.

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.