Proteasome regulation by O-glycosylation

通过 O-糖基化调节蛋白酶体

• 批准号: 7499426
• 负责人: Jeffrey E Kudlow
• 金额: $ 11.48万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7499426
• 关键词: 26S proteasome; ATP phosphohydrolase; Acetylglucosamine; Amino Acids; Apoptosis; Apoptotic; Biochemical; Cells; Enzymes; Epithelial Cells; Glucosamine; Glucose; Goals; In Vitro; Induction of Apoptosis; Label; Laboratories; Lead; Link; Modification; Muscle; Muscle Proteins; O-GlcNAc transferase; Organelles; Pathway interactions; Peptide Hydrolases; Peptides; Play; Post-Translational Protein Processing; Property; Proteasome Inhibition; Proteins; Recombinants; Regulation; Research Design; Role; Streptozocin; TP53 gene; Transcription Coactivator; Transgenic Mice; analog; chemotherapeutic agent; glucose metabolism; glycosylation; in vivo; inhibitor/antagonist; molecular mass; multicatalytic endopeptidase complex; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; protein degradation; wasting

The laboratory has shown that protein modification with O-linked N-acetylglucosamine (O-GlcNAc) plays a direct role in the function of transcriptional activators and repressors. This modification, which results from glucose metabolism, also modulates the function of the proteasome, the major organelle involved in intracellular degradation of proteins. The chymotryptic activity of 26S proteasomes, but not 20S proteasomes against 4 amino acid peptides (LLVY) is blocked by incubation of the proteasome with O-GlcNAc transferase (OGT). In addition, the ATPase activity of intact proteasomes is blocked by OGT. Physiologically inactivated proteasomes from NRK cells treated with high glucose or glucosamine can be reactivated by recombinant O-GlcNAcase, the enzyme that removes this modification. Labeling studies on purified proteasomes with [3H]-GlcNAc indicate that the modified protein(s) have a molecular mass of about 45 kDa and that this substrate resides in the 19S regulatory cap of the proteasome. Since the proteasome degrades pro-apoptotic factors such as p53 and many of its downstream targets, inhibition of proteasome function might lead to the accumulation of these factors with the induction of apoptosis. The chemotherapeutic agent and GlcNAc analog, streptozotocin, also induces apoptosis through its property as a non-competitive inhibitor of the O-GlcNAcase. The proposed studies are designed to determine the biochemical linkage between the O-GlcNAc pathway and the proteasome. The ability of O-GlcNAc to block proteasomal function may also couple glucose metabolism to amino acid release from muscle wasting. The specific aims are as follows: General goal: Determine the role of O-GlcNAc in proteasomal function. 1. Determine the effect of O-GlcNAc transferase (OGT) and O-GlcNAcase on proteasome function in vitro using these enzymes to reversibly modify proteins in the proteasome in vitro. 2. Identify proteasome- associated protein(s) that contain the O-GlcNAc modification and regulate proteasome function in a reversible manner. 3. Determine how O-GlcNAcylation of the proteasome 19S regulatory subunit modifies the function of the proteasomal peptidase and ATPases. 4. Using transgenic mice, determine the effect of proteasome blockade in vivo on epithelial cell apoptosis and muscle protein wasting.

该实验室表明，用O连接的N-乙酰葡萄糖（O-GLCNAC）修饰蛋白质会播放A 在转录激活器和阻遏物的功能中的直接作用。这种修改是由 葡萄糖代谢，还调节蛋白酶体的功能，涉及的主要细胞器 蛋白质的细胞内降解。 26S蛋白酶体的胰蛋白酶活性，而不是20S蛋白酶体 蛋白酶体与O-GlCNAC一起孵育4个氨基酸肽（LLVY）。 转移酶（OGT）。另外，完整蛋白酶体的ATPase活性被OGT阻断。 来自高葡萄糖或葡萄糖治疗的NRK细胞的生理灭活蛋白酶体可以是 由重组O-Glcnacase重新激活，该酶消除了这种修饰。标记研究 具有[3H] -GlCNAC的纯化蛋白酶体表明，修饰的蛋白质的分子质量约为 45 kDa，该基材位于蛋白酶体的19S调节帽中。由于蛋白酶体 降解促凋亡因素，例如p53及其许多下游靶标，抑制蛋白酶体 功能可能会导致这些因素的积累，并诱导凋亡。这 化学治疗剂和GlcNAC类似物，链蛋白酶链霉素也可以通过其特性诱导凋亡 O-Glcnacase的非竞争性抑制剂。拟议的研究旨在确定 O-GLCNAC途径与蛋白酶体之间的生化连接。 O-GLCNAC阻止的能力 蛋白酶体功能还可以将葡萄糖代谢与氨基酸浪费释放。这 具体目的如下：一般目标：确定O-GLCNAC在蛋白酶体功能中的作用。 1。 确定O-GLCNAC转移酶（OGT）和O-Glcnacase对体外蛋白酶体功能的影响 使用这些酶在体外可逆地改变蛋白酶体中的蛋白质。 2。确定蛋白酶体 - 相关蛋白质，其中包含O-GlCNAC修饰并调节A中的蛋白酶体功能 可逆的方式。 3。确定蛋白酶体19S调节亚基的O-Glcnacylation如何修饰 蛋白酶体肽酶和ATPases的功能。 4。使用转基因小鼠，确定 蛋白酶体在体内封锁上皮细胞凋亡和肌肉蛋白浪费。