Proteasome regulation by O-glycosylation

通过 O-糖基化调节蛋白酶体

• 批准号: 7234137
• 负责人: Jeffrey E Kudlow
• 金额: $ 27.53万
• 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/7234137
• 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

DESCRIPTION (provided by applicant): 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 proteasomeassociated 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.

描述（由申请人提供）： 实验室已经表明，蛋白质修饰与O-连接的N-乙酰葡糖胺（O-GlcNAc）在转录激活因子和抑制因子的功能中起着直接的作用。这种由葡萄糖代谢引起的修饰也调节蛋白酶体的功能，蛋白酶体是参与细胞内蛋白质降解的主要细胞器。26 S蛋白酶体（但不是20 S蛋白酶体）对4种氨基酸肽（LLVY）的胰凝乳蛋白酶活性通过蛋白酶体与O-GlcNAc转移酶（OGT）孵育而被阻断。此外，完整蛋白酶体的ATP酶活性被OGT阻断。来自用高葡萄糖或葡糖胺处理的NRK细胞的生理失活的蛋白酶体可以被重组O-GlcNAc酶（去除这种修饰的酶）重新激活。用[3 H]-GlcNAc对纯化的蛋白酶体进行的标记研究表明，修饰的蛋白质具有约45 kDa的分子量，并且该底物位于蛋白酶体的19 S调节帽中。由于蛋白酶体降解促凋亡因子如p53及其许多下游靶标，蛋白酶体功能的抑制可能导致这些因子的积累，诱导凋亡。化疗剂和GlcNAc类似物链脲佐菌素也通过其作为O-GlcNAc酶的非竞争性抑制剂的性质诱导细胞凋亡。拟定的研究旨在确定O-GlcNAc途径和蛋白酶体之间的生物化学联系。O-GlcNAc阻断蛋白酶体功能的能力也可能将葡萄糖代谢与肌肉萎缩的氨基酸释放偶联。具体目标如下：总体目标：确定O-GlcNAc在蛋白酶体功能中的作用。1.使用O-GlcNAc转移酶（OGT）和O-GlcNAc酶在体外可逆修饰蛋白酶体中的蛋白质，测定这些酶对体外蛋白酶体功能的影响。2.鉴定含有O-GlcNAc修饰并以可逆方式调节蛋白酶体功能的蛋白酶体相关蛋白。3.确定蛋白酶体19 S调节亚基的O-GlcNAc酰化如何改变蛋白酶体肽酶和ATP酶的功能。4.使用转基因小鼠，确定体内蛋白酶体阻断对上皮细胞凋亡和肌肉蛋白质消耗的影响。