Proteasome regulation by O-glycosylation

通过 O-糖基化调节蛋白酶体

• 批准号: 7097359
• 负责人: Jeffrey E Kudlow
• 金额: $ 28.35万
• 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/7097359
• 关键词: N acetylglucosamine; acylation; adenosinetriphosphatase; apoptosis; enzyme mechanism; epithelium; genetically modified animals; glycosyltransferase; laboratory mouse; matrix assisted laser desorption ionization; peptidases; proteasome; protein structure; yeast two hybrid system

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）： 该实验室表明，用O连接的N-乙酰葡萄糖（O-GLCNAC）修饰蛋白质在转录激活剂和阻遏物的功能中起着直接作用。这种修饰是由葡萄糖代谢引起的，还调节了蛋白酶体的功能，蛋白酶体是参与蛋白质内降解的主要细胞器。 26S蛋白酶体的胰蛋白酶活性，而不是针对4种氨基酸肽（LLVY）的20s蛋白酶体通过与O-GLCNAC转移酶（OGT）孵育的阻断。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通过重组O-GlcNACase（去除这种修饰的酶）重新激活。对具有[3H] -GLCNAC的纯化蛋白酶体的标记研究表明，修饰的蛋白质的分子质量约为45 kDa，并且该底物位于蛋白酶体的19S调节帽中。由于蛋白酶体会降解促凋亡因子，例如p53及其许多下游靶标，因此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。化学治疗剂和GlcNAC类似物链蛋白酶链球菌素也可以通过其特性作为O-Glcnacase的非竞争力抑制剂诱导凋亡。拟议的研究旨在确定O-GLCNAC途径和蛋白酶体之间的生化连接。 O-GLCNAC阻断蛋白酶体功能的能力也可能将葡萄糖代谢与氨基酸从肌肉浪费中释放出来。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。使用这些酶在体外确定O-GLCNAC转移酶（OGT）和O-GlCNACase对蛋白酶体功能的影响，以在体外逆转地修饰蛋白酶体中的蛋白质。 2。鉴定蛋白酶环共同的蛋白质，这些蛋白包含O-GLCNAC修饰并以可逆的方式调节蛋白酶体功能。 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，确定体内蛋白酶体阻断对上皮细胞凋亡和肌肉蛋白浪费的影响。