Post-translational mechanisms of UDP-glucuronosyltransferase enzyme function

UDP-葡萄糖醛酸基转移酶功能的翻译后机制

• 批准号: RGPIN-2017-03808
• 负责人: Collier, Abby
• 金额: $ 2.04万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=682699
• 关键词: Post; translational; mechanisms; UDP; glucuronosyltransferase

The UDP-glucuronosyltransferases are an enzyme superfamily that catalyzes the metabolic reaction glucuronidation. This is an elimination reaction that is critical for balancing of steroids and other essential vitamins and nutrients in the body as well as detoxifying chemicals and drugs. Multiple enzymes belonging to the family exist in mammals, fish, birds, insects and invertebrates, differing within and across species. Regulation of UGT activity is complex e.g. enzymes can be present but inactive. Each also has individual genetic and environmental control that can alter activity, causing toxicity and disease. Preliminary data implicates glycosylation in controlling UGT activity and we hypothesize this is fundamental for the enzymes' catalytic reaction. We will investigate the importance of translation, post-translational protein structural changes, namely glycosylation, and endoplasmic reticulum processes (chaperoning, capacity for anchoring) on UGT function. To investigate this we will determine the roles of protein production as well as protein modifications using in vitro cell-free systems. Then using whole cells we will study UGTs protein assembly and processing inside the cell. Preliminary data shows that processing chaperones calnexin and calreticulin are important for UGT protein assembly, because interference with these chaperones alters UGT expression. Also, the absolute capacity inside the cell for UGT protein content and within the ER lumen is unknown, as is how long each UGT exists (protein lifetime) before it is degraded and recycled. We will use fluorescent technologies and expression systems to label UGT enzymes in living cells and determine the absolute capacity [space] for the enzyme within the cell as amount of protein might be related to how well the protein can perform its detoxification roles. Finally, because the specific protein modification called glycosylation is known to influence UGT function, we will study the effects of two types of glycosylation N-linked, which happens early in protein production and O-linked than tends to happen later and be more structural on the ability of the UGT proteins to metabolize and eliminate essential internal and environmental cehmicals. Understanding the regulation of glucuronidation can identify pathways amenable to manipulation for disease prevention or treatment, aid drugs development and use and improve chemical safety in humans and other species.

UDP-葡萄糖醛酸基转移酶是催化代谢反应葡萄糖醛酸化的酶超家族。这是一种消除反应，对于平衡体内类固醇和其他必需维生素和营养素以及解毒化学物质和药物至关重要。属于该家族的多种酶存在于哺乳动物、鱼类、鸟类、昆虫和无脊椎动物中，在物种内和物种间存在差异。UGT活性的调节很复杂，例如酶可以存在但无活性。每一种都有单独的遗传和环境控制，可以改变活动，导致毒性和疾病。初步数据表明糖基化控制UGT活性，我们假设这是酶催化反应的基础。我们将研究翻译的重要性，翻译后蛋白质结构的变化，即糖基化，和内质网过程（伴侣，锚定能力）对UGT功能。为了研究这一点，我们将使用体外无细胞系统确定蛋白质生产以及蛋白质修饰的作用。然后使用整个细胞，我们将研究UGT蛋白组装和细胞内的加工。初步数据显示，加工伴侣钙连接蛋白和钙网蛋白对于UGT蛋白组装是重要的，因为干扰这些伴侣改变UGT表达。此外，细胞内和ER腔内UGT蛋白含量的绝对容量是未知的，每个UGT在降解和回收之前存在多长时间（蛋白质寿命）也是未知的。我们将使用荧光技术和表达系统来标记活细胞中的UGT酶，并确定细胞内酶的绝对容量[空间]，因为蛋白质的量可能与蛋白质执行其解毒作用的程度有关。最后，由于已知称为糖基化的特定蛋白质修饰会影响UGT功能，我们将研究两种类型的糖基化N-连接的影响，其发生在蛋白质产生的早期，O-连接的影响比以后发生的更倾向于UGT蛋白质代谢和消除必需的内部和环境微生物的能力。了解葡萄糖醛酸化的调节可以确定适合疾病预防或治疗的途径，帮助药物开发和使用，并提高人类和其他物种的化学安全性。