Structural Analysis of NAT Acetylation, Substrate Specificity and Polymorphisms

描述（由申请人提供）：芳胺n -乙酰基转移酶（NATs）解毒作为烹饪，农药，烟草或染料副产品摄入的芳胺膀胱癌物质。它们通过将乙酰辅酶a的乙酰基转移到芳胺上来实现这一功能。与对照组相比，膀胱癌患者的芳胺加合物水平升高，其中一些已被证明是NATs的不良底物。作为本提案的一部分，我们定义了NAT底物特异性的决定因素，以揭示为什么某些芳胺致癌物不能被解毒。NAT是一种高度多态性的蛋白质，很大比例的人群携带NAT变体，在体内的催化活性降低。我们和其他人最近已经证明，这种变体可以通过泛素-蛋白酶体途径迅速降解。我们的长期目标之一是使用NATs作为模型系统，以确定如何在细胞中识别泛素化靶标，并阐明导致它们递送到蛋白酶体的机制。这项研究将为识别和消除异常蛋白的质量控制途径提供基础信息。主要涉及以下三个方面。1)建立NAT底物特异性的通用模型。核磁共振和稳态动力学实验用于确定NAT底物特异性的决定因素，这些知识用于产生可以乙酰化额外芳胺致癌物的变体。2)确定导致NAT组成泛素化的机制。在之前的工作中，我们证明了NAT组成泛素化与其聚集状态有关，我们的初步数据表明这种泛素化发生在内质网。在本研究中，我们确定了NATs是如何被识别为异常的，它们的泛素化的决定因素，以及它们是否通过适用于其他聚集或错误折叠的蛋白质的共同途径进行加工。3)定义乙酰化如何影响NAT结构和表面性质。这项研究的成功结果可能有助于预防膀胱癌，并为与错误折叠或聚集蛋白相关的疾病（包括神经退行性疾病和癌症）提供新的治疗策略。这项研究对nat相关的癌变具有治疗意义，特别是膀胱癌，以及与错误折叠或聚集蛋白相关的疾病，包括神经退行性疾病。NAT解毒化学物质被称为芳胺，基于人群的研究已经将NAT活性降低和逃避NAT解毒的芳胺与膀胱癌联系起来。我们确定了某些芳胺是如何逃避NAT解毒的，以及为什么有些人的NAT蛋白在发挥蛋白质功能之前就被破坏了。

DESCRIPTION (provided by applicant): Arylamine N-acetyltransferases (NATs) detoxify arylamine bladder carcinogens that are ingested as cooking, pesticide, tobacco, or dye byproducts. They perform this function by transferring to the arylamine an acetyl group from acetyl-CoA. Bladder cancer patients exhibit elevated levels of arylamine-adducts compared to control groups, some of which have been demonstrated to be poor substrates for NATs. As part of this proposal, we define the determinants of NAT substrate specificity to reveal why certain arylamine carcinogens are unable to be detoxified. NATs are highly polymorphic proteins and a large percentage of the population harbor NAT variants with reduced catalytic activity in vivo. We, and others, have recently demonstrated such variants to be rapidly degraded through the ubiquitin-proteasome pathway. One of our long-term goals is to use NATs as a model system to determine how ubiquitylation targets are identified in cells and to elucidate the mechanism(s) that lead to their delivery to the proteasome. This research will provide fundamental information on quality control pathways that exist to recognize and eliminate aberrant proteins. The following three general areas are pursued. 1) Develop a general model for NAT substrate specificity. NMR and steady state kinetics experiments are used to define the determinants of NAT substrate specificity and this knowledge used to generate variants that can acetylate additional arylamine carcinogens. 2) Determine the mechanisms that lead to NAT constitutive ubiquitylation. In previous work, we demonstrated NAT constitutive ubiquitylation to be linked to its aggregation state and our preliminary data indicate such ubiquitylation to occur at the endoplasmic reticulum. In the proposed research, we determine how NATs are recognized as aberrant, the determinants of their ubiquitylation, and whether they are processed through a common pathway that applies to other aggregated or mis-folded proteins. 3) Define how acetylation affects NAT structure and surface properties. The successful outcome of this research could aid in the prevention of bladder cancer and lead to new therapeutical strategies for diseases associated with mis-folded or aggregated proteins, including neurodegenerative diseases and cancer. Relevance of this research to public health This research has therapeutical implications for NAT-associated carcinogenesis, especially bladder cancer, as well as diseases associated misfolded or aggregated proteins, including neurodegenerative diseases. NATs detoxify chemicals known as arylamines and population-based studies have connected reduced NAT activity and arylamines that evade NAT detoxification to bladder cancer. We determine how certain arylamines evade NAT detoxification and why the NAT proteins of some people are destroyed before they can perform their protein function.