Catalytic and regulatory mechanisms of human Tryptophan Dioxygenase

人色氨酸双加氧酶的催化和调节机制

• 批准号: 9107183
• 负责人: Syun-Ru Yeh
• 金额: $ 40.92万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9107183
• 关键词: Achievement; Active Sites; Alzheimer' s Disease; Amines; Binding; Biology; Catalysis; Chemistry; Collaborations; Crystallography; Degradation Pathway; Development; Dioxygen; Dioxygenases; Documentation; Ensure; Enzymes; Foundations; Freezing; Goals; Helix-Loop-Helix Motifs; Heme; Hemeproteins; Hepatic; Homeostasis; Human; Huntington Disease; Immunity; Iron; Kinetics; Knowledge; Kynurenine; Link; Liver; Malignant Neoplasms; Mental Depression; Methods; Microscopic; Molecular; Molecular Conformation; Mutagenesis; Neurodegenerative Disorders; Occupations; Optics; Pathway interactions; Peptides; Play; Positioning Attribute; Preparation; Property; Proteins; Protocols documentation; Public Health; Reaction; Regulation; Research; Role; Spectrum Analysis; Structure; Testing; Therapeutic; Therapeutic Intervention; Tryptophan; Ubiquitin; Ubiquitination; Water; analog; base; biophysical analysis; design; enzyme structure; flexibility; inhibitor/antagonist; insight; novel; public health relevance; simulation; therapeutic target; three dimensional structure; tumor; tumor growth

 DESCRIPTION (provided by applicant): Human tryptophan dioxygenase (hTDO) is an important hemeprotein that catalyzes the initial and rate-limiting step of the kynurenine (KYN) pathway-the conversion of Trp to N-formyl kynurenine (NFK). It is well-accepted that hTDO plays a critical role in controlling Trp homeostasis in liver. More recently, it was discovered tha hTDO is also expressed in a variety of human tumors to suppress antitumor immunity, thereby promoting aggressive tumor growth. In addition to cancer, hTDO has been implicated in a wide spectrum of neurodegenerative diseases, such as Alzheimer's and Huntington's diseases, as well as depression. hTDO has hence been recognized as an important therapeutic target. Despite its importance, the progress has been limited by the poor knowledge of the molecular properties of the enzyme. Recently, we have made several major breakthroughs in (i) developing a new protocol allowing the preparation of high quality hTDO protein, allowing the documentation of the first glimpse of the structural features critical for the dioxygenase chemistry, as well as an unusual activity of the ferric enzyme, (ii) solving the first substrate-bound 3D-structure of hTDO, as well as trapping reaction intermediates in single crystals and solved their structures, which unveiled several previously unidentified structural features that ar critical for controlling the catalytic activity and cellular degradation of the enzyme, one of the most rapidly degraded enzymes in liver, and (iii) obtaining the first evidence supporting the cellular degradation of hTDO via a ubiquitination-linked proteasomal degradation pathway. These important achievements allow us to build novel hypotheses and propose a comprehensive approach from a unique perspective to test them. The main objective of this project is to delineate the structure, function and regulation of hTDO, as part of our long term goal in comprehending heme dioxygenase chemistry and TDO biology. The specific aims of our project are: (i) identify the novel dioxygenase intermediates of hTDO. (ii) Determine the dioxygen activation mechanism of the unique ferric reaction. (iii) Delineate the product release mechanism of hTDO. (iv) Delineate the exosite-dependent regulation mechanisms of hTDO. We anticipate that fulfilling the objectives of this project will result in new insights into the struture, function and regulation of this essential enzyme, and provide an important blueprint for the design of hTDO-selective inhibitors as therapeutics for cancer, neurodegenerative diseases and depression.

 描述（由申请人提供）：人色氨酸双加氧酶（hTDO）是一种重要的血红素蛋白，催化犬尿氨酸（KYN）途径的初始和限速步骤-色氨酸转化为N-甲酰犬尿氨酸（NFK）。普遍认为hTDO在控制肝脏中Trp稳态中起关键作用。最近，发现hTDO也在多种人类肿瘤中表达以抑制抗肿瘤免疫，从而促进侵袭性肿瘤生长。除了癌症之外，hTDO还涉及广泛的神经变性疾病，例如阿尔茨海默病和亨廷顿病，以及抑郁症。hTDO因此被认为是重要的治疗靶点。尽管它的重要性，进展一直受到限制的知识贫乏的分子特性的酶。最近，我们已经在以下方面取得了几个重大突破：（i）开发允许制备高质量hTDO蛋白的新方案，允许记录对双加氧酶化学至关重要的结构特征的第一一瞥，以及铁酶的不寻常活性，（ii）解决hTDO的第一个底物结合的3D结构，以及在单晶中捕获反应中间体并解析其结构，这揭示了几个先前未鉴定的结构特征，这些结构特征对于控制酶的催化活性和细胞降解至关重要，酶是肝脏中降解最快的酶之一，和（iii）获得支持hTDO通过泛素化连接的蛋白酶体降解途径的细胞降解的第一证据。这些重要的成就使我们能够建立新的假设，并从独特的角度提出一个全面的方法来测试它们。本研究的主要目的是阐明血红素双加氧酶的结构、功能和调控，作为我们理解血红素双加氧酶化学和TDO生物学的长期目标的一部分。我们的项目的具体目标是：（i）鉴定hTDO的新型双加氧酶中间体。(ii)确定了独特的三价铁反应的分子氧活化机理。(iii)阐明了hTDO的产物释放机制。(iv)阐明hTDO的外来位点依赖性调节机制。我们预计，实现该项目的目标将导致对这种必需酶的结构，功能和调节的新见解，并为设计hTDO选择性抑制剂作为癌症，神经退行性疾病和抑郁症的治疗药物提供重要的蓝图。