Catalytic and regulatory mechanisms of human Tryptophan Dioxygenase

 描述(申请人提供)：人色氨酸双加氧酶(HTDO)是一种重要的血红蛋白，催化犬尿氨酸(KYN)途径的起始和限速步骤-色氨酸转化为N-甲酰犬尿氨酸(NFK)。众所周知，hTDO在控制肝脏内色氨酸的动态平衡中起关键作用。最近，人们发现hTDO也在多种人类肿瘤中表达，以抑制抗肿瘤免疫，从而促进肿瘤的侵袭性生长。除了癌症，hTDO还与一系列神经退行性疾病有关，如阿尔茨海默氏症和亨廷顿氏病，以及抑郁症。因此，hTDO已被公认为一个重要的治疗靶点。尽管它很重要，但由于对该酶的分子性质知之甚少，这一进展一直受到限制。最近，我们在以下方面取得了几项重大突破：(I)开发了一种新的方案，可以制备高质量的hTDO蛋白质，使人们能够第一次看到对双加氧酶化学至关重要的结构特征，以及铁酶的异常活性，(Ii)解决了hTDO的第一个底物结合的三维结构，以及在单晶中捕获反应中间体并解决了它们的结构，这揭示了几个以前未知的结构特征，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，以及(Iii)首次获得支持hTDO通过泛素化连接的蛋白酶体降解途径的细胞降解的证据。这些重要的成就使我们能够构建新的假设，并从独特的角度提出一种全面的方法来检验它们。这个项目的主要目标是描述hTDO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)确定独特的铁反应的氧气活化机理。(Iii)说明hTDO的产品释放机制。(4)阐明hTDO的外源依赖调控机制。我们预计，完成该项目的目标将导致对这种关键酶的结构、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。

 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.