Selenodeidinase Processing by the Proteasome System

描述（由申请人提供）：2型碘甲状腺原氨酸脱碘酶（D2）是催化甲状腺激素作用的第一步（甲状腺素（T4）转化为活性激素3，5，3 '-三碘甲状腺原氨酸（T3））的关键调节性硒酶。支持这一建议的研究已经导致了新的结论，这种酶的活性调节post-acidonally之间的动态平衡的泛素化，导致D2失活和蛋白酶体降解，去泛素化，重新激活it. We的目标是确定这两个过程中的蛋白质和监管步骤，并把这个新认识到的机制，为维持T3稳态到生理背景。D2是一种内质网（ER）整合膜蛋白，选择性泛素化并靶向蛋白酶体系统，这一过程称为ER相关降解（ERAD）。我们鉴定了ERAD相关的UBC 6和UBC 7作为D2的重要泛素缀合物（E2）。我们假设D2泛素化是在被一种尚未鉴定的D2特异性泛素连接酶（E3）识别后启动的，该连接酶与这些E2相关。具体目标我将确定关键的E3，他们的接触网站和他们的功能。D2在基础条件下不断被泛素化，但这被T4催化加速。这些过程中的关键步骤的身份是未知的。底物诱导的泛素化是一个类似的，但更快的反应比基础泛素化或有不同的E2和D2特异性E3参与？由于我们正在研究的过程是复杂的，特异性目的II将使用体内系统，包括哺乳动物双杂交系统和肽，可以阻止E3结合，以确认体外结果和定义T4相关事件。使用酵母双杂交系统，我们确定了两个密切相关的D2结合泛素特异性加工蛋白酶（UBP），专门去泛素化和重新激活D2。在棕色脂肪组织中，冷暴露诱导其中之一的快速增加，激活泛素化D2并比从头合成更快地提供活性酶。目前对这些UBPs知之甚少，而Specific Aim III将确定它们的D2相互作用机制，发育表达以及T3，cAMP和其他激素的调节。公共卫生相关性：甲状腺激素是所有脊椎动物发育和控制人类能量消耗的关键激素。本项目旨在了解甲状腺激素作用调节的新机制。一旦完成，我们将更有能力开发药理学工具，以在特定组织的基础上调节甲状腺激素的作用，这与肥胖、胰岛素抵抗和代谢综合征极为相关。

DESCRIPTION (provided by applicant): The type 2 iodothyronine deiodinase (D2) is a pivotal regulatory selenoenzyme catalyzing the first step in thyroid hormone action, the conversion of thyroxine (T4) to the active hormone, 3,5,3'-triiodothyronine (T3). Studies supported by this proposal have led to the novel conclusion that the activity of this enzyme is regulated post-translationally by a dynamic balance between ubiquitination, causing D2 inactivation and proteasomal degradation, and deubiquitination, which reactivates it. Our goals are to identify the proteins and regulatory steps in these two processes, and place this newly recognized mechanism for maintaining T3 homeostasis into a physiological context. D2 is an endoplasmic reticulum (ER) integral membrane protein selectively ubiquitinated and targeted to the proteasomal system, a process termed ER-associated degradation (ERAD). We identified the ERAD-related UBC6 and UBC7 as important ubiquitin conjugases (E2s) for D2. We hypothesize that D2 ubiquitination is initiated after recognition by an as yet unidentified D2-specific ubiquitin ligase (E3) that associates with these E2s. Specific Aim I will identify the critical E3s, their contact sites and their function. D2 is constantly ubiquitinated under basal conditions but this is accelerated by T4 catalysis. The identity of the critical steps in these processes is unknown. Is substrate-induced ubiquitination a similar, but faster, reaction than basal ubiquitination or are there different sets of E2s and D2-specific E3 involved? Since the process we are investigating is complex, Specific Aim II will use in vivo systems including mammalian two hybrid systems and peptides which can block E3 binding to confirm the in vitro results and define T4 related events. Using a yeast-two-hybrid system, we identified two closely related D2-binding ubiquitin-specific processing proteases (UBPs) that specifically deubiquitinate and reactivate D2. In brown adipose tissue, cold exposure induces a rapid increase one of these, activating ubiquitinated D2 and providing active enzyme more rapidly than does de novo synthesis. Little is known of these only recently identified UBPs and Specific Aim III will define their D2 interaction mechanism, developmental expression and regulation by T3, cAMP and other hormones. PUBLIC HEALTH RELEVANCE: Thyroid hormone is a critical hormone for all vertebrate development and control of energy expenditure in humans. This project is to understand new mechanisms involved in the regulation of thyroid hormone action. Once completed, we will be better equipped to develop pharmacological tools to regulate thyroid hormone action on a tissue-specific basis, which is extremely relevant for obesity, insulin resistance and the metabolic syndrome.