SELENODEIODINASE PROCESSING BY THE PROTEASOME SYSTEM

描述：（改编自申请人的摘要）这是一项新提案， 确定泛素中限速步骤的调节机制 蛋白酶体系统导致1型、2型和3型的受控降解 硒代脱碘酶D2是调节甲状腺第一步的关键酶 激素作用，激素原T4转化为活性激素T3。 这一过程发生在脑、垂体和棕色脂肪的细胞内， 产生的T3构成了核受体结合的主要部分， 激素.此外，在人类中，与成年啮齿动物不同，D2 也可以产生相当大部分的血浆T3， 在骨骼肌中的组织表达。多年来， 底物对D2的翻译后调节是一种主要的调节机制， 确定活性酶的组织水平的步骤。最近的研究 表明D2的降解发生在蛋白酶体中，其半衰期相当长， 短（<1小时），其降解加速2倍，暴露于 基底，并且该过程需要基底与D2 在其活性中心具有硒代半胱氨酸或半胱氨酸的酶。到 定义此过程中的监管步骤，研究者将使用几个 互补系统。这些包括在HEK-293细胞中的瞬时表达， 含有突变UB-1酶的温度敏感CHO细胞系。第三 策略将是利用D2可以在酵母中表达的事实， 在泛素-蛋白酶体降解途径中有充分记载的突变， 可以表达野生型和表位标记蛋白， 评价D2蛋白水平的活性变化。最后 研究者将采用无细胞系统， D2代谢中泛素蛋白酶体系统的特定组分 并将结果与D1和D3的结果进行比较，D1和D3具有显著更长的 半衰期从生理学的角度来看， 在饥饿期间，人骨骼肌中D2的加速蛋白水解， 疾病可以解释“低T3综合征”的快速发作。“这是一 这是一种公认但知之甚少的现象，发生在每个人身上。 食物限制或严重的系统性疾病。

DESCRIPTION: (Adapted from the applicant's abstract) This is a new proposal to determine the mechanisms regulating the rate-limiting steps in the ubiquitin proteasome system leading to the controlled degradation of the Types 1, 2 and 3 selenodeiodinases. D2 is a critical enzyme regulating the first step in thyroid hormone action, the conversion of the prohormone T4 to the active hormone, T3. This process occurs intracellularly in brain, pituitary and brown fat in which the T3 produced constitutes a major fraction of the nuclear receptor-bound hormone. Furthermore, it appears that, in humans, unlike in adult rodents, D2 may also generate a significant fraction of plasma T3 by virtue of its wide tissue expression in skeletal muscle. It has been known for a number of years that post-translational regulation of D2 by substrate is a major regulatory step in determining the tissue level of the active enzyme. Recent studies have shown that degradation of D2 occurs in proteasomes, that its half-life is quite short (<1 hr), that its degradation is accelerated 2-fold by exposure to substrate and that this process requires interaction of substrate with a D2 enzyme which has either selenocysteine or cysteine in its active center. To define the regulatory steps in this process, the investigator will use several complementary systems. These include transient expression in HEK-293 cells and a temperature-sensitive CHO cell line containing a mutant UB-1 enzyme. A third strategy will be to exploit the fact that D2 can be expressed in yeast with well-documented mutations in the ubiquitin-proteasome degradation pathway and that it is possible to express both wild type and epitope-labeled protein to evaluate changes in activity those in D2 protein levels. Lastly, the investigator will employ cell-free systems which will allow the study of specific components of the ubiquitin proteasome system in the metabolism of D2 and compare results with those for Dl and D3 which have significantly longer half-lives. From a physiological perspective, it is quite conceivable that the accelerated proteolysis of D2 in human skeletal muscle during starvation or illness could explain the rapid onset of the "low T3 syndrome." This is a well-recognized but poorly understood phenomenon which occurs in every human under food restriction or with significant systemic illness.