Selenodeiodinase processing by the proteasome system

蛋白酶体系统处理硒代碘化酶

• 批准号: 6795500
• 负责人: PHILIP REED LARSEN
• 金额: $ 4.03万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6795500
• 关键词: Europe; SDS polyacrylamide gel electrophoresis; autoradiography; catalyst; endoplasmic reticulum; enzyme substrate; immunoprecipitation; iodide peroxidase; physiologic stressor; proteasome; protein localization; protein metabolism; protein sequence; protein structure function; proteolysis; recombinant DNA; starvation; thyronines; transfection; ubiquitin

DESCRIPTION (provided by applicant) This collaborative study will be primarily performed in Hungary at the Institute of Experimental Medicine as an extension of NIH grant #RO1 DK-58538 to reveal novel molecular aspects of the selective proteolysis of the type 2 deiodinase (D2), the major enzyme activating thyroxine. It is proposed that as an extension of the parent grant these studies will identify D2 structures which are responsible for the metabolic instability of this key rate-limiting enzyme of thyroid hormone action serving as triggers for ubiquitination. In addition, D2 degradation will be investigated from a novel point of view. The hypothesis will be tested whether the intracellular localization of D2 plays a role in its degradation. An experimental model will be established in mammalian cells that enables the systematic testing of the functional role of different D2 domains in the half-life of type 2 deiodinase. Expression constructs will be created using standard recombinant DNA techniques suitable for fusion of D2 fragments to the carboxy terminus of Sec62. The stable and endoplasmic reticulum (ER) inserted Sec62 with both of its termini in the cytosol will drive D2 with no transmembrane domain into its native compartment, the ER. The amino terminus of Sec62 will be FLAG tagged and its carboxy terminus will be used for fusion with the amino terminus of different D2 and fragments, lacking the D2 transmembrane domain. Second, D2 fragments will be systematically tested to identify fragments involved in decreasing half-life of the D2/Sec62 fusion protein. As controls, D1/Sec62 controls will be used since D1 is a long-lived protein and is not expected to change Sec62 half-life. Constructs will be transiently transfected into HEK-293 cells and the half-life determined by pulse chase with 35S Met/Cys. FLAG-immunoprecipitation will be followed by SDS-PAGE and autoradiograpy. We will also investigate D2 degradation from a novel point of view studying whether the intracellular localization of D2 plays a role in the selected proteolysis of D2. By fusing D2 to a stable, plasma membrane located protein, such as the type 1 deiodinase, we will direct D2 into a different cell compartment and investigate, whether the D2 degradation is dependent on the ER based ERAD system or independent from its intracellular localization and is able to change the half-life of the D2/NIS fusion protein. Since the "low T3 syndrome" is associated with impaired T3 generation and in certain mesotheliomas overexpress D2, it is critical to understand the rate-limiting steps in D2 proteolysis.

描述(由申请人提供) 这项合作研究将主要在匈牙利的实验医学研究所进行，作为NIH赠款#RO1 DK-58538的延伸，以揭示2型脱碘酶(D2)选择性蛋白质分解的新分子方面，D2是激活甲状腺激素的主要酶。有人建议，作为父母拨款的延伸，这些研究将确定D2结构，这些结构负责这种甲状腺激素作用的关键限速酶的代谢不稳定性，该酶是泛素化的触发因素。此外，将从新的角度研究D2的降解。这一假说将被检验D2的细胞内定位是否在其降解过程中发挥作用。将在哺乳动物细胞中建立一个实验模型，使系统地测试不同D2结构域在2型脱碘酶半衰期中的功能作用。将使用适合于将D2片段融合到Sec62的羧基末端的标准重组DNA技术来创建表达构建体。稳定的内质网(ER)插入的Sec62的两端都位于胞浆中，它将驱动没有跨膜结构域的D2进入其天然的内质网。Sec62的氨基末端将被标记，其羧基末端将用于与不同的D2和片段的氨基末端融合，缺少D2跨膜结构域。其次，将对D2片段进行系统测试，以确定与缩短D2/Sec62融合蛋白半衰期有关的片段。作为对照，将使用D1/Sec62对照，因为D1是一种长寿蛋白质，预计不会改变Sec62的半衰期。将构建的载体瞬时导入HEK-293细胞，用35S Met/Cys脉冲追踪法测定半衰期。FLAG免疫沉淀后将进行十二烷基硫酸钠-PAGE和放射自显影。我们还将从一个新的角度研究D2的降解，研究D2在细胞内的定位是否在D2的选择性蛋白分解中发挥作用。通过将D2与稳定的、位于质膜上的蛋白质融合，如1型脱碘酶，我们将D2引导到不同的细胞室，并研究D2的降解是依赖于基于ER的ERAD系统还是独立于其细胞内定位，并能够改变D2/NIS融合蛋白的半衰期。由于“低T_3综合征”与T_3生成受损和某些间皮瘤过度表达D2有关，因此了解D_2蛋白分解的限速步骤是至关重要的。