3D ELECTRON MICROSCOPY OF PYRUVATE DEHYDROGENASE

丙酮酸脱氢酶的 3D 电子显微镜

• 批准号: 6530645
• 负责人: JAMES K STOOPS
• 金额: $ 27.76万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 2004-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6530645
• 关键词: Saccharomyces cerevisiae; X ray crystallography; active sites; binding proteins; chemical cleavage; computer simulation; cryoelectron microscopy; dihydrolipoamide dehydrogenase; dimer; enzyme complex; enzyme structure; image processing; immunoglobulin structure; macroglobulins; methylamines; protease inhibitor; protein structure function; pyruvate dehydrogenase; structural biology

alpha2-Macroglobulin (alpha2M), a general proteinase inhibitor ubiquitous in the plasma of vertebrates, is believed to serve as a general proteinase scavenger thereby protecting blood and tissue proteins from degradation. A better understanding of the structure- function relationships of human alpha2M will result from the determination of the structure of its various complexes by stain and cryo-electron microscopy, and image processing. The native and transformed molecules (formed upon reaction with a proteinase) are comprised of dimers. The determination of the 3-D structure of the native dimer (protomeric unit) will elucidate the structural organization of the native molecule and offer further insight in the mechanism of proteinase entrapment. The 3-D structure of alpha2M with the truncated bait domain compliments this study since this variant alpha2M is not capable of trapping the proteinase. Bait domain cleavage is the initial step in the reaction Of alpha2M with a proteinase. A 3-D structure of anti-bait domain Fab-labelled native alpha2M will locate this important functional site and make it possible to compare it with the location of this epitope in the transformed structure. Further insight into the mechanism of the transformation of native to the activated structure will be obtained by determining the 3-D structures of monoclonal Fab-labelled native, half-transformed and transformed alpha2Ms. Mammalian pyruvate dehydrogenase complex (PDC) plays a critical role in regulating cellular fuel utilization in the heart and, therefore, studies of the structure-function relationships of the PDC may expand the treatment options for heart disease. Saccharomyces cerevisiae PDC activity is regulated by the pyruvate dehydrogenase component (E1). The 3-D structure (determined by cryo-electron microscopy) of E1 and its binding subunit beta associated with dihydrolipoamide acetyltransferase core complex (E2) will elucidate its disposition on the core and may give insight into its structural organization. The disposition of functional sites associated with the PDC will be elucidated by determining the 3-D structure of these sites labelled with a gold cluster. They include the lipoyl domain of the binding protein (BP) and the catalytic sites of the dihydrolipoamide dehydrogenase (E3). 3-D reconstructions of recombinant intact E2 and the E2 core with BP E3 and E2 BP E3 E1 bound should elucidate the structural organization of this large complex. A 3-D reconstruction of the beef kidney intact PDC will permit a comparison of the mammalian and yeast complexes.

α2-巨球蛋白（α2M），一种通用蛋白酶抑制剂 普遍存在于脊椎动物的血浆中，被认为是一种 一般蛋白酶清除剂，从而保护血液和组织 蛋白质免受降解。 更好地理解结构—— 人类 alpha2M 的功能关系将由 通过染色和测定其各种配合物的结构 冷冻电子显微镜和图像处理。 本土和 转化分子（与蛋白酶反应形成）是 由二聚体组成。 3-D结构的测定 天然二聚体（原体单元）将阐明结构 天然分子的组织并提供进一步的见解 蛋白酶包埋机制。 alpha2M 的 3-D 结构 截断的诱饵结构域补充了这项研究，因为这个变体 alpha2M 不能捕获蛋白酶。 诱饵结构域裂解 是α2M与蛋白酶反应的第一步。 3-D 抗诱饵结构域 Fab 标记的天然 alpha2M 将定位 这个重要的功能站点并使其可以与 该表位在转化结构中的位置。 更远 洞察本土向本土转化的机制 通过确定 3-D 结构可以获得激活的结构 单克隆 Fab 标记的天然、半转化和转化 阿尔法2女士。哺乳动物丙酮酸脱氢酶复合物 (PDC) 发挥着重要作用 在调节心脏细胞燃料利用方面发挥着关键作用， 因此，对PDC结构与功能关系的研究 可能会扩大心脏病的治疗选择。酵母属 酿酒酵母 PDC 活性受丙酮酸脱氢酶调节 组件（E1）。 3-D 结构（由冷冻电子确定） E1及其结合亚基β的显微镜检查） 二氢硫辛酰胺乙酰转移酶核心复合物（E2）将阐明其 核心的配置，可以深入了解其结构 组织。 与相关功能位点的配置 PDC 将通过确定这些位点的 3-D 结构来阐明 标有金色簇。 它们包括硫辛酰结构域 结合蛋白（BP）和二氢硫辛酰胺的催化位点 脱氢酶（E3）。 重组完整 E2 和的 3-D 重建 E2 核心与 BP E3 和 E2 BP E3 E1 结合应该阐明 这个大型综合体的结构组织。 3D 重建 牛肾完整的 PDC 将允许对哺乳动物和 酵母复合物。

项目成果

Reverse transcriptase and protease inhibitors mutational viral load in HIV infected pregnant women with transmitted drug resistance in Argentina.

• DOI: 10.37201/req/022.2021
• 发表时间: 2021-08
• 期刊: Revista espanola de quimioterapia : publicacion oficial de la Sociedad Espanola de Quimioterapia
• 作者: Cecchini D;Sfalcin J;Zapiola I;Gómez A;Fernández Giuliano S;Mammana L;Seravalle A;Rodríguez C;Fay F;Bouzas MB
• 通讯作者: Bouzas MB