Analysis of the glutaryl-CoA dehydrogenase multiprotein complex

遗传性代谢紊乱1型谷氨酸尿症（GA 1）是由同源四聚体线粒体基质酶戊二酰辅酶A脱氢酶（GCDH）缺乏引起的。GCDH缺乏导致病理性代谢产物的积累，即谷氨酸（GA）和3-羟基谷氨酸（3OHGA），它们通过有机阴离子转运蛋白（OAT）1跨膜转运。自己的准备工作表明，GCDH还与电子转移黄素蛋白β亚基（ETFB）和二氢硫辛酰胺琥珀酰转移酶（DLST）形成异聚体复合物。更高分子量的其他异聚GCDH复合物的检测表明，进一步的，但身份不明，蛋白质与GCDH相互作用。我们假设这些蛋白质-蛋白质相互作用的多酶复合物（ES）可能会影响GCDH活性的调节，我们的项目的目的是提高GCDH作为这些线粒体高分子量多酶复合物的成员的作用的理解，和致病性氨基酸取代定位在这些复合物的GCDH蛋白的表面上的影响。该项目的主要重点是a）使用共沉淀、蛋白质互补测定（PCA）和BRET分析，验证和表征GCDH与ETFB、DLST或与其他最近鉴定的GCDH结合线粒体基质蛋白的异聚体相互作用; B）通过进行相互作用蛋白与野生型或突变型GCDH的共表达来分析这些相互作用的功能影响，和随后的催化活性测量，以及测试定位在GCDH蛋白表面上的突变对其的影响; c）通过应用蓝色天然凝胶电泳，随后对选择的条带进行质谱分析，并通过线粒体基质蛋白文库的PCA筛选，鉴定新的GCDH相互作用蛋白; d）通过靶向siRNA敲低鉴定线粒体GCDH降解蛋白酶，及其对突变GCDH稳定性的影响。我们的目的是研究Gcdh肾近曲小管细胞中OAT 1通过含PDZ的衔接蛋白进行细胞内分选和再分布的机制。缺陷小鼠和极化肾细胞系中。这些研究可能导致药理学靶点的鉴定，以增加GA 1患者体内病理代谢物的运输和排泄。

The inherited metabolic disorder glutaric aciduria type 1 (GA1) is caused by the deficiency of the homotetrameric mitochondrial matrix enzyme glutaryl-CoA dehydrogenase (GCDH). GCDH deficiency leads to the accumulation of pathologic metabolites, namely glutaric (GA) and 3-hydroxyglutaric (3OHGA) acids, which are translocated across membranes amongst others via the organic anion transporter (OAT) 1.Own preparatory work showed that GCDH forms also heteromeric complexes with the electron transfer flavoprotein beta-subunit (ETFB), and dihydrolipoamide succinyltransferase (DLST). The detection of additional heteromeric GCDH complexes of higher molecular weight indicates that further, yet unidentified, proteins interact with GCDH. We hypothesize that these protein-protein interactions in multienzym complex(es) may affect the regulation of GCDH activity.The aim of our project is to improve the understanding of the role of GCDH as a member of these mitochondrial high molecular weight multienzyme complexes, and the effects of pathogenic amino acid substitutions localized on the surface of the GCDH protein on these complexes. The main focus of the project is a) the verification and characterization of heteromeric interactions of GCDH with ETFB, with DLST, or with other recently identified GCDH-binding mitochondrial matrix proteins, using co-precipitation, protein complementation assays (PCA), and BRET analyses; b) analyses of the functional impact of these interactions by performing co-expression of interacting proteins with wild-type or mutant GCDH, and subsequent measurements of the catalytic activity, as well as testing for the effects of mutations localized on the GCDH protein surface on this; c) identification of new GCDH-interacting proteins by applying blue native gel electrophoresis followed by mass spectrometry of selected bands, and by PCA screen of a mitochondrial matrix protein library; d) identification of mitochondrial GCDH-degrading protease(s) through targeted siRNA knock down, and their implications on the stability of mutant GCDH.Furthermore, we aim to examine the mechanisms of intracellular sorting and redistribution of OAT1 by PDZ-containing adaptor proteins in renal proximal tubule cells of Gcdh-deficient mice, and in a polarized kidney cell line. These investigations may lead to the identification of pharmacological targets to increase the transport and excretion of pathologic metabolites out of the body of GA1 patients.