Analysis of the glutaryl-CoA dehydrogenase multiprotein complex

遗传性代谢紊乱戊二酸尿1型(GA1)是由线粒体同源四聚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戊二酸(GA)和3-羟基戊二酸(3OHGA)的积累，这些酸通过有机阴离子转运体(OAT)1跨膜转运。一些前期工作表明，GCDH还与电子转移黄素蛋白β亚单位(ETFB)和二氢硫辛酰胺琥珀酰基转移酶(DLST)形成异构体复合体。检测到更多相对分子质量更高的异构体GCDH复合体表明，进一步的、尚未鉴定的蛋白质与GCDH相互作用。我们假设这些多酶复合体(ES)中的蛋白质-蛋白质相互作用可能影响GCDH活性的调节。本项目的目的是加深对GCDH作为线粒体高相对分子质量多酶复合体的一员的作用的理解，以及定位于GCDH蛋白表面的致病氨基酸取代对这些复合体的影响。该项目的主要重点是a)利用共沉淀、蛋白质互补分析(PCA)和Bret分析，验证和表征GCDH与ETFB、DLST或其他最近发现的与GCDH结合的线粒体基质蛋白的异构体相互作用；b)通过与野生型或突变型GCDH相互作用蛋白的共表达，分析这些相互作用的功能影响，随后测量催化活性，以及测试GCDH蛋白表面定位的突变对此的影响；C)通过蓝色天然凝胶电泳联用质谱仪和主成分分析筛选线粒体基质蛋白文库，鉴定新的GCDH相互作用蛋白；d)通过靶向siRNA敲除鉴定线粒体GCDH降解酶(S)，及其对突变体GCDH稳定性的影响。此外，我们的目的是研究含有PDZ接头蛋白的OAT1在GCDH缺陷小鼠肾近端小管细胞和极化肾细胞系中的细胞内分选和重新分布的机制。这些研究可能导致确定药物靶点，以增加GA1患者体内病理性代谢物的转运和排泄。

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.