Analysis of the glutaryl-CoA dehydrogenase multiprotein complex

• Applicant: Professor Dr. Chris Mühlhausen
• Subject Area: Pediatric and Adolescent Medicine
• Term: from 2011 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 211332540

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.

• DFG Programme: Research Grants