Transmembrane domain interactions in carnitine palmitoyltransferase I

Abstract

Membrane proteins are involved in a wide range of vital cellular processes, responsible for relaying signals and cargo across cell membranes. A deeper molecular understanding of their function is essential to elucidate the mechanisms of numerous diseases and medical conditions. As an example, the carnitine palmitoyltransferase 1 (CPT1) enzymes are responsible for the regulation of mitochondrial fatty acid oxidation thus are central to cell function. Previous work had shown that the transmembrane (TM) domains of the mitochondrial outer membrane protein CPT1 have a significant influence on the enzyme’s kinetics, which is different in the two catalytically active isoforms, CPT1A and CPT1B. It was also shown that TM-domains, and specifically TM2, are involved in driving the oligomerisation of the full length CPT1A. For this reason, the study of TM-TM oligomerisation and their roles in the function of CPT1 are fundamentally important in the design of pharmacological strategies aimed at the modulation of the activities of these enzymes in conditions such as diabetes. The main focus of this PhD research was to systematically investigate the homo- and hetero-oligomerisation of the TM domains to help better understand the structurefunction relationship for CPT1 membrane proteins. In this project, different techniques were used from the areas of chemistry, molecular biology, and biophysics (in vivo oligomerisation assays, chemical crosslinking, circular dichroism and analytical ultracentrifugation), in order to examine these interactions. The membrane spanning sequences of rCPT1A and rCPT1B were found to selfassociate, and the order of oligomerisation was also determined. Sequence motifs likely to be responsible for the interactions of these TM domains were identified using in silico modelling. Mutagenesis analyses confirmed the role of suggested GxxxG(A) motifs in the homo-oligomerisation of rCPT1A TM2 domains. The heterooligomerisation of TM1-TM2 domains was also studied, and was found to be significant in both isoforms. The work presented in this thesis shows that the membrane spanning regions of the CPT1 enzymes are capable of interacting through both homo- and hetero-oligomerisation. The results strongly suggest that these interactions may play a significant role in the complex formation of the full length enzymes and provide further evidence that CPT1 may function as a channel in the outer mitochondrial membrane