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Biochemical Investigation of Ceramide Transfer Protein Regulation
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De novo synthesis of sphingomyelin in the trans-Golgi requires the ceramide transfer protein (CERT) to deliver ceramide, a key sphingolipid precursor, from its site of synthesis in the endoplasmic reticulum (ER) to the Golgi apparatus. CERT contains an N-terminal pleckstrin homology (PH) domain which interacts with phosphatidylinsolitol-4-phosphate (PI4P) for targeting to the Golgi, a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain responsible for extracting and transferring ceramide, and a serine-rich (SR) motif adjacent to the PH domain which receives multiple phosphorylations resulting in inhibition of PI4P binding and ceramide transfer. CERT also contains an uncharacterized middle region (MR) that contains a two phenylalanines in an acid tract (FFAT) motif and a putative coiled-coil (CC) motif. The FFAT motif is responsible for interaction with VAP-A, an integral ER membrane protein, for tethering to the ER. It has been previously reported that phopshomimic mutation of all 10 Ser/Thr residues to Glu (CERT-10E-FL) within the SR motif down-regulates CERT activity, which requires the presence of both the PH and START domains. The crystal structure of the PH/START complex reveals the START domain interacts with the PH domain and inhibits PI4P binding, whereas the isolated PH domain has little effect on ceramide transfer by the START domain. Here we provide biochemical evidence that the middle region of CERT contributes to inhibition of ceramide transfer by the START domain, and that the CC motif within the middle region of CERT is required for 10E inhibition of CERT activity. Additionally, small angle x-ray scattering data suggest CERT-10E-FL adopts an elongated and semi-rigid conformation that is associated with inhibition of CERT activity, and the formation of this conformation requires the putative CC motif. In addition to investigating the biochemical basis of 10E inhibition of CERT activity, we also carried out biochemical investigations of CERT activity enhancement. First, we probed the role of PI4P in the enhancement of ceramide transfer. Preliminary data indicate PI4P is not specifically required for enhancement of ceramide transfer of wild-type CERT (CERT-WT-FL), rather acceptor membrane targeting is primarily responsible for enhancement of CERT-WT-FL activity. Secondly, we investigated a CERT S315E phosphomimic mutant and its ability to increase binding affinity for VAP-A regardless of SR motif phosphomimic state. The S315E mutation increases VAP-A binding affinity in the full-length protein, but not in a short FFAT motif peptide suggesting S315E acts to expose the FFAT motif for VAP-A binding. Finally, we present progress on investigations of an interaction between the CERT PH domain and a Chlamydial inclusion membrane protein (IncD).
Table of Contents
Introduction -- Materials and methods -- Investigating the Role of the Middle Region (MR) in the function and regulation of the Ceramide Transfer Protein (CERT) -- The putative Coiled-Coil Motif of CERT is Required for Proper Phosphorylation Inhibition -- Investigating the mechanism for PI4P Enhancement of Ceramide Transfer by CERT -- Understanding the Structural basis of how the S315E Mutation enhances CERT Binding to the VAP-A Protein -- Investigating a potential Interaction Between Chlamydial Inclusion Membrane Protein D and CERT -- Final Discussion -- Appendix we -- Appendix II
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Ph.D. (Doctor of Philosophy)