Molecular Dissection of a Candidate Ceramide Sensor

Abstract

Sphingomyelin is an essential component of cellular membranes that contribute to the barrier function of the plasma membrane, signaling and molecular sorting. Ceramides are precursors of sphingomyelins and they are produced de novo in the ER. Ceramides are also associated with growth arrest, senescence and apoptosis. Thus, cells must control their ceramides to keep the balance between proliferation and growth arrest/apoptosis. The bulk of newly synthesized ceramides is converted to sphingomyelin in the Golgi by sphingomyelin synthases (SMS). Mammalian cells contain two SMS isoforms, SMS1 in the Golgi and SMS2 at the plasma membrane. A closely related third enzyme, sphingomyelin synthase-related protein (SMSr) is not a conventional SM synthase but synthesizes ceramide-phosphoethanolamine. SMSr orthologs are found in all members of the animal kingdom, even though some do not produce SM. We recently suggested that SMSr is a candidate ceramide sensor whose primary role is to monitor ER ceramide levels to protect cells against the potential risk of apoptosis during sphingolipid biosynthesis. The present thesis aims to investigate the molecular mechanisms by which SMSr regulates ceramide homeostasis. We found that SMSr is a novel and specific substrate of caspase-6, a non-conventional effector caspase implicated in Alzheimer’s Huntington’s disease. In cells treated with staurosporine or FasL, SMSr undergoes caspase-6-dependent cleavage at a conserved aspartate located between its N-terminal SAM domain and the first membrane span. Next, we uncovered a striking structural and functional similarity between SMSr-SAM and the SAM domain of diacylglycerol kinase DGKδ, a central regulator of lipid signaling at the plasma membrane. We demonstrated that SMSr-SAM drives self-assembly of the enzyme into ER-resident trimers and hexamers. Mutations that destabilize SMSr oligomers caused a partial redistribution of the enzyme to the Golgi. Conversely, treatment of cells with curcumin, a drug disrupting ceramide and calcium homeostasis in the ER, stabilizes SMSr oligomers and promotes retention of the enzyme in the ER. Moreover, we report on the successful application of single-molecule photobleaching as approach to monitor homo-typic oligomerization of SMSr in its native cellular environment. By imaging GFP-tagged SMSr proteins as single-fluorescent spots in the ER of intact cells, we were able to trace photobleaching of protein complexes to monitor their oligomeric states. In agreement with our biochemical data, this analysis shows that the SAM domain of SMSr drives self-assembly of the enzyme in the ER and that curcumin promotes SMSr oligomerization. Our study documents, for the first time, that single-molecule photobleaching can be used to resolve changes in the oligomeric state of an ER-resident membrane protein, hence establishing a valuable complementary method to unravel the mechanism by which SMSr mediates ceramide homeostasis in the ER. Collectively, our findings in this thesis have resulted in a better understanding and a toolbox for further investigation of the candidate ceramide sensor SMSr.