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Mind the Gap: Mechanism and Impact of Ceramide Trafficking at the ER-Mitochondrial Interface

Final Report Summary - MECETERMITI (Mind the Gap: Mechanism and Impact of Ceramide Trafficking at the ER-Mitochondrial Interface)

BACKGROUND - Ceramides are obligatory precursors for sphingolipid biosynthesis but also potent mediators of apoptosis. However, the mechanism by which ceramides exert their apoptogenic activity is largely unexplored. We previously identified sphingomyelin synthase-related protein SMSr as a candidate ceramide sensor in the ER. Acute disruption of SMSr function causes an accumulation of ER ceramides and their mislocalization to mitochondria, triggering a mitochondrial pathway of apoptosis. Targeting a bacterial ceramidase to mitochondria rescues SMSr-deficient cells from apoptosis, indicating that translocation of ER ceramides to mitochondria is essential for committing cells to death. Indeed, mistargeting ER ceramides to mitochondria with engineered lipid transfer proteins is sufficient to trigger cell death. How ER ceramides can reach mitochondria and promote outer membrane permeabilisation to execute apoptosis remains to be established.

ACHIEVEMENTS - In this project, I used a photoactivatable and clickable ceramide analogue, pacCer, to search for ceramide transport machinery at ER-mitochondrial junctions as well as down-stream effectors involved in the execution phase of ceramide-mediated cell death. Proteins in direct contact with pacCer can be tagged by photo-affinity labelling and then clicked with a reporter molecule to allow their visualization and identification. Besides CERT and a novel putative ceramide transfer protein, pacCer labelling of cell lysates led to the identification of two porins as the principal candidate ceramide binding proteins in mitochondria. Interestingly, both proteins have previously been implicated as critical players in the cytosolic release of mitochondrial apoptogenic proteins. To investigate whether these porins play a role as down-stream ceramide effectors in mitochondrial apoptosis, I will analyse the consequences of their removal on the fate of SMSr-depleted cells and isolate ceramide-binding defective mutants to study their ability to support ceramide-mediated cell death.

POTENTIAL IMPACT - A principal cause of failure in the treatment of cancer is that cancer cells develop resistance to chemo- and radiotherapy, leading to recurrence of the disease or even death. Sphingolipid molecules can modulate the ability of malignant cells to grow and resist anticancer regimens, with some molecules promoting tumorigenesis and others acting as tumour-suppressors, e.g. ceramides. Several anti-cancer regimens cause an increase in endogenous ceramide levels through de novo synthesis, leading to G0/G1 arrest and cell death. Moreover, blocking ceramide export from the ER by knocking down ceramide transfer protein CERT sensitises various types of cancer cells to cytotoxic agents. Such tumour-suppressor activity of ceramides has prompted development of different formulations of synthetic ceramides in anticancer therapies, but the poor solubility of these compounds restricts their biocompatibility. Therefore, attention is increasingly focused on possibilities to influence cellular sphingolipid balances from within. In this project, I established new experimental approaches to unravel the mechanisms by which newly synthesized ceramides can commit cells to death. Precise knowledge of the underlying molecular principles may open up new avenues for controlling the fate of diseased cells, in particular those causing cancer.

Marie Curie Intra European fellow: Dr. John Mina
Host laboratory: Molecular Cell Biology Division, University of Osnabrück, Germany
Contact: john.mina[at]biologie.uni-osnabrueck.de
Website: www.holthuis-lab-uos.de