Structural Insights into Non-vesicular Trafficking of Lipids by Lipid Transfer Proteins and Bacterial Toxin-Antitoxin Systems
Phosphorylated sphingolipid Ceramide-1-Phosphate (C1P) is a key regulator of cell growth, survival, migration and inflammatory responses, but non-vesicular mechanisms involved in its intracellular sensing, transfer and presentation remained unexplored till recently. We have identified a widely-expressed protein, CPTP (Ceramide-1-Phosphate Transfer Protein) in humans, which specifically transfers C1P. Our co-crystal structures established that C1P binding occurs via a surface-localized phosphate head-group recognition center connected to a hydrophobic pocket. Down-regulation of CPTP dramatically alters C1P steady-state levels, decreasing at the plasma membrane while elevating at the trans-Golgi where C1P is produced by ceramide kinase. The elevated C1P triggers proinflammatory eicosanoid generation associated with cellular inflammation, thus highlighting its physiological importance. We have also carried out structure-function studies on lipid transfer proteins involved in cell survival such as “accelerated cell death 11 (ACD11)” protein from Arabidopsis and “heterokaryon incompatibility C2 protein (HET-C2)” from the fungus Podospora anserina which has provided insights on roles of lipid transfer process in cell survival.
Almost all bacteria and many archaea contain genes (encoding toxins) whose expression inhibit cell growth and may lead to cell death when overproduced, reminiscent of apoptotic genes in eukaryotes. These toxins are co-expressed and neutralized with their cognate antitoxins from TA (toxin-antitoxin) operons in normally growing cells. MazF (toxin) / MazE (antitoxin) system is one of the most extensively characterized TA systems. Under stress conditions, labile anti-toxins (MazE) are readily degraded by proteases allowing MazF (toxin) to cleave mRNA in a sequence specific manner. Recently, we solved the structure of Bacillus MazF in complex with ssRNA containing its target site as well as in complex with its cognate antitoxin MazE. This study has provided for the first time structural basis of recognition and cleavage of mRNA by MazF in a sequence specific manner during stress conditions and also demonstrated how antitoxin inactivates the toxin in normally growing cells.