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Structural determination and mechanistic understanding of membrane proteases

Final Activity Report Summary - MEMBRANE PROTEASES (Structural Determination and Mechanistic understanding of Membrane proteases.)

Cellular signalling is an important biological process in eukaryotic cells through which they communicate. Regulation of such signalling is critical since unchecked signalling can lead to cancer. Recent identification of intra-membrane proteases has revealed a new strategy for cellular regulation where transmembrane proteins, which are inactive in their membrane bound form, are activated by intra-membrane proteolytic cleavage. Proteolysis results in the release of cytoplasmic, luminal or extracellular domains that move to new locations where they can carry out their biological function. Different intra-membrane protease families, classified as serine, aspartyl or metalloprotease have been identified in all life kingdoms.

The primary objective of the project was the determination of intra-membrane proteases structures, along with their mechanistic understanding. My focus was on two of these families, namely rhomboid, an intra-membrane serine protease, and gamma secretase, an intra-membrane aspartyl protease. Rhomboids are widely distributed in all organisms and are involved in diverse processes, such as quorum sensing in bacteria, differentiation and growth factor signalling in eukaryotes. I was successful in obtaining two-dimensional and three-dimensional crystals of a prokaryotic homologue of rhomboid protease from escehrichia coli GlpG. The three-dimensional crystals of GlpG provided us with a structure that would enable us to carry out structure-function studies and understand the mechanism of proteolysis. Gamma secretase was a multi-subunit membrane protein complex, involved in cell signalling and generation of Aß (beta) peptide in Alzheimer disease. The major focus was to obtain good amounts of homogenous protein, suitable for cryo-electron microscopy, which would lay a platform for future structural work. Any structural information on intra-membrane proteases would be invaluable for rational design of drugs.