Caspase-independent mechanisms of GzmH-induced target cell death

点击翻译按钮获取中文摘要

Recently, our findings demonstrated the importance of recombinant GzmH as a novel cytotoxic effector protease. Although we were unable to find a cell death related substrate, our study was strongly suggestive of target cell death defined by caspase independent damage inflicted upon DNA and mitochondria. This initial study sets the scene for GzmH as an important protease in innate immunity. The study also lays down the foundations for the search into the pro-apoptotic substrates that govern the cell death pathway triggered by this chymotrypsin-like protease, the goal of this project. To achieve our aim, we intent to use state of the art methods. Firstly, with the use of protease substrate libraries, comprised of a selection of linker regions of known human cytosolic proteins, we will attempt to better decipher the cleavage specificity of GzmH; but also its cytotoxic action, by searching for peptides that are cleaved by GzmH and which are derived from pro- and anti-apoptotic proteins. Additional methods such as “proteome-wide identification of protease cleavage specificity” or “CLIP-PICS” employ proteome-wide peptide libraries as proteolytic substrate screens for the characterization of protease active site specificity. The technique should allow us to selectively isolate neo amino (N)-terminal peptides, generated following treatment with GzmH, from target cell lysates. The recovered N-terminal peptides can then be identified by sequencing and various mass spectrometric methods. Secondly, the search for GzmH related substrates has prompted us to attempt the structural analysis of GzmH. Our recombinant, non glycosylated and untagged GzmH is a perfect candidate for crystallization, not only because we are able to produce high quantities of inclusion body based protein from E. coli, but also because pure recombinant GzmH lacks N-linked carbohydrates which facilitates crystallization. The three-dimensional crystal structure of active GzmH would be a helpful tool with which to gain a better understanding of the protease’s overall structure and its substrate specificity but also for the development highly specific inhibitors. Indeed, since the animal models of choice do not possess a functional equivalent to the human GzmH gene, functional studies on GzmH by knock-out methodology are not possible. As we learn more about the specificity of GzmH, the design of tailored GzmH inhibitors should dramatically improve our understanding of GzmH cytotoxicity.