The mitochondrial intermembrane space protein Smac/DIABLO in signal transduction and immune response

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Smac is a mitochondrial intermembrane space protein that is released and that facilitates caspase-activation during apoptosis. A number of other, potential, cytosolic functions of Smac can be inferred based on experimental cytosolic expression of Smac and on the use of small-molecule ‘Smac-mimetics’ but there is very little evidence of a function of Smac-protein outside cell death. It is becoming evident that the apoptosis signalling apparatus can also be activated only to a low level, where some signalling events can be detected but the cell stays alive. We have recently found that such ‘sub-lethal’ mitochondrial apoptosis signalling (‘minority MOMP’) is a feature of infection of human and mouse cells with any of several bacterial and viral pathogens tested. Our data suggest that this signalling is a mechanism of cell alert and plays a role in anti-microbial host defence. Our recent data further suggest that Smac is important in mitochondrial sub-lethal apoptosis signalling. We have tested for a role of Smac in infection in vitro with Helicobacter pylori and have found evidence that Smac is in this infection released during minority MOMP, is required for the activation of alternative NF-kappaB and contributes to cytokine production by epithelial cells. We propose that Smac-release is a general feature of minority MOMP, and that Smac has a signalling function in the detection of the infection of non-professional immune cells by pathogens. In this project we will test this hypothesis. We will analyse the relative release of Smac and cytochrome c during situations of minority MOMP and will establish whether it is individual mitochondria or more of the mitochondrial network releasing these proteins. We will further endeavour to distinguish the relative roles of cytochrome c and Smac for the biological effects of minority MOMP. We will investigate Smac-dependent signalling in the cytosol, focussing on events consecutive to the neutralisation of inhibitor of apoptosis proteins and will more thoroughly test for the secretion of cytokines and bacterial containment. We have made a Smac-deficient mouse line and will use intestinal organoids from these mice and perform infections of the mice with Helicobacter felis and Chlamydia muridarum to understand the contribution of Smac to the immune response to bacterial infection. Our data suggest that Smac has signalling functions outside apoptotic cell death in the initiation of an immune reaction, and we hope to be able here to work out signalling pathways and physiological roles of such functions.