Granule serine proteases and cysteine cathepsins of immune cells regulate physiological and pathological processes through the cleavage of substrate proteins. These highly reactive enzymes are stored in specialized lysosomes and are released into other cellular and extracellular compartments to perform their homeostatic and antimicrobial functions. Proteases are also accidentally released into the cytosol after granule leakage or extracellularly after cell death causing cellular and tissue damage. Serpins are the largest family of serine and cysteine protease inhibitors and are essential for the regulation of proteolysis in humans as shown by inborn diseases caused by inherited serpin gene defects. In vertebrate evolution, serpin genes were duplicated, providing redundancy and opportunity for the evolution of new regulatory functions to control proteolysis and maintain homeostasis and immunity.
We have previously identified essential functions of clade B serpins in the inhibition of leukocyte serine proteases in cell death, inflammation, and infection. In particular, we have shown the importance of certain protease-serpin axes in a cell-specific manner. We have also found that serpin deficiencies are associated with higher release of cytokines and delayed resolution of inflammation and infection. Particularly, we have shown that the neutrophil serine protease cathepsin G preferentially activates gasdermin D leading to enhanced release of pro-inflammatory IL-1ß in absence of cytosolic serpins. However, molecular mechanisms leading to cathepsin G-mediated cell death and to release of other cytokines by uncontrolled proteases remain to be discovered. Using an unbiased proteomic screen, we have now identified novel target proteins that are specifically cleaved by neutrophil proteases and that may function as molecular bottlenecks in cell death, inflammatory signaling pathways, and immune responses to pathogens. To address these knowledge gaps we propose the following specific aims:
Aim 1. Identification of molecular targets of cathepsin G mediated cell death
Aim 2. Characterization of new mechanisms of inflammation regulated by serpins, neutrophil proteases, and their targets, notably in the context of SARS-CoV-2 infection
Based on strong preliminary findings from an unbiased proteomic screen, we will use our expertise in cell death, inflammation and infectious disease to generate new advances in the field. Using in vitro assays as well as genetically modified mouse models, we will test specific hypothesis in the regulation of inflammatory and innate immune responses that will shed light on yet unexplored path in cell death and uncontrolled cytokine responses.
