DESCRIPTION (provided by applicant): Caspases are cysteine aspartases found in all eukaryotic cells as zymogens that require induced proximity and/or proteolytic cleavage for activation. Activated caspases function as both regulators and executioners of cell death in response to developmental cues or environmental stress (e.g. apoptosis) and infections (e.g. pyroptosis and necroptosis). Activated caspases can have equally profound systemic effects by controlling inflammatory responses through cytokine maturation as well as induction of cell proliferation and migration in the wound-healing response (e.g. the "phoenix rising" phenomena). Therefore there has been a significant amount of research on the mechanism of caspase activation and the function of active caspases since aberrant activation could have catastrophic consequences for the cell. It has long been presumed that the zymogens of caspases (procaspases) had no function and were only present to allow for a quick and decisive means for a cell to induce cell death or an inflammatory response. While studying the role of the effector caspases-3 and -7 on mitochondrial function following cytochrome c release, we serendipitously discovered novel functions for procaspase-3 and procaspase-7 that shed new light on why cells carry killer molecules. Mouse embryo fibroblasts (MEFs) that are deficient in casp3 display enhanced adhesion and altered migration velocity that is associated with increased fibronectin secretion. Introduction of procaspase-3 or a procaspase-3 that is catalytically inactive reverses this phenotype. Casp7-deficient MEFs have altered morphology and loss of directionally-persistent migration. These events appear to be associated with altered actin cytoskeleton organization and are also independent of caspase activity. Thus effector procaspases are not only poised killer molecules, they have cellular functions in viable cells. The first two Specific Aims of this application are designed to determine how these effector procaspases regulate cell adhesion, migration and secretion. Additionally we have found that the survival advantage observed with casp3-deficient MEFs is dependent on cell adhesion. Casp3-deficient cells are not protected against loss of adherence to extracellular matrix (anoikis). This suggests that the non-apoptotic functions of procaspase-3 can also influence the cellular response to apoptotic signals. This would have a significant impact on current models of feed-forward loops that have been proposed to explain how an effector caspase could influence events that occur upstream of caspase activation. Therefore the final Specific Aim of this application will re-examine the mechanism by which loss of casp3 alters cell survival. Completion of these Aims will have a paradigm-shifting effect on how we think about procaspases and may require a re-interpretation of data generated with caspase-deficient cells.

PUBLIC HEALTH RELEVANCE: Effector caspases are involved in dismantling a cell that undergoes a specialized form of cell death called apoptosis and are present in cells in an inactive form called procaspases. We have found novel roles for two procaspases and propose to further define how these proteins control cell adhesion and movement when they are not activated to kill cells. Understanding how procaspases regulate these other biologic functions could have important implications for therapeutic approaches to diseases including metastatic cancer and wound-healing disorders.