Systemic determination of legumain physiological roles

Legumain is an evolutionary conserved cysteine protease with narrow substrate specificity. It is ubiquitously expressed in various cell and tissue types of animals, plants and other eukaryotes. Although it is primarily located in the endo/lysosomal compartments, legumain can be translocated to the cytosol, nucleus and extracellular space. In the last decade, numerous studies have connected legumain activity with several clinically relevant pathological processes such as cancer, neurodegeneration, atherosclerosis and acute cardiovascular events. However, only a handful of legumain physiological substrates has been identified so far and the exact mechanisms through which legumain influences biological processes remain largely unknown. In the course of our preliminary work using COFRADIC degradomic methodology, we were able to identify the largest set of legumain substrates reported to date and this finding puts us in a unique position to determine the pathways through which legumain affects physiology of cells and tissues on molecular level. Interestingly, majority of our identified legumain substrates are cytoplasmic proteins involved in regulation of gene transcription, RNA stability and intracellular signalling. Our finding therefore indicates the presence of completely novel legumain functions. The aim of the proposed work is to append the legumain substrate data with blood plasma peptidomic approach and to determine how proteolytic cleavage of legumain substrates influences processes like RNA metabolism and cellular signalling. To reveal that, we will perform a complex combination of proteomic, degradomic, phosphoproteomic and transcriptomic analysis of WT and leg-/- mice tissue samples. Obtained data will be bioinformatically integrated which will help us connect most relevant cleaved substrates with affected downstream processes and pathways. In the final stage, we will also develop cell-based assays, which will validate cleavages of selected substrates with specific cellular processes. We are convinced that such comprehensive analysis preformed in in vivo experimental setting will provide an unprecedented new insight into legumain biological function. This knowledge will also be able to expand the use of legumain targeting in clinical applications such as development of novel diagnostic, prognostic and therapeutic approaches.