Journal Article

In search of partners: linking extracellular proteases to substrates

Overall, Christopher M., Blobel, Carl P.

2007

Overview

Key Points Proteases function as molecular switches in signalling circuits on the cell surface and in the extracellular milieu. In light of the many proteases that are encoded by the genome, and the even larger number of bioactive substrates, it is crucial to identify which substrates individual enzymes cleave and which proteases cleave a particular substrate. Four general approaches are discussed that are commonly used to link proteases and relevant substrates: biochemistry, cell biology, proteomics and animal models (with a focus on mouse models). Biochemical studies use purified enzymes and substrates, and provide valuable information on which peptide and proteins a protease can cleave, the substrate's cleavage sites, and the inhibitor profile for small molecules and naturally occurring protease inhibitors. Cell-biological assays help determine the function of enzymes in the context of an intact cell. Gain- and loss-of-function experiments can link enzymes and substrates and can help build hypotheses about enzyme function. Regulation of enzymes by activators and inhibitors of signal transduction as well as by transcriptional activation can be evaluated. Degradomics studies of proteolysis use liquid chromatography or gel-based approaches for the mass spectrometric analysis of proteolysis. Degradomics enables the identification of hundreds or thousands of proteins in complex proteomes that have been moulded by proteolysis. Through isotope tagging, changes in the abundance levels of multiple peptides of a sample enables the identification of cleaved native substrates in cell-based systems to define the substrate degradome of a protease. Mouse models allow an analysis of a protease's function in the context of an intact organism and help establish its expression pattern and relevance in development, adult homeostasis and disease models. Loss-of-function models help evaluate the contribution of enzymes to development and disease in vivo , whereas gain-of-function models yield insights into the consequences of dysregulated enzyme activity. The combined application of these different approaches provides insights that exceed the sum of the individual approaches, and help resolve questions that arise from individual approaches. For example, they can help resolve the question: which of several candidate enzymes are relevant for processing a substrate in different cells and tissues or under different circumstances during development and in disease? Extracellular proteases regulate cell function through proteolytic switching of signalling circuits and through shedding of membrane proteins. It is crucial to link proteases to their substrates to understand their functions in development and disease, and to validate proteases as drug targets. Proteases function as molecular switches in signalling circuits at the cell surface and in the extracellular milieu. In light of the many proteases that are encoded by the genome, and the even larger number of bioactive substrates, it is crucial to identify which proteases cleave a particular substrate and which substrates individual proteases cleave. Elucidating the substrate degradomes of proteases will help us to understand the function of proteases in development and disease and to validate proteases as drug targets.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: Analysis / Animals / Arthritis / Biochemistry / Biomedical and Life Sciences / Cancer Research / Cell Biology / Cellular signal transduction / Chemokines / Cytokines / Developmental Biology / Enzyme kinetics / Extracellular matrix / Humans / Libraries / Life Sciences / Peptide Hydrolases - chemistry / Peptide Hydrolases - genetics / Peptide Hydrolases - metabolism / Peptides / Proteins / Proteolysis / Proteomics / Proteomics - methods / review-article / Stem Cells / Substrate Specificity / Substrates / Tumor necrosis factor-TNF