A PROTEASE can be defined as an enzyme that hydrolyzes peptide bonds. Proteases can be divided into endopeptidases or proteinases, which cleave internal peptide bonds in proteins, and exopeptidases, which cleave terminal peptide bonds. Exopeptidases can be further subdivided into aminopeptidases and carboxypeptidases, depending on which end of the protein amino acids are cleaved off. Proteases can be classified as aspartic proteases (e.g., cathepsins D and E, pepsin, renin), cysteine proteases (e.g., cathepsins B, L, S, K, Q, calpains, and caspases), metalloproteases (e.g., gelatinases A and B), serine proteases (e.g., plasminogen activators, plasmin, and chymase), and threonine proteases (e.g., proteasome), depending on the nature of the active site. Selective inhibitors can be used to distinguish among these classes of proteases. Protease activity is regulated in vivo by altering the rate of their synthesis and degradation, activation of (pre-)proforms, and binding with endogenous inhibitors.
Initially, proteases were considered as hydrolytic enzymes that were associated with protein catabolism, but it is now widely accepted that the highly specific hydrolysis of peptide bonds can regulate a wide range of biological processes (e.g., via processing of bioactive peptides) in all living organisms. This highly specific substrate cleavage is referred to as proteolytic processing, which regulates the activity and the compartmentalization of many proteins and therefore of many cellular processes (Barrett et al. 1998; Lopez-Otin and Overall 2002). Dysregulation of protease expression and in particular of their activity is involved in various pathological conditions, such as cardiovascular and neurodegenerative diseases, arthritic diseases, infection, and cancer. Therefore, proteases are attractive potential therapeutic targets.
Various techniques are used to determine the presence of proteases in tissues. Northern blotting analysis and RT-PCR are applied to quantify mRNAs in tissue extracts, whereas in situ hybridization (ISH) is used to localize mRNA in cell preparations or tissue sections. However, transcriptional activity does not necessarily reflect the amount and activity of the protein product of a certain gene. Western blots and immunohistochemistry (IHC) are used to determine the amount and localization of the protease protein but do not provide information about the activity of a protease because proteases are synthetized in an inactive proform or preproform that requires proteolytic processing for activation. Moreover, endogenous protease inhibitors can bind proteases and inhibit them. Biochemical techniques have been developed to detect protease activity in tissue extracts. However, homogenization of tissues for these assays does not allow localization of enzyme activity. In addition, extraction procedures can artifactually activate enzymes or cause interactions of active enzymes with their respective inhibitors when they are localized in different compartments in intact tissues. Therefore, techniques to localize specific proteolytic activity in cell preparations or tissue sections may provide crucial additional information on the exact role played by proteases in various physiological and pathological conditions.