Protease degradomics: A new challenge for proteomics

Key Points

• Proteases initiate, modulate and terminate many important cellular functions by highly specific and limited substrate cleavage. This mechanism, called proteolytic processing, allows the precise cellular control of several biological processes, including DNA replication, cell-cycle progression, cell proliferation, wound healing, immunity, angiogenesis and apoptosis.

• The hierarchical importance of proteases in a system — a crucial issue in drug development — is influenced by specific activity of the protease, redundancy, expression levels, temporal–spatial distribution, zymogen activation, protease turnover and inhibition properties. Organism-wide degradomics approaches — which utilize genomic and proteomic techniques — are therefore required to identify the members of the ~500 protease human degradome that are expressed by a cell or tissue in disease, and to determine the complete natural substrate repertoire — the so-called “substrate degradome” — for each protease.

• Protease profiling using DNA microarray chips provides a general view of the protease transcriptome, but messenger RNA expression levels do not reflect protease protein abundance or activity, which can be determined with protease-specific and protease-activity protein chips. Substrate chips analyse the net proteolytic potential of the entire functional protease degradome towards a particular substrate without identifying the active proteases that are involved. This is important information, as the net cleavage of a particular substrate determines the biological response.

• Chemical proteomics uses labelled-irreversible protease inhibitors to isolate or identify active proteases in complex mixtures by two-dimensional (2D) gel electrophoresis or by using protease-activity chips with matrix-assisted laser desorption-ionization–time-of-flight (MALDI–TOF) or MALDI–quadrupole–TOF (MALDI–Q–TOF) mass-spectrometric identification of the captured proteases. In vivo applications of activity inhibitor probes include determination of protease function by chemical knockouts or intravital imaging of proteolytic activity.

• Isotope-coded affinity tags (ICATs) that are built on an irreversible protease-inhibitor scaffold can be used for targeted ICAT, a mass-spectrometric technique that is used for the identification and quantification of proteases that are differentially expressed by cells or tissues in distinct pathological conditions or physiological states.

• To identify protease substrates, peptide- or inhibitor-library approaches that determine peptide-bond preference can be complemented by analysis of substrate accumulation in protease-knockout mice, and 2D gel-MALDI–TOF or MALDI–Q–TOF mass-spectrometric analysis of proteolytic degradation products in protease-treated cell or tissue protein extracts. Yeast two-hybrid techniques of substrate identification by exosite-domain binding (exosite scanning) and inactive catalytic domain capture (ICDC) have now been adapted for proteomic screens on columns or chips.

• ; The recent identification of many bioactive molecules — including cytokines, cell-adhesion molecules and receptors — and intracellular targets (such as transcription factors and kinases) as new protease substrates that are precisely processed by proteolytic activity is redefining our views on the roles of several protease families in many physiological and pathological processes.

Abstract

Degradomics — the application of genomic and proteomic approaches to identify the protease and protease-substrate repertoires, or 'degradomes', on an organism-wide scale — promises to uncover new roles for proteases in vivo. This knowledge will facilitate the identification of new pharmaceutical targets to treat disease. Here, we review emerging degradomic techniques and concepts.