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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.

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Figure 1: Relationship of degradomics to the fields of proteomics and genomics, and of the degradome to the proteome and genome.
Figure 2: Degradomics approaches: activity profiling.
Figure 3: Exosite scanning and inactive catalytic domain capture (ICDC): substrate scanning by yeast two-hybrid screens and immobilization techniques.

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Acknowledgements

C.M.O. is supported by a Canada Research Chair in Metalloproteinase Biology, and by grants from the Canadian Institutes for Health Research, the National Cancer Institute of Canada, the Protein Engineering Network of Centers of Excellence and the Canadian Arthritis Network of Centers of Excellence. C.L-O. is supported by the Comision Interministerial de Ciencia y Tecnologia, Spain, and by the European Union.

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Correspondence to Christopher M. Overall .

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DATABASES

Flybase

Rhomboid

LocusLink

α-defensin

Fas ligand

galectin-3

IL-1α

IL-1β

IL-8

IL-18

γ-interferon

MEKK-1

MMP-2

MT1-MMP

tumour-necrosis factor-α

Swiss-Prot

Caspase-1

caspase-11

β-catenin

cathepsin B

cathepsin C

cathepsin G

cathepsin L

cathepsin S

chymase

FAK

GAL4

granzyme A

granzyme B

neutrophil elastase

p53

pRb

STAT-1

FURTHER READING

Bioactive MMP substrates

Chris Overall's laboratory 

Encyclopedia of Life Sciences

confocal microscopy

liquid chromatography 

MEROPS version 5.7

Glossary

PROTEASE

An enzyme that cleaves proteins by the catalysis of peptide-bond hydrolysis. On the basis of their catalytic mechanism, proteases belong to one of five classes (aspartic, cysteine, metallo, serine, or threonine).

PROTEOLYTIC PROCESSING

Proteolysis that is distinct from degradation in that it represents highly specific and limited substrate cleavage, which results in a specific change of protein function.

MATRIX METALLOPROTEINASES

A family of 23 endoproteinases in humans that are encoded by 24 genes. These are characterized by a HEXXHXXGXXH zinc-binding motif, a cysteine-switch mechanism of proenzyme latency, an ability to cleave extracellular-matrix and bioactive molecules, and inhibition by tissue inhibitors of metalloproteinases (TIMPs).

MASS SPECTROMETRY

A technique that precisely measures sample mass from the analysis of mass-to-charge ratio (m/z).

PEPTIDE FINGERPRINTING

A mass spectrometric technique of protein identification that matches tryptic peptide masses of an unknown protein with those that are generated in silico for all the proteins in a database.

ADAM AND ADAMTS

Cell surface (ADAM) or secreted (ADAMTS) metalloproteinases that are related to MMPs, but that have a different multidomain structure, which includes a cysteine-rich disintegrin domain and thrombospondin modules.

INTRAVITAL IMAGING

Visualization of biological processes in intact animals or organ systems.

ISOTOPE CODED AFFINITY TAGS

(ICAT). ICAT probes have different masses, but are chemically identical. They incorporate a reactive cysteine, a biotin moiety, and eight deuteriums in place of eight hydrogens, and they are used to specifically label, by mass-difference, identical proteins in two separate samples for the identification and semiquantitative comparison of abundance.

EXOSITE

A substrate-binding site that lies outside the active-site cleft of a protease and that is located on specialized substrate-binding modules/domains. Exosites generate diversity in substrate specificity and produce increased rates of cleavage.

EXOSITE SCANNING

On the basis of the hypothesis that proteins that bind protease exosites might be substrates, this is a technique for substrate identification that uses recombinant exosite domains as bait (see Ref. 8).

INACTIVE CATALYTIC DOMAIN CAPTURE

(ICDC). A technique used for the identification of protease substrates. This uses mutated, proteolytically inactive catalytic domains to capture potential substrates without cleavage and subsequent release.

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López-Otín , C., Overall , C. Protease degradomics: A new challenge for proteomics. Nat Rev Mol Cell Biol 3, 509–519 (2002). https://doi.org/10.1038/nrm858

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