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  • Review Article
  • Published:

In search of partners: linking extracellular proteases to substrates

Nature Reviews Molecular Cell Biology volume 8, pages 245–257 (2007)Cite this article

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?

Abstract

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.

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Figure 1: Human and murine protease degradomes.
Figure 2: Degradomics discovery of protease substrates.
Figure 3: Mouse models in protease research.
Figure 4: Linking enzymes and substrates: the big picture.

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Acknowledgements

C.M.O. is supported by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology, with research grants from the Canadian Institutes of Health Research, the National Cancer Institute of Canada (with funds raised by the Canadian Cancer Association), and the Canadian Breast Cancer Research Alliance special program grant on metastasis, as well as with a Centre Grant from the Michael Smith Research Foundation. C.P.B. is funded by grants from the National Institutes of Health, from the National Institute of General Medical Sciences and from the Eye Institute, and by a sponsored Research Agreement from Novartis, Basel, Switzerland.

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Authors and Affiliations

  1. Departments of Oral Biological & Medical Sciences, The UBC Centre for Blood Research, CBCRA Program in Breast Cancer Metastasis, and Biochemistry & Molecular Biology, University of British Columbia, 4.401 Life Sciences Center, Vancouver, V6T 1Z3, British Columbia, Canada

    Christopher M. Overall

  2. Arthritis and Tissue Degeneration Program, Caspary Research Building, Room 426, Hospital for Special Surgery, 535 East 70th Street, New York, 10021, New York, USA

    Carl P. Blobel

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  1. Christopher M. Overall
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Correspondence to Carl P. Blobel.

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FURTHER INFORMATION

Carl P. Blobel's homepage

Christopher M. Overall's homepage

CancerDegradome

Human, Mouse and Rat Degradomes

International Proteolysis Society

MEROPS: the Peptidase Database

Nomenclature Committee of the International Union of Biochemistry and Molecular Biology — Peptidase Nomenclature

Prediction of Protease Specificity (PoPS)

Glossary

Extracellular matrix

A complex extracellular network of structural proteins, including collagens, glycoproteins and proteoglycans, that supports cell adhesion and migration, and that transmits information through interactions with cell receptors.

Processing

Proteolysis that is distinct from degradation in that it represents a highly specific and efficient, yet limited, activity. Nonetheless, cleaving a protein at only one or two sites can result in a specific change of protein function.

Ectodomain

The extracellular portion of a plasma-membrane protein. In secretory vesicles, the topologically equivalent compartment is the lumen.

Chemokines

A large group of cytokines that elicit chemotactic responses from leukocytes and some other cells that express specific G-protein-coupled chemokine receptors.

Matrix metalloproteinases

(MMPs). A family of 23 metzincin proteases in humans capable of degrading extracellular matrix proteins and of processing many bioactive molecules.

Neoprotein

A protein with a new function generated from a protease-cleavage product that is functionally different from the parent protein.

Degradome

The complete set of proteases that are expressed at a specific time by a cell, tissue or an organism. The degradome of a protease is its substrate repertoire.

Anti-target

A molecule with essential roles in normal cell and tissue function, or life; the down modulation of an anti-target results in clinically unacceptable side effects, initiation of disease or deleteriously alters disease progression.

Extracellular protease

An enzyme that has a catalytic domain in the extracellular compartment or in the lumen of a secretory compartment, both of which are topologically equivalent.

Proteomics

Investigations and techniques for elucidating the proteome.

Degradomics

All genomics, proteomics and systems biology investigations and techniques regarding the genetic, structural and functional identification and characterization of the proteases, inactive homologues, protease substrates and protease inhibitors that are present in an organism.

Zymography

A method for determining protease substrates. Substrates are separated by non-denaturing gel electrophoresis and are incubated with a protease. Negative staining of the stained substrate gel reveals enzymatic activity because the protease has degraded the substrate in the gel.

Activity-based probe

Mechanism-based inhibitor that has been modified by incorporating detection moieties, such as fluorophores, biotin and radioactive elements, to specifically target and visualize individual proteases or a family of proteases in complex samples.

Non-prime residue

A residue in the substrate that is N-terminal to the proteolytic cleavage site is called a non-prime (P) residue, and in some proteases forms part of the recognition motif for substrate cleavage.

Prime residue

A residue in the substrate that is C-terminal of the proteolytic cleavage site is called a prime (P′) residue and in some proteases forms part of the recognition motif for substrate cleavage.

ADAM proteases

A disintegrin and metalloprotease (ADAM) proteases are multifunctional membrane proteins with crucial roles in ectodomain shedding of other membrane proteins, such as the ligands of the epidermal-growth-factor receptor.

Tissue inhibitors of metalloproteinases (TIMPs)

A family of four specific inhibitors of matrix metalloproteinases and some ADAM proteases that are expressed by most mammalian cells.

Aptamers

Aptamers are chemically synthesized (usually short) strands of oligonucleotides (DNA or RNA) that can adopt highly specific three-dimensional conformations. Aptamers are designed to have appropriate binding affinities and specificities towards certain target molecules.

Exosite

A substrate-binding site that lies outside the active-site cleft of a protease. It is usually located on substrate-binding modules or domains and can function to accelerate the rate of substrate cleavage.

Anti-neoepitope antibody

An antibody that specifically recognizes the free amino or carboxyl groups of the amino-acid residues from a cleaved scissile bond that forms the new N and C termini of the cleaved product.

Proteome

The expressed set of proteins that are encoded by the genome and that are expressed by a particular cell or tissue.

Peptide mapping

By proteomically comparing the abundance ratios of multiple peptides from a substrate with their location in the protein sequence, the domain that is proteolytically released can be predicted, as can the general location of the cleavage site.

Terminopes

The N and C termini of a protein are chemically distinguished from the remainder of the intact molecule. Terminopes are generated following proteolytic cleavage. They might also be immunologically recognized as an antibody epitope, called a neoepitope.

Singletons

In proteomics analyses of two peptide samples that are labelled with different isotopes, a singleton is a single ion peak that is detected in the mass spectrometry spectrum without its comparative isotopic counterpart owing to the absence of that parent protein or peptide in one sample. This might occur because of reduced expression or following the cleavage of an intact protein or in the generation of a unique N- or C-terminal peptide following cleavage.

Driver lines

A system, such as the Cre–Lox system, for creating conditional gene deletions.

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Overall, C., Blobel, C. In search of partners: linking extracellular proteases to substrates. Nat Rev Mol Cell Biol 8, 245–257 (2007). https://doi.org/10.1038/nrm2120

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