Explore the vast range of titles available.

Clinical trial results with matrix metalloproteinase (MMP) inhibitors have been poor. So, which MMPs are validated drug targets with a crucial role in disease pathogenesis and which MMPs are actually proteins that have unacceptable deleterious effects when inhibited (anti-targets)? The matrix metalloproteinases (MMPs) mediate homeostasis of the extracellular environment. They have multiple signalling activities that are commonly altered during tumorigenesis and that might serve as intervention points for anticancer drugs. However, there are many criteria to consider in validating MMPs as drug targets and for the development of MMP inhibitors. The inhibition of some MMPs could have pro-tumorigenic effects (making them anti-targets), counterbalancing the benefits of target inhibition. These effects might partially account for the failure of MMP inhibitors in clinical trials. What are the major challenges in MMP target validation and MMP-inhibitor-drug development?

Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8-/- versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and validated evidence for the existence of the protease web, a network that affects the activity of most proteases and thereby influences the functional state of the proteome and cell activity.

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.

[This corrects the article DOI: 10.1371/journal.pone.0000312.].

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo–N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro ; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.

Many proteases are important drug targets, but identification of their substrates remains challenging. By using polymers to selectively isolate N-terminal peptides generated by proteolysis of complex samples, Kleifeld et al . identify substrates of clinically relevant proteases with broad specificity. Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.

The spatiotemporal proteome of the intervertebral disc (IVD) underpins its integrity and function. We present DIPPER, a deep and comprehensive IVD proteomic resource comprising 94 genome-wide profiles from 17 individuals. To begin with, protein modules defining key directional trends spanning the lateral and anteroposterior axes were derived from high-resolution spatial proteomes of intact young cadaveric lumbar IVDs. They revealed novel region-specific profiles of regulatory activities and displayed potential paths of deconstruction in the level- and location-matched aged cadaveric discs. Machine learning methods predicted a ‘hydration matrisome’ that connects extracellular matrix with MRI intensity. Importantly, the static proteome used as point-references can be integrated with dynamic proteome (SILAC/degradome) and transcriptome data from multiple clinical samples, enhancing robustness and clinical relevance. The data, findings, and methodology, available on a web interface ( http://www.sbms.hku.hk/dclab/DIPPER/ ), will be valuable references in the field of IVD biology and proteomic analytics. The backbone of vertebrate animals consists of a series of bones called vertebrae that are joined together by disc-like structures that allow the back to move and distribute forces to protect it during daily activities. It is common for these intervertebral discs to degenerate with age, resulting in back pain and severely reducing quality of life. The mechanical features of intervertebral discs are the result of their proteins. These include extracellular matrix proteins, which form the external scaffolding that binds cells together in a tissue, and signaling proteins, which allow cells to communicate. However, how the levels of different proteins in each region of the disc vary with time has not been fully examined. To establish how protein composition changes with age, Tam, Chen et al. quantified the protein levels and gene activity (which leads to protein production) of intervertebral discs from young and old deceased individuals. They found that the position of different mixtures of proteins in the intervertebral disc changes with age, and that young people have high levels of extracellular matrix proteins and signaling proteins. Levels of these proteins decreased as people got older, as did the amount of proteins produced. To determine which region of the intervertebral disc different proteins were in, Tam, Chen et al. also performed magnetic resonance imaging (MRI) of the samples to correlate image intensity (which represents water content) with the corresponding protein signature. The data obtained provides a high-quality map of how the location of different proteins changes with age, and is available online under the name DIPPER. This database is an informative resource for research into skeletal biology, and it will likely advance the understanding of intervertebral disc degeneration in humans and animals, potentially leading to the development of new treatment strategies for this condition.

Proteomics techniques can be applied in drug target identification and validation, but data interpretation can be complicated by the identification of proteins in unexpected cellular locations. Here, Butler and Overall discuss the importance of recognizing that many intracellular proteins may have physiological functions in the extracellular compartment, and its implications for drug discovery. Proteomics has revealed that many proteins are present in unexpected cellular locations. Moreover, it is increasingly recognized that proteins can translocate between intracellular and extracellular compartments in non-conventional ways. This increases gene pleiotrophy as the diverse functions of the protein that the gene encodes are dependent on the cellular location. Given that trafficking drug targets may exist in various forms — often with completely different functions — in multiple cellular compartments, careful interpretation of proteomics data is needed for an accurate understanding of gene function. This Perspective is intended to inspire the investigation of unusual protein localizations, rather than assuming that they are due to mislocalization or artefacts. Given a fair chance, proteomics could reveal novel and unforeseen biology with important ramifications for target validation in drug discovery.

A method to analyze the sequence of C-terminal peptides using a combination of a specific enrichment approach and mass spectrometry is described, allowing the study of C-terminal proteolytic processing on a global scale. Also in this issue, Van Damme et al . describe a related method for simultaneous C- and N-terminal peptide analysis. As proteome-wide C-terminal sequence analysis has been largely intractable, we developed a polymer-based enrichment approach to profile protein C-terminal peptides by mass spectrometry and identified hundreds of C-terminal peptides in the Escherichia coli proteome. We isotopically labeled GluC protease–digested and undigested samples and identified GluC substrates and their cleavage sites by quantification of neo–C-terminal peptides. Our method thus enables global annotation of protein C-terminal posttranslational modifications, including proteolytic truncations.

We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. Protease- and protease inhibitor–specific microarray analyses (CLIP-CHIP) of laser-dissected leukocyte infiltrated and noninfiltrated pancreatic islets and confirmatory quantitative real time PCR and protein analyses identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. Interestingly, the peri-islet BM and underlying IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies.