Post-Translational Modification-Dependent Activity of Matrix Metalloproteinases.

Due to their capacity to process different proteins of the extracellular matrix (ECM), matrix metalloproteinases (MMPs) were initially described as a family of secreted proteases, functioning as main ECM regulators. However, through proteolytic processing of various biomolecules, MMPs also modulate intra- and extracellular pathways and networks. Thereby, they are functionally implicated in the regulation of multiple physiological and pathological processes. Consequently, MMP activity is tightly regulated through a combination of epigenetic, transcriptional, and post-transcriptional control of gene expression, proteolytic activation, post-translational modifications (PTMs), and extracellular inhibition. In addition, MMPs, their substrates and ECM binding partners are frequently modified by PTMs, which suggests an important role of PTMs in modulating the pleiotropic activities of these proteases. This review summarizes the recent progress towards understanding the role of PTMs (glycosylation, phosphorylation, glycosaminoglycans) on the activity of several members of the MMP family.

Matrix Metalloproteinase 10 Degradomics in Keratinocytes and Epidermal Tissue Identifies Bioactive Substrates With Pleiotropic Functions.

Matrix metalloproteinases (MMPs) are important players in skin homeostasis, wound repair, and in the pathogenesis of skin cancer. It is now well established that most of their functions are related to processing of bioactive proteins rather than components of the extracellular matrix (ECM). MMP10 is highly expressed in keratinocytes at the wound edge and at the invasive front of tumors, but hardly any non-ECM substrates have been identified and its function in tissue repair and carcinogenesis is unclear. To better understand the role of MMP10 in the epidermis, we employed multiplexed iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) and monitored MMP10-dependent proteolysis over time in secretomes from keratinocytes. Time-resolved abundance clustering of neo-N termini classified MMP10-dependent cleavage events by efficiency and refined the MMP10 cleavage site specificity by revealing a so far unknown preference for glutamate in the P1 position. Moreover, we identified and validated the integrin alpha 6 subunit, cysteine-rich angiogenic inducer 61 and dermokine as novel direct MMP10 substrates and provide evidence for MMP10-dependent but indirect processing of phosphatidylethanolamine-binding protein 1. Finally, we sampled the epidermal proteome and degradome in unprecedented depth and confirmed MMP10-dependent processing of dermokine in vivo by TAILS analysis of epidermis from transgenic mice that overexpress a constitutively active mutant of MMP10 in basal keratinocytes. The newly identified substrates are involved in cell adhesion, migration, proliferation, and/or differentiation, indicating a contribution of MMP10 to local modulation of these processes during wound healing and cancer development. Data are available via ProteomeXchange with identifier PXD002474.

In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates.

Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the ProteomeXchange Consortium with identifier PXD001198.

Distinct extracellular-matrix remodeling events precede symptoms of inflammation.

Identification of early processes leading to complex tissue pathologies, such as inflammatory bowel diseases, poses a major scientific and clinical challenge that is imperative for improved diagnosis and treatment. Most studies of inflammation onset focus on cellular processes and signaling molecules, while overlooking the environment in which they take place, the continuously remodeled extracellular matrix. In this study, we used colitis models for investigating extracellular-matrix dynamics during disease onset, while treating the matrix as a complete and defined entity. Through the analysis of matrix structure, stiffness and composition, we unexpectedly revealed that even prior to the first clinical symptoms, the colon displays its own unique extracellular-matrix signature and found specific markers of clinical potential, which were also validated in human subjects. We also show that the emergence of this pre-symptomatic matrix is mediated by subclinical infiltration of immune cells bearing remodeling enzymes. Remarkably, whether the inflammation is chronic or acute, its matrix signature converges at pre-symptomatic states. We suggest that the existence of a pre-symptomatic extracellular-matrix is general and relevant to a wide range of diseases.

Cell density-dependent proteolysis by HtrA1 induces translocation of zyxin to the nucleus and increased cell survival.

Proteases modulate critical processes in cutaneous tissue repair to orchestrate inflammation, cell proliferation and tissue remodeling. However, the functional consequences and implications in healing impairments of most cleavage events are not understood. Using iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) we had characterized proteolytic signatures in a porcine wound healing model and identified two neo-N termini derived from proteolytic cleavage of the focal adhesion protein and mechanotransducer zyxin. Here, we assign these proteolytic events to the activity of either caspase-1 or serine protease HtrA1 and analyze the biological relevance of the resultant zyxin truncations. By cellular expression of full-length and truncated zyxin proteins, we demonstrate nuclear translocation of a C-terminal zyxin fragment that could also be generated in vitro by HtrA1 cleavage and provide evidence for its anti-apoptotic activities, potentially by regulating the expression of modulators of cell proliferation, protein synthesis and genome stability. Targeted degradomics correlated endogenous generation of the same zyxin fragment with increased cell density in human primary dermal fibroblasts. Hence, this newly identified HtrA1-zyxin protease signaling axis might present a novel mechanism to transiently enhance cell survival in environments of increased cell density like in wound granulation tissue.

Exploring Extracellular Matrix Degradomes by TMT-TAILS N-Terminomics.

Global characterization of protein N termini provides valuable information on proteome dynamics and diversity in health and disease. Driven by the progress in mass spectrometry-based proteomics, novel approaches for the dedicated investigation of protein N termini and protease substrates have been recently developed. Terminal amine isotopic labeling of substrates (TAILS) is a quantitative proteomics approach suitable for high-throughput and system-wide profiling of protein N termini in complex biological matrices. TAILS employs isotopic labeling of primary amines of intact proteins in combination with an amine-reactive high molecular weight polymer (HPG-ALD) for depletion of internal tryptic peptides and high enrichment of protein N termini by negative selection. Thereby, TAILS allows simultaneous identification of the natural N termini, protease-generated neo-N termini, and endogenously modified (e.g., acetylated) N termini. In this chapter, we provide a protocol for tandem mass tag (TMT)-TAILS analysis and further discuss specific considerations regarding N-terminome data interpretation using Proteome Discoverer™ software.

Comparative Degradomics of Porcine and Human Wound Exudates Unravels Biomarker Candidates for Assessment of Wound Healing Progression in Trauma Patients.

Impaired cutaneous wound healing is a major complication in elderly people and patients suffering from diabetes, the rate of which is rising in industrialized countries. Heterogeneity of clinical manifestations hampers effective molecular diagnostics and decisions for appropriate therapeutic regimens. Using a customized positional quantitative proteomics workflow, we have established a time-resolved proteome and N-terminome resource from wound exudates in a clinically relevant pig wound model that we exploited as a robust template to interpret a heterogeneous dataset from patients undergoing the same wound treatment. With zyxin, IQGA1, and HtrA1, this analysis and validation by targeted proteomics identified differential abundances and proteolytic processing of proteins of epidermal and dermal origin as prospective biomarker candidates for assessment of critical turning points in wound progression. Thus, we show the possibility of using a fine-tuned animal wound model to bridge the translational gap as a prerequisite for future extended clinical studies with large cohorts of individuals affected by healing impairments. Data are available via ProteomeXchange with identifier PXD006674.