The proteolytic landscape of osteoarthritic cartilage

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The initiating causes of osteoarthritis (OA), a disease affecting all joint tissues, are complex. Regardless of the initiating cause, relentless, cell-mediated proteolytic degradation of articular cartilage extracellular matrix (ECM) follows, accompanied by cellular dysfunction, impairing joint function. Proteolytic fragments stimulate further catabolism, promote joint inflammation by acting as damage-associated molecular patterns (DAMPs) and are implicated as a cause of joint pain. Dysregulated proteolysis also has a role in cartilage aging since the senescence-associated secretory phenotype is associated with increased protease production. Despite the significance of proteolysis for OA and although some proteolytic events and pathways in OA are well-understood, much of the work on proteolysis to date has used candidate approaches to interrogate specific proteases or select ECM components. An unbiased strategy that would systematically elucidate the complete landscape of OA cartilage proteolysis has not been previously undertaken. Here, we will map proteolysis on a proteome-wide scale, identifying precise cleavage sites within the parent molecules directly within human knee OA cartilage. We hypothesize that cartilage from knees undergoing surgical replacement, which often show considerable regional variation in severity, contains most cleavages occurring through the natural history of OA and allows detailed categorization of the proteolytic landscape of OA cartilage relevant to its timeline and other factors. This important knowledge is currently lacking. The preliminary data demonstrates the experimental feasibility of a new approach to this unmet need and illustrates its potential to be potentially transformative for OA research. Specifically, we will apply advances in proteomics workflows and bioinformatics collectively termed N- terminomics, in which protein N-termini are labeled, enriched, identified by high resolution mass spectrometry and annotated by position in the parent molecules. We will apply the N-terminomics method Terminal Amine Isotopic Labeling of Substrates (TAILS), to knee OA and non-OA cartilage to gather all cleavages and create a searchable public database of their proteolytic landscapes (or degradomes), establishing a research resource that is currently unavailable (Aim 1); quantitatively compare proteolytic cleavage in OA vs non-OA cartilage, mild vs severely diseased OA cartilage, male vs female OA, as well as young with old control cartilage (Aim 2); and define the proteome-wide substrate repertoire of two proteases causally important in OA and identified as drug targets, ADAMTS5 and MMP13, to fully define their contribution to the proteolytic landscape of OA (Aim3). Impact: By providing a resource and platform for future OA research, proteome-wide analysis of cartilage degradation will provide prospective biomarkers for diagnosis, severity and drug response, help to resolve the protease activities contributing to OA progression, and identify potential targets and pathways for new therapy.