Degradomics of proteolytic mechanisms in post-traumatic osteoarthritis

Cleveland Clinic Lerner Com-Cwru, Cleveland OH

Investigators

Abstract

Osteoarthritis (OA) is usually diagnosed when joint damage is well under way. Its early detection and treatment continue to be elusive, requiring greater knowledge of pathogenic processes and potential drug targets active early in the disease. Proteolytic destruction of cartilage extracellular matrix (ECM) is a hallmark of OA that contributes to loss of joint function; proteolytic products provoke further catabolism, drive inflammation and incite pain. Despite the interest in proteases as drug targets in OA, the scale, timeline and impact of proteolysis and the contributing proteases are largely unknown. The few biomarkers that detect proteolytic cleavages in the joint are widely used, but more are needed, especially for incipient/early OA. These unmet needs matter acutely for post-traumatic OA (PTOA), since a high proportion of the growing number of patients with significant joint injuries are at risk of OA at a young age but cannot be identified early enough. Prior research investigated only select proteases and cartilage components, providing a very limited view of joint breakdown. This lack of a proteome- wide definition of the proteolytic landscape of OA, its timeline and molecular mechanisms has hindered early, active clinical management of PTOA. Degradomics, a specialized proteomics approach tailored for proteolysis, has the potential to fill these gaps in knowledge and unmet needs, but has not been applied to PTOA. The objective of this proposal is to precisely define proteolytic events on the proteome scale (i.e., the degradome) and proteolytic mechanisms along the timeline of PTOA, taking advantage of its sharply defined starting point in a previously healthy joint. Our recent analysis of human OA cartilage and synovial fluid degradomes demonstrated substantial proteolytic information content from cartilage, other joint tissues and circulating proteins in synovial fluid. We developed a rigorous approach for defining the precise contributions of individual proteases to joint breakdown in OA and created a new web-based resource for sharing these data with the research community. We have piloted quantitative degradomics comparison of healthy and OA synovial fluid. In addition, we have established spectral libraries of knee OA synovial fluid for data-independent acquisition mass spectrometry (DIA-MS), as a starting point for an innovative quantitative degradomics technology. These accomplishments and strong preliminary data laid the groundwork for our three objectives, 1. To quantitatively define proteolytic impact and mechanisms in PTOA by quantitative analysis of synovial fluid degradomes in humans with anterior cruciate ligament (ACL) injury and an experimental equine model of PTOA. 2. To precisely define specific proteolytic contributions by key proteases to PTOA/OA, 3. Develop proteolysis- oriented DIA-MS as a high-throughput, scalable technology, to be applied initially for quantitative degradomics of synovial fluid, but with the eventual goal of identifying PTOA proteolytic signatures in blood. Impact: We expect to deliver time- and protease-resolved degradomic signatures of PTOA, new insights on osteoarthritis mechanisms and new technology for early diagnosis, risk assessment and drug development.