Taking the bait: exploiting new tools to capture, identify, and visualize active proteinases in osteoarthritic cartilage destruction

骨关节炎（OA）是一种影响英国约1000万人的疾病（VersusArthritis，2022）。公众对这种疾病的认识通常是准确的-对骨关节炎的理解是身体受影响关节周围软骨的破坏。然而，将骨关节炎视为关节过度使用或作为一般衰老过程的固有部分的想法是有缺陷的。过去的研究表明，OA中的软骨破坏是由基质金属蛋白酶（MMP）驱动的。随后的研究揭示了丝氨酸蛋白酶在导致关节软骨破坏的细胞过程中的深刻作用（威尔金森等人，2019），更重要的是，丝氨酸蛋白酶和MMP在软骨退化中的协同关系（威尔金森等人，2017 a;威尔金森等人，2017 b;威尔金森等人，2021;法尔科纳等人，2019年）。随着这些研究的进展，下一个合乎逻辑的步骤是表征关节不同区域内蛋白酶的活性，以及监测OA各个阶段蛋白酶的活性。因此，本项目的目的是设计和利用基于活性的探针（ABPs），以形成一个连贯的图片丝氨酸蛋白酶活性在骨关节炎关节。这些探针是化学过程的最终产物，其导致活性蛋白酶通过“弹头”（或诱饵）结合的有吸引力的靶标。此外，这些探针的内容物，例如生物素标签或荧光团，可以允许分别通过质谱法结合和表征蛋白酶或通过荧光成像表征蛋白酶活性。该项目将有三个不同的阶段，其中将通过使用ABP监测蛋白酶活性：首先，在培养的细胞群中，其次，在滑液中，最后，在OA小鼠模型中。我们对这些澄清程序的有效性充满热情，以促进对人体骨关节炎进展的科学理解，以及丝氨酸蛋白酶和基质金属蛋白酶在人体软骨分解中的作用。

Osteoarthritis (OA) is a disease that affects approximately 10 million people in the UK (VersusArthritis, 2022). Public knowledge of this disease is generally accurate - with a firm understanding of osteoarthritis as the breakdown of the cartilage surrounding the affected joints in the body. However, the idea of osteoarthritis as stemming from joint overuse or as an inherent part of the general aging process is flawed. Past research has shown that cartilage breakdown in OA is driven by matrix metalloproteinases (MMPs). Subsequent research reveals a profound role for serine proteinases in the cellular processes leading to the destruction of cartilage in the joint (Wilkinson et al, 2019) and, more importantly, a synergistic relationship between serine proteinases and MMPs in the deterioration of cartilage (Wilkinson et al., 2017a; Wilkinson et al., 2017b; Wilkinson et al., 2021; Falconer et al., 2019). With these advances in research, the next logical step is to characterize the activity of proteinases within different areas of the joint as well as monitor the activity of proteinases throughout the various stages of OA. Therefore, the aim of this project is to design and utilize activity-based probes (ABPs) to form a coherent picture of serine proteinase activity within the osteoarthritic joint. These probes are the final product of a chemical process that results in an attractive target for active proteinases to bind to via a "warhead" (or bait). In addition, the contents of these probes, such as a biotin tag or fluorophore, can allow for the binding and characterization of proteinases through mass spectrometry or the characterization of proteinase activity via fluorescent imaging, respectively. There will be three distinct stages to the project wherein proteinase activity will be monitored via the use of ABPs: firstly, in cultured cellular populations, secondly, in synovial fluid, and finally, in OA mouse models. We are enthusiastic about the effectiveness of these investigatory procedures to advance the scientific understanding of osteoarthritis progression in the human body as well as the role of serine proteinases and matrix metalloproteinases in the breakdown of human cartilage.