The proteolytic landscape of osteoarthritic cartilage

骨关节炎（OA）是一种影响所有关节组织的疾病，其发病原因很复杂。无论 引发关节软骨细胞外基质（ECM）的持续细胞介导的蛋白水解降解 随后，伴随细胞功能障碍，损害关节功能。蛋白水解片段进一步刺激 catastrophic，促进关节炎症作为损害相关的分子模式（DAMP）， 被认为是关节疼痛的原因蛋白质水解失调也在软骨老化中起作用， 衰老相关的分泌表型与蛋白酶产量增加有关。尽管 蛋白水解对OA的重要性，尽管OA中的一些蛋白水解事件和途径已被充分理解， 迄今为止，许多关于蛋白质水解的工作已经使用候选方法来询问特定的蛋白酶， 选择ECM部件。一个公正的战略，将系统地阐明完整的景观， OA软骨蛋白水解以前没有进行过。在这里，我们将在蛋白质组范围内绘制蛋白质水解图。 规模，确定直接在人膝关节OA软骨内的母体分子内的精确切割位点。 我们假设，接受手术置换的膝关节软骨，通常表现出相当大的 严重程度的区域差异，包含了OA自然史中发生的大多数裂缝， 与其时间轴和其他因素相关的OA软骨蛋白水解景观的详细分类。这 目前缺乏重要的知识。初步数据表明，实验的可行性， 新的方法来满足这一未满足的需求，并说明其潜力是潜在的变革OA研究。 具体来说，我们将应用蛋白质组学工作流程和生物信息学的进展，统称为N- 末端组学，其中蛋白质N-末端被标记、富集、通过高分辨率质谱鉴定 并通过在母体分子中的位置进行注释。我们将应用N-末端组学方法Terminal Amine 基质同位素标记（TAILS），用于膝关节OA和非OA软骨，以收集所有裂隙并创建 他们的蛋白水解景观（或degradomes）的可搜索公共数据库，建立研究资源 目前还无法获得（目标1）;定量比较OA与非OA软骨中蛋白水解裂解，轻度 与严重患病的OA软骨相比，男性与女性OA相比，以及年轻与老年对照软骨相比（目标2）;以及 定义了两种蛋白酶的蛋白质组范围内的底物库，这两种蛋白酶在OA中具有重要的因果关系，并被鉴定为药物 靶点，ADAMTS 5和MMP 13，以充分确定它们对OA蛋白水解景观的贡献（Aim 3）。 影响：通过为未来的OA研究提供资源和平台， 降解将为诊断、严重程度和药物反应提供前瞻性生物标志物，有助于解决 蛋白酶活性，促进OA的进展，并确定新的治疗的潜在目标和途径。

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.