The proteolytic landscape of osteoarthritic cartilage

骨关节炎软骨的蛋白水解景观

• 批准号: 10370498
• 负责人: SUNEEL S APTE
• 金额: $ 21.25万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-10 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10370498
• 关键词: Affect; Age; Aging; Amines; Area; Arthralgia; Bioinformatics; Biological Markers; Cartilage; Catabolism; Cells; Collagen; Complex; Data; Databases; Degenerative polyarthritis; Detection; Development; Diagnosis; Disease; Disease Pathway; Drug Targeting; Encyclopedias; Event; Extracellular Matrix; Female; Fractionation; Functional disorder; Future; Goals; Hip Osteoarthritis; Histologic; Histopathologic Grade; Human; Impairment; Individual; Isotope Labeling; Joints; Knee; Knee Osteoarthritis; Knowledge; Label; Liquid Chromatography; Maps; Mass Spectrum Analysis; Mediating; Methods; Mission; Molecular; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Natural History; Operative Surgical Procedures; Parents; Pathogenesis; Pathway interactions; Pattern; Peptide Hydrolases; Peptides; Persons; Pharmaceutical Preparations; Phenotype; Physiological; Positioning Attribute; Process; Production; Proteins; Proteolysis; Proteome; Proteomics; Reagent; Research; Resolution; Resources; Role; Running; Sampling; Severities; Shotguns; Site; Specificity; Systems Analysis; Testing; TimeLine; Tissues; United States; United States National Institutes of Health; Variant; Woman; Work; adduct; aggrecan; arthropathies; articular cartilage; biomarker development; cartilage degradation; clinically relevant; cohort; collagenase 3; data resource; high reward; improved; instrument; joint function; joint inflammation; male; men; mortality; new therapeutic target; novel; novel strategies; novel therapeutics; online resource; prospective; protein degradation; public database; response; senescence; sex; specific biomarkers; tandem mass spectrometry; virtual

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.

骨关节炎（OA）是一种影响所有关节组织的疾病，其引发原因很复杂。无论 关节软骨细胞外基质 (ECM) 的始因、无情、细胞介导的蛋白水解降解 随后，伴随着细胞功能障碍，损害关节功能。蛋白水解片段进一步刺激 分解代谢，通过作为损伤相关分子模式（DAMP）促进关节炎症，并且 被认为是关节疼痛的原因。蛋白水解失调也在软骨衰老中发挥作用，因为 衰老相关的分泌表型与蛋白酶产生的增加有关。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了解， 迄今为止，许多有关蛋白水解的工作都使用候选方法来询问特定的蛋白酶或 选择 ECM 组件。一种公正的策略，将系统地阐明整个领域的情况 以前未进行过 OA 软骨蛋白水解。在这里，我们将在蛋白质组范围内绘制蛋白质水解图谱 规模，直接识别人膝骨关节炎软骨内母体分子内的精确切割位点。 我们假设进行手术置换的膝盖软骨通常表现出相当大的 严重程度存在区域差异，包含 OA 自然史中发生的大多数裂解，并允许 与时间线和其他因素相关的 OA 软骨蛋白水解景观的详细分类。这 目前缺乏重要的知识。初步数据证明了实验的可行性 解决这一未满足需求的新方法，并说明了其对 OA 研究具有潜在变革性的潜力。 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步，统称为 N- 术语组学，其中蛋白质 N 末端通过高分辨率质谱进行标记、富集和鉴定 并通过母体分子中的位置进行注释。我们将应用N末端组学方法末端胺 底物同位素标记 (TAILS)，对膝关节 OA 和非 OA 软骨进行收集，收集所有劈裂并创建 其蛋白水解景观（或降解组）的可搜索公共数据库，建立研究资源 当前不可用（目标 1）；定量比较 OA 与非 OA 软骨的蛋白水解裂解，轻度 与严重患病的 OA 软骨、男性 OA 与女性 OA 以及年轻人与老年对照软骨（目标 2）；和 定义两种在 OA 中起重要作用并被鉴定为药物的蛋白酶的整个蛋白质组底物库 目标 ADAMTS5 和 MMP13，以充分定义它们对 OA 蛋白水解景观的贡献 (Aim3)。 影响：通过为未来 OA 研究、软骨蛋白质组分析提供资源和平台 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 蛋白酶活性有助于 OA 进展，并确定新疗法的潜在靶点和途径。