Mechanistic Studies on Meprin Metalloproteases and Meprin-Substrate Interactions in Tissue Injury

项目摘要/摘要。我的研究项目专注于了解细胞和 金属蛋白酶介导的组织损伤进展的分子机制。 Meprs由α和β两个亚基组成，形成两种蛋白质亚型：Meprin A(α-α或α-β)和Meprin B(β-β)具有不同和重叠的底物。Meprins在灌木丛边缘表现得最为丰富 近端肾小管和小肠的膜。Meprs也在白细胞中表达。 (单核细胞和巨噬细胞)、足细胞、皮肤、内皮细胞和癌细胞。梅普林斯一直是 与包括肾脏在内的炎症和纤维化相关疾病的病理生理学有关 疾病、炎症性肠病、肺纤维化、神经退行性疾病(如阿尔茨海默病)，以及 癌症。MEPRINβ基因的单核苷酸多态(SNP)被证明与病情严重程度有关 某些疾病，如糖尿病、肾脏疾病和癌症。我的研究小组结合使用了 分子生物学和蛋白质组学方法鉴定梅普林底物并表征相互作用 在梅普林异构体和它们的底物之间。这些结合了体内对meprin基因敲除的研究。 建立小鼠模型，以确定梅普林的活动如何影响疾病的进展。已知的美普林底物 包括细胞外基质蛋白、炎症调节剂(例如促炎细胞因子[IL-1β， IL-6、IL-18、MCP-1和抗炎蛋白Ac-SDKP)、细胞信号蛋白(例如蛋白激酶A和 蛋白激酶C)、缺氧反应的介体(如骨肉瘤-9)、紧密连接蛋白(如 Claudin 5、occludin、E-cadherin和Z0-1)细胞骨架蛋白(例如绒毛蛋白和肌动蛋白)和 与AD斑块有关(例如，髓系细胞上表达的淀粉样前体蛋白和触发受体 2)。底物的多样性表明，在不同的环境下， 在不同的条件和不同的器官中。重要的是要了解这些机制以促进 开发诊断和治疗工具。在这项建议的五年期间，我们将进行 三个方面的研究；(I)确定meprinβ基因中的SNPs如何影响其与底物的相互作用 和生理脱落酶，(Ii)决定meprin与底物相互作用如何调节信号 低氧、炎症和细胞外基质代谢中的途径和影响反应，以及(Iii)评估 MEPRIN和MEPRIN裂解产物作为生物标志物用于早期检测的诊断工具的开发 疾病。拟议的研究将超越基础(体外和体内)，在以下基础上获得见解 与梅普林斯相关的遗传易感性。翻译研究也建议应用这一点。 开发适用于糖尿病肾损伤和阿尔茨海默病的诊断工具的知识 (AD)，推进精准医疗的重要一步。此外，该奖项将促进指导 培训对象来自代表性不足的少数群体，从而促进生物医学劳动力的多样性。

PROJECT SUMMARY/ ABSTRACT. My research program focuses on understanding the cellular and molecular mechanisms underlying the progression of tissue injury mediated by meprin metalloproteases. Meprins comprise of two subunits, α and β, which form two protein isoforms, meprin A (α-α or α-β) and meprin B (β-β) with distinct and overlapping substrates. Meprins are most abundantly expressed in the brush-border membranes of proximal kidney tubules and small intestines. Meprins are also expressed in leukocytes (monocytes and macrophages), podocytes, skin, endothelial cells, and cancer cells. Meprins have been implicated in the pathophysiology of inflammatory- and fibrosis-associated diseases that include kidney disease, inflammatory bowel disease, lung fibrosis, neurodegenerative disease (e.g. Alzheimer’s disease), and cancer. Single nucleotide polymorphisms (SNPs) in the meprin β gene were shown to associate with severity of certain diseases such as diabetic kidney disease and cancer. My research group uses a combination of molecular biology and proteomic approaches to identify meprin substrates and characterize the interactions between meprin isoforms and their substrates. These are coupled with in vivo studies with meprin knockout mouse models to determine how meprin activity impacts the progression of disease. Known meprin substrates include extracellular matrix (ECM) proteins, modulators of inflammation (e.g. proinflammatory cytokines [IL-1β, IL-6, IL-18, MCP-1; and anti-inflammatory proteins Ac-SDKP), cell signaling proteins (e.g. protein kinase A and protein kinase C), mediators of the hypoxia response (e.g. osteosarcoma-9), tight junction proteins (e.g. claudin 5, occludin, E-cadherin, and Z0-1) cytoskeletal proteins (e.g. villin and actin) and proteins that contribute to plaques in AD (e.g. amyloid precursor protein and triggering receptor expressed on myeloid cells 2). The diversity of meprins substrates suggests that complex mechanisms are involved under different conditions and in different organs. It’s important to gain understanding of these mechanisms to facilitate development of diagnostic and therapeutic tools. For the five year period of this proposal, we will conduct studies in three areas; (i) to determine how SNPS in the meprin β gene impact its interactions with substrates and physiological sheddases, (ii) determine how meprin interactions with substrates modulate signaling pathways and impact responses in hypoxia, inflammation, and ECM metabolism, and (iii) to evaluate the use of meprin and meprin cleavage products as biomarkers for development of diagnostic tools for early detection of disease. The proposed research will transcend basic (in vitro and in vivo) to gain insights on the basis for genetic predispositions associated with meprins. Translational studies are also proposed to apply this knowledge in development of diagnostic tools applicable to diabetic kidney injury and Alzheimer’s disease (AD), an important step in advancing precision medicine. Furthermore, this award will facilitate mentoring of trainees from underrepresented minority populations and thus promote diversity of the biomedical workforce.