Functional and biochemical studies of ADAM19

描述(申请人提供)：金属蛋白酶去整合素(ADAMS)是一种 膜锚定糖蛋白家族，在受精过程中起关键作用， 神经发生，以及在发育过程中EGF受体配体的激活。这个 这项建议的主要目标是揭示围产期高血压的发病机制。 缺乏广泛表达的ADAM 19的小鼠的致死表型，强调 评估ADAM19在蛋白质胞外结构域脱落中的预测作用。 蛋白质胞外结构域的脱落导致多种结构蛋白的释放 以及来自质膜的功能多样化的蛋白质，最近已经 作为一种关键的翻译后调节功能的机制出现 释放出的蛋白质。胞外结构域脱落对于正确的信号转导是必不可少的 通过EGF受体配体，Notch介导的侧向抑制，限制肿瘤坏死因子 受体介导的炎症反应，并调节几个方面的 轴突引导。因为第一个发现的脱氧核糖酶，即肿瘤坏死因子α 转换酶，是亚当，因为跛行抑制研究目前排除了 已知的基质金属蛋白酶(MMPs)是大多数蛋白质的脱水酶，Adams 成为最好的外区脱落酶候选者。我们的工作假设是 缺乏ADAM19的小鼠的表型至少部分是由于一种缺陷 一种或多种蛋白质的胞外结构域脱落。这样做的具体目的是 项目包括： 1)对缺乏ADAM19的小鼠进行仔细的组织病理学分析 以揭示观察到的围产期死亡的原因。这将包括一个 肺、心脏和骨髓中细胞类型的鉴定 ADAM19高表达的细胞，体内研究评估 ADAM19在这些细胞和组织中的功能。 2)解决功能冗余和补偿的可能性 亚当19和其他亚当斯在发育过程中通过产生缺乏其他 除ADAM19外，还有相关的ADAMS。 3)在缺乏ADAM 19或的细胞中发现胞外结构域脱落的潜力 其他ADAMS与野生型细胞使用有偏见和无偏见的方法进行比较 有偏见的方法将集中在已知脱落的蛋白质上，并可能 与缺乏ADAM 19的小鼠的表型有关，如EGFR配体、肿瘤坏死因子 和肿瘤坏死因子受体家族成员。这种无偏见的方法将比较蛋白质 分泌到野生型和亚当19基因敲除细胞的上清中。这 研究将为ADAM19的功能提供令人兴奋的见解 在成年小鼠身上发育，并将揭示缺陷的机制 在亚当19世纪导致围产期死亡。我们的结果很可能会有 ADAM19在人类疾病中的功能暗示，并可能发现新的 由一种疾病引起的疾病的治疗干预目标 管理不当或有缺陷的ADAMI9或其他相关的ADAMS。

DESCRIPTION (provided by applicant): Metalloprotease-disintegrins (ADAMs) are a family of membrane anchored glycoproteins that have key roles in fertilization, neurogenesis, and in activation of EGF receptor ligands during development. The main goal of this proposal is to uncover the mechanism underlying the perinatal lethal phenotype of mice lacking the widely expressed ADAM 19, with an emphasis on evaluating the predicted role of ADAM19 in protein ectodomain shedding. Protein ectodomain shedding leads to the release of a variety of structurally and functionally diverse proteins from the plasma membrane, and has recently emerged as a critical post-translational mechanism of regulating the function of the released proteins. Ectodomain shedding is essential for proper signaling via EGF receptor-ligands, Notch-mediated lateral inhibition, limiting TNF receptor mediated inflammatory reactions, and regulating several aspects of axonal guidance. Because the first identified sheddase, the TNFalpha convertase, is an ADAM, and because lIMP inhibition studies rule out currently known matrix metalloproteases (MMPs) as sheddases for most proteins, ADAMs have emerged as the best candidate ectodomain sheddases. Our working hypothesis is that the phenotype of mice lacking ADAM19 is due, at least in part, to a defect in ectodomain shedding of one or more proteins. The specific aims of this project are: 1) Perform a careful histopathological analysis of mice lacking ADAM19 in order to uncover the cause of the observed perinatal lethality. This will include an identification of the cell types in the lung, heart, and in bone marrow derived cells where ADAM19 is highly expressed, and in vivo studies to evaluate the function of ADAM19 in these cells and tissues. 2) Address the possibility of functional redundancy and compensation between ADAM 19 and other ADAMs during development by generating mice lacking other related ADAMs in addition to ADAM19. 3) Uncover potential aelects in ectodomain shedding in cells lacking ADAM 19 or other ADAMs compared to wildtype cells using a biased and unbiased approach The biased approach will focus on proteins that are known to be shed and could contribute to the phenotype of mice lacking ADAM 19, such as EGFR-ligands, TNF- and TNF-receptor family members. The unbiased approach will compare proteins secreted into the supernatant of wildtype and ADAM 19 knockout cells. This study will provide exciting insights into the function of ADAM19 during development and in adult mice, and will uncover the mechanism by which a defect in ADAM 19 leads to perinatal lethality. Our results are likely to have implications for the function of ADAM19 in human disease, and may uncover novel targets for therapeutic interventions in diseases that are caused by a misregulated or defective ADAMI9 or by other related ADAMs.