The physiological role of the cytoplasmic domain of a disintegrin and metalloproteinase 17 (ADAM17)

项目摘要/摘要 表皮生长因子受体和炎性细胞因子配体的蛋白水解性释放 肿瘤坏死因子-α(TNFa)受细胞表面金属蛋白酶ADAM17(去整合素)的严格调控 和金属蛋白酶17)。ADAM17依赖的EGFR激活对正常的心脏、骨骼和皮肤是必不可少的 和眼睛的发育，以及在成年期间维持皮肤和肠道屏障，但也可以 有助于癌症的发展。对小鼠的遗传学研究表明，ADAM17发挥着一种非 ADAM17在这些通路中的多余作用，突出了ADAM17在生理和病理上的重要性。老鼠 缺乏ADAM17损害了EGFR信号转导，其表型类似于缺乏EGFR的小鼠 围产期致命性、出生时睁开眼睛和心脏瓣膜缺陷。此外，免疫细胞中缺乏ADAM17的小鼠 可预防感染性休克和炎症性关节炎等依赖肿瘤坏死因子的病理。一个标志性的 ADAM17的特点是它能够对许多生理刺激做出快速反应。这种快速激活 通过一种需要其跨膜结构域而不是细胞质结构域的机制发生。这个 ADAM17的跨膜区与失活的菱形蛋白1和2(IRHOM1/2)相互作用以支持 ADAM17调控的关键方面，如成熟度、催化活性和底物选择性。然而， ADAM17胞质结构域的功能相关性仍然难以捉摸。这部分是因为 在细胞中过表达的ADAM17主要产生失活的前-ADAM17，但很少或没有成熟的-ADAM17。 为了避免这种过度表达的现象，我描述了一个小鼠品系，其中ADAM17 利用CRISPR/Cas9截断内源ADAM17基因的胞质结构域。此截断 导致ADAM17蛋白水平显著降低，部分功能表型丧失。这是第一次 据我们所知，遗传证据表明ADAM17细胞质结构域在功能上是相关的，并可能发挥作用 在调节ADAM17水平方面的作用。此外，细胞生物学数据表明，ADAM17细胞质 结构域是ADAM17/IRHOM2依赖底物释放所必需的，这表明ADAM17细胞质 结构域是ADAM17/IRHOM2相互作用的关键调节因子。我的目标是定义细胞质序列 控制其水平并识别与ADAM17胞浆结构域相互作用的分子的ADAM17 ADAM17稳定性。我还将定义ADAM17/IRHOM2-所需的ADAM17细胞质结构域序列- 依赖于催化活性。这些研究承诺提供新的和令人兴奋的洞察力，以监管 ADAM17的胞质结构域。了解ADAM17胞质结构域如何调节ADAM17 蛋白质水平为确定EGFR依赖癌症的新治疗靶点和 依赖肿瘤坏死因子的自身免疫性疾病。

Project Summary/Abstract The proteolytic release of ligands of the epidermal growth factor-receptor (EGFR) and the inflammatory cytokine tumor necrosis factor-a (TNFa) is tightly regulated by the cell surface metalloprotease ADAM17 (A disintegrin and metalloproteinase 17). ADAM17-dependent activation of the EGFR is essential for normal heart, bone, skin and eye development, and for the maintenance of the skin and intestinal barrier during adulthood, but can also contribute to the development cancer. Genetic studies in mice have revealed that ADAM17 plays a non- redundant role in these pathways, highlighting the physiological and pathological importance of ADAM17. Mice lacking ADAM17 have impaired EGFR signaling and phenotypically resemble mice lacking the EGFR in their perinatal lethality, open eyes at birth and heart valve defects. Moreover, mice lacking ADAM17 in immune cells are protected from septic shock and TNFa-dependent pathologies such as inflammatory arthritis. A hallmark feature of ADAM17 is its ability to rapidly turn on in response to many physiological stimuli. This rapid activation occurs through a mechanism that requires its transmembrane domain, but not its cytoplasmic domain. The transmembrane domain of ADAM17 interacts with the inactive Rhomboid proteins 1 and 2 (iRhom1/2) to support key aspects of ADAM17 regulation such as maturation, catalytic activity, and substrate selectivity. However, the functional relevance of the ADAM17 cytoplasmic domain has remained elusive. This is in part because overexpressing ADAM17 in cells mainly gives rise to the inactive pro-ADAM17, but little or no mature-ADAM17. In order to circumvent this overexpression artifact, I have characterized a mouse line in which the ADAM17 cytoplasmic domain was truncated at the endogenous ADAM17 locus using CRISPR/Cas9. This truncation resulted in strongly reduced ADAM17 protein levels and a partial loss of function phenotype. This is the first genetic evidence, to our knowledge, that the ADAM17 cytoplasmic domain is functionally relevant and may play a role in regulating the levels of ADAM17. Furthermore, cell-biological data show that the ADAM17 cytoplasmic domain is required for ADAM17/iRhom2-dependent substrate release, suggesting that the ADAM17 cytoplasmic domain is a key mediator of ADAM17/iRhom2 interactions. My goal is to define the cytoplasmic sequences in ADAM17 that control its levels and identify ADAM17 cytoplasmic domain interacting molecules that mediate ADAM17 stability. I will also define the ADAM17 cytoplasmic domain sequence required for ADAM17/iRhom2- dependent catalytic activity. These studies promise to provide new and exciting insights into the regulation of ADAM17 by its cytoplasmic domain. Understanding how the ADAM17 cytoplasmic domain regulates ADAM17 proteins levels offers the possibility of identifying novel therapeutic targets for EGFR-dependent cancers and TNFa-dependent autoimmune disorders.