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

解整合素和金属蛋白酶 17 (ADAM17) 胞质结构域的生理作用

基本信息

批准号：
10090459
负责人：
Jose Lora
金额：
$ 4.6万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10090459
关键词：
AREG gene Address Adult Affect Animals Autoimmune Diseases Autoimmunity Binding Binding Sites Biological Biological Assay Birth Bone Development CRISPR/Cas technology Cell membrane Cell surface Cells Co-Immunoprecipitations Cytoplasmic Tail DTR gene Data Defect Development Disease Disintegrins Embryonic Development Epidermal Growth Factor Receptor Epiphysial cartilage Excision Eye Eye Development Genetic Genetic study Goals Heart Valves Immune Immunoprecipitation Impairment Inflammatory Inflammatory Arthritis Knock-in Mouse Ligands Maintenance Malignant Neoplasms Mass Spectrum Analysis Mediating Mediator of activation protein Membrane Metalloproteases Morphologic artifacts Mus Mutagenesis Mutate Mutation Pathogenesis Pathologic Pathology Pathway interactions Peptide Hydrolases Perinatal Perinatal mortality demographics Phenotype Phorbol Esters Physiologic pulse Physiological Play Post-Translational Regulation Proteins Proteomics Receptor Signaling Regulation Resistance Role Septic Shock Signal Pathway Signal Transduction Small Interfering RNA Stimulus TNF gene Testing Time Transmembrane Domain cardiogenesis cytokine experimental study in vivo insight intestinal barrier knock-down loss of function mutant new therapeutic target novel overexpression programs response rhomboid skin barrier skin organogenesis tumor

项目摘要

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.

项目总结/摘要表皮生长因子受体（EGFR）和炎性细胞因子配体的蛋白水解释放肿瘤坏死因子-α（TNF α）受细胞表面金属蛋白酶ADAM 17（一种去整合素）的严格调节和金属蛋白酶17）。EGFR的ADAM 17依赖性激活对正常心脏、骨骼、皮肤至关重要和眼睛的发育，并在成年期维持皮肤和肠道屏障，但也可以有助于癌症的发展。在小鼠中的遗传研究表明，ADAM 17在小鼠中起着非- 这些途径中的冗余作用，突出了ADAM 17的生理和病理重要性。小鼠缺乏ADAM 17的小鼠EGFR信号传导受损，表型上类似于缺乏EGFR的小鼠。围产期死亡、出生时睁眼和心脏瓣膜缺陷。此外，免疫细胞中缺乏ADAM 17的小鼠保护免于感染性休克和TNF α依赖性病理学如炎性关节炎。一个标志 ADAM 17的特征是能够响应许多生理刺激而迅速开启。这种快速激活通过需要其跨膜结构域而不是其胞质结构域的机制发生。的 ADAM 17的跨膜结构域与失活的Rhomboid蛋白1和2（iRhom 1/2）相互作用，以支持 ADAM 17调控的关键方面，如成熟，催化活性和底物选择性。但 ADAM 17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ADAM 17主要产生无活性的前ADAM 17，但很少或没有成熟的ADAM 17。为了避免这种过度表达的假象，我已经表征了一种小鼠品系，其中ADAM 17 使用CRISPR/Cas9在内源性ADAM 17基因座处截短胞质结构域。这种截断导致ADAM 17蛋白水平的强烈降低和功能表型的部分丧失。这是第一据我们所知，有遗传学证据表明，ADAM 17胞质结构域在功能上是相关的，调节ADAM 17水平的作用。此外，细胞生物学数据表明，ADAM 17细胞质结构域是ADAM 17/iRhom 2依赖性底物释放所必需的，这表明ADAM 17细胞质结构域是ADAM 17/iRhom 2相互作用的关键介质。我的目标是定义细胞质序列， ADAM 17，控制其水平，并确定ADAM 17胞质结构域相互作用分子，介导 ADAM 17稳定性我还将定义ADAM 17/iRhom 2所需的ADAM 17胞质结构域序列。依赖催化活性。这些研究有望提供新的和令人兴奋的见解， ADAM 17由其胞质结构域。了解ADAM 17胞质结构域如何调节ADAM 17 蛋白质水平提供了鉴定EGFR依赖性癌症的新治疗靶点的可能性， TNF α依赖性自身免疫性疾病。