Illuminating the role of chloride channel regulator CLCA4 in TMEM16 Family potentiation and disease

摘要： 钙激活的氯离子通道调节剂CLCA4已经被认为是一种 治疗呼吸道和胃肠道炎症性疾病的药物靶点，包括哮喘、COPD、囊肿性疾病 纤维化、感染性腹泻、克罗恩病、溃疡性结肠炎和一些癌症。然而，一份详细的 对它的生物学功能的了解远远落后于它与这些疾病的联系，这主要是由于 完全缺乏生化知识和优质抗体试剂。我们最近取得了突破性的进展 一个相关家族成员CLCA1的研究进展表明它是一种自裂解的金属蛋白酶 结合并增强钙激活的氯离子通道TMEM16A。在我们的初步数据中，我们 证明CLCA4以类似的方式作用于另一个不同的TMEM16家族成员TMEM16B。因此， 我们正准备在我们对CLCA4在健康和健康中的功能的理解方面取得重大进展 疾病。这一试点项目将开发促进这一工作所需的知识库和工具。在目标1中，我们将 研究CLCA4活性的调节机制，全面验证TMEM16B是 唯一受CLCA4调控的TMEM16家族成员。在目标2中，我们将开发一种新的单克隆组 可使用的针对成熟的、裂解形式的CLCA4(N-CLCA4和C-CLCA4)的抗体试剂 以几种实验形式，并对健康和患病的呼吸道和肠道进行初步分析 组织和生物样本随身携带。该项目的长期目标(超出该试点R03)是开发 全面了解CLCA4在健康和疾病中的作用，我们认为这是 被严重低估了。这项试点的成功完成将为调查提供知识和工具 研究CLCA4在特定疾病中的作用，并在后续项目中开发有针对性的治疗方法。

ABSTRACT: The calcium-activated chloride channel regulator CLCA4 has been associated with and suggested as a drug target for inflammatory diseases of the airway and gastrointestinal tract including asthma, COPD, cystic fibrosis, infectious diarrhea, Crohn's disease, ulcerative colitis, and some cancers. However, a detailed understanding of its biological functions has lagged far behind its association with these diseases, largely due to complete lack of biochemical knowledge and quality antibody reagents. We recently made groundbreaking advances investigating a related family member, CLCA1, by showing that it is a self-cleaving metalloprotease that binds to and potentiates the calcium activated chloride channel TMEM16A. In our preliminary data we show that CLCA4 acts in a similar manner on another distinct TMEM16 family member, TMEM16B. Therefore, we are uniquely poised to generate significant advances in our understanding of CLCA4 function in health and disease. This pilot project will develop the knowledge base and tools required to facilitate this. In Aim 1, we will investigate the mechanism of regulation of CLCA4 activity and comprehensively validate that TMEM16B is the sole TMEM16 family member modulated by CLCA4. In Aim 2, we will develop a novel panel of monoclonal antibody reagents specific for the mature, cleaved forms of CLCA4 (N-CLCA4 and C-CLCA4) that can be used in several experimental formats, and carry out initial analysis of healthy and diseased airway and intestinal tissue and biological samples with them. The long-term goal of this project (beyond this pilot R03) is to develop a comprehensive mechanistic understanding of CLCA4 function in health and disease, which we believe is sorely underappreciated. Successful completion of this pilot will provide the knowledge and tools to investigate the functional roles of CLCA4 in specific diseases and to develop targeted therapeutics in subsequent projects.