Development of tools and knowledge to facilitate the investigation of chloride channel regulator CLCA2 in rare human diseasses

摘要： 钙激活的氯离子通道调节剂CLCA2已经被认为是一种 针对呼吸道和胃肠道炎症性疾病的药物靶点，包括罕见的囊性疾病 纤维化(CF)。然而，对它的生物学功能的详细了解远远落后于它的联系 对于这些疾病，很大程度上是由于完全缺乏生化知识和优质的抗体试剂。我们 最近在研究其他家庭成员CLCA1和CLCA4方面取得了突破性的进展，显示了 它们是自我切割的金属蛋白水解酶，能增强钙激活的氯离子通道TMEM16A 和TMEM16B。在我们的初步数据中，我们显示CLCA2还可以增强钙- 激活了人细胞中的氯通道。因此，我们正独一无二地准备取得重大进展 在我们对CLCA2在健康和疾病中的功能的理解中。这一试点项目将发展知识 促进这一点所需的基础和工具。在目标1中，我们将研究clca2的调控机制。 并全面鉴定受CLCA2调控的TMEM16家族成员。在目标2中，我们将 开发一组新的针对成熟的、裂解形式的CLCA2(N- CLCA2和C-CLCA2)，可以在几种实验格式中使用，并对 健康和患病的CF呼吸道组织及其细胞样本。该项目的长期目标(超越 本试点R03)旨在对CLCA2在健康和健康中的作用进行全面的机械理解 疾病，这可能为治疗CF和其他粘膜阻塞性疾病提供新的治疗途径，因为我们 在我们的初步数据中展示了。成功完成这一试点将为以下工作提供知识和工具 研究CLCA2在CF中的功能作用，并有可能开发靶向治疗 后续项目，以及深入了解CLCA2与哮喘、COPD、感染性腹泻、克罗恩病 疾病、溃疡性结肠炎和一些癌症。

ABSTRACT: The calcium-activated chloride channel regulator CLCA2 has been associated with and suggested as a drug target for inflammatory diseases of the airway and gastrointestinal tract including the rare disease cystic fibrosis (CF). 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 fellow family members, CLCA1 and CLCA4, by showing that they are self-cleaving metalloproteases that potentiate the calcium activated chloride channels TMEM16A and TMEM16B, respectively. In our preliminary data we show that CLCA2 can also potentiate a calcium- activated chloride channel in human cells. Therefore, we are uniquely poised to generate significant advances in our understanding of CLCA2 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 CLCA2 activity and comprehensively identify the TMEM16 family member modulated by CLCA2. In Aim 2, we will develop a novel panel of monoclonal antibody reagents specific for the mature, cleaved forms of CLCA2 (N- CLCA2 and C-CLCA2) that can be used in several experimental formats, and carry out initial analysis of healthy and diseased CF airway tissues and cell samples with them. The long-term goal of this project (beyond this pilot R03) is to develop a comprehensive mechanistic understanding of CLCA2 function in health and disease, which could provide new therapeutic routes to treat CF and other muco-obstructive diseases, as we demonstrate in our preliminary data. Successful completion of this pilot will provide the knowledge and tools to investigate the functional roles of CLCA2 in CF, with the potential to develop targeted therapeutics in subsequent projects, as well as provide insight into CLCA2 links in asthma, COPD, infectious diarrhea, Crohn’s disease, ulcerative colitis, and some cancers.