STRUCTURAL REGULATION OF CLCA1 ACTIVITY

描述(由申请人提供)：本项目的总体目标是了解CLCA1激活的结构基础及其在健康和疾病中的呼吸道生物学中的作用，以便 将这些知识转化为哮喘和慢性阻塞性肺病的治疗，这是目前尚未得到满足的需求。CLCA1是钙激活的氯离子通道(CaCCs)的有效调节剂，也是粘液细胞化生的中心介体，这一过程导致粘液过度生产。在本项目中，我们将研究CaCCs CLCA激活的结构和生化基础，并探讨CLCA1介导的通道激活在粘液细胞化生中所起的作用。在我们的初步结果中，我们证明了CLCA蛋白包含一个共同的切割位点，该切割位点被位于CLCA自身N端的一个独特的锌金属蛋白酶结构域所识别。此外，我们还证明了hCLCA1激活CaCCs需要这种自我切割。这些数据表明，CLCA1是以全长“非活性”形式合成的，需要自我切割才能产生“活性”形式的蛋白质。这个项目将集中于金属蛋白酶域活性调节的结构和生化分析，因为它是产生活性形式所必需的。然后，我们将通过确定全长“不活跃”和“活跃”形式的CLCA1的结构来表征激活时发生的结构变化。最后，我们将讨论CLCA1在CACC激活中的功能作用以及该通道在粘液细胞化生中的作用。了解CLCA1活性是如何由其自身的金属蛋白酶结构域调控的，以及这一调控的下游功能后果将有助于设计用于抗粘液治疗的CLCA1抑制剂。

DESCRIPTION (provided by applicant): The overall goal of this project is to understand the structural basis of CLCA1 activation and its role in airway biology in health and disease, in order to translate this knowledge into treatments for asthma and COPD, a current unmet need. CLCA1 is a potent modulator of calcium-activated chloride channels (CaCCs) and also a central mediator in mucous cell metaplasia, the process that leads to mucus overproduction. In this project we will investigate the structural and biochemical basis of CLCA activation of CaCCs, and investigate the role that CLCA1-mediated channel activation plays in mucous cell metaplasia. In our preliminary results, we demonstrate that CLCA proteins contain a consensus cleavage site that is recognized by a unique zinicin metalloprotease domain located within the N-terminus of CLCA itself. Furthermore, we show that this self- cleavage is required for hCLCA1 to activate CaCCs. These data suggest that CLCA1 is synthesized in a full- length "inactive" form and that self-cleavage is required to produce an "active" form of the protein. This project will focus on the structural and biochemical analysis of regulation of the metalloprotease domain activity, since it is necessary to produce the active form. We will then characterize the structura changes that occur upon activation by determining the structures of the full-length "inactive" and "active" forms of CLCA1. Finally, we will address the functional role of CLCA1 features in CaCC activation and the role of the channel in mucous cell metaplasia. Understanding how CLCA1 activity is regulated by its own metalloprotease domain and the downstream functional consequences of this regulation will facilitate the design of CLCA1 inhibitors for anti- mucus therapeutics.