STRUCTURAL REGULATION OF CLCA1 ACTIVITY

描述（由申请人提供）：本项目的总体目标是了解CLCA 1激活的结构基础及其在健康和疾病中的气道生物学作用， 将这些知识转化为哮喘和COPD的治疗方法，这是目前尚未满足的需求。CLCA 1是钙激活氯离子通道（CaCC）的有效调节剂，也是粘液细胞化生（导致粘液过度产生的过程）的中心介质。在这个项目中，我们将研究CLCA激活CaCC的结构和生化基础，并研究CLCA 1介导的通道激活在粘液细胞化生中的作用。在我们的初步结果中，我们表明，CLCA蛋白含有一个共识裂解位点，这是由一个独特的zinicin金属蛋白酶结构域位于CLCA本身的N-末端内确认。此外，我们表明，这种自我切割所需的hCLCA 1激活CaCC。这些数据表明，CLCA 1是以全长“无活性”形式合成的，并且需要自切割来产生“活性”形式的蛋白质。本项目将集中于金属蛋白酶结构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CLCA 1的全长“非活性”和“活性”形式的结构来表征激活后发生的结构变化。最后，我们将讨论CLCA 1功能在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CLCA 1活性如何受其自身金属蛋白酶结构域的调节以及这种调节的下游功能后果将有助于设计用于抗粘液治疗的CLCA 1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.