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ORCID

http://orcid.org/0000-0002-1113-4798

Date of Award

Spring 5-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

In the airway, proper activity of the anion channel cystic fibrosis transmembrane conductance regulator (CFTR) contributes to innate immune defense by maintaining a hydratedand alkaline mucus layer through the conductance of chloride and bicarbonate ions. This allows potentially pathogenic microorganisms to be trapped, quickly killed, and cleared via mucociliary clearance, thus preventing microbial colonization of the lungs. In cystic fibrosis (CF), this activity is impaired, resulting in repeated pulmonary infections that damage the lung and, if severe and prolonged, may lead to premature death without lung transplantation. Available therapies remain focused on targeted rescue of the CFTR mutation. However, given the thousands of mutations found in this patient population, individualized rescue of each would be difficult. This dissertation explores the potential of an alternative and mutation-agnostic strategy to restore healthy airway properties by activating an alternate anion channel in the lung, the calcium activated chloride channel (CaCC), TMEM16A. TMEM16A conducts both chloride and bicarbonate, making it an ideal surrogate for CFTR activity. Our group has identified a secreted xiv potentiator of TMEM16A, calcium activated chloride channel regulator 1 (CLCA1), that directly engages TMEM16A on the cell surface and prevents its internalization on the order of minutes, thereby increasing TMEM16A surface density and anion currents through the cell. A detailed molecular understanding of the interaction between CLCA1 and TMEM16A will be important towards developing a CF therapy that can be modeled after the interaction. Therefore, the goal of this dissertation was two-fold: 1) uncover the molecular determinants governing the interaction between CLCA1 and TMEM16A, and 2) examine the effects of CLCA1 on TMEM16A currents and mucociliary properties of CF airway. Towards developing a model of this interaction, this study presents the X-ray crystal structure of the human CLCA1 vWA domain to 2.0 Å, the first structure of any CLCA family member. Human TMEM16A was purified and a structural model of the CLCA1-TMEM16A interaction pursued by single-particle cryo-electron microscopy. To understand the specificity of the interaction, the remaining human CLCA family members, CLCA2 and CLCA4, were investigated functionally and structurally, as both can potentiate CaCC. CLCA4 specifically potentiates anion currents through the CaCC TMEM16B and not through TMEM16A, indicating that CLCA proteins potentiate specific TMEM16 channels. To translate these discoveries to disease, CLCA1 was applied to CF airway of multiple genotypes. Treatment with CLCA1 demonstrated the ability to potentiate TMEM16A currents and restore mucociliary clearance. This effect on TMEM16A, however, was not observed in normal CFTR-sufficient airway epithelial cells, and evidence indicates that this discrepancy may be due to different TMEM16A isoforms expressed in normal versus CF airway epithelial cells. Together, these studies indicate that CLCA1 vWA domain may restore healthy mucus properties to CF airway and highlights the exciting potential for universal CF treatment modeled after the CLCA1 vWA domain/TMEM16A interaction.

Language

English (en)

Chair and Committee

Tom J. Brett

Committee Members

Jennifer Alexander-Brett, Gaya K. Amarasinghe, Jeffrey Haspel, Colin G. Nichols, Peng Yuan

Available for download on Sunday, May 15, 2022

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