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Advisory Committee Chair

Eric J Sorscher

Advisory Committee Members

Jeong Hong

Maaike Everts

James Collawn

Document Type

Dissertation

Date of Award

2018

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The traditional CFTR functional defect subcategories (i.e. protein misfolding, gating, conductance, etc.) are sometimes inadequate for determining patients most likely to benefit from emerging modulator treatment. We employ the P67L variant as emblematic of ways a thorough biochemical analysis of a rare CF defect can guide new therapeutic approaches, help predict drug response, and facilitate clinical precision. Historically, P67L has been designated a class IV conductance abnormality (i.e., malformed ion pore). In contrast, our studies demonstrate P67L disrupts CFTR protein maturation, prompts misfolding, causes gating defects, and exhibits wild-type like conductance. We show that P67L CFTR is robustly rescued by lumacaftor or ivacaftor, both in cell lines and primary airway epithelia. To further elucidate the impact of this mutation on protein biogenesis, we introduced low-temperature correction, second-site suppressors, and other informative mutations in concert with P67L. Experiments of this type provide new insight regarding the CFTR misfolding pathway and means by which drugs such as lumacaftor mediate a beneficial effect. We next applied the recently published CFTR cryo-EM structure to elucidate mechanisms by which mutations such as P67L disrupt protein conformation, including the importance of a less well-studied CFTR structural element (the lasso helix) during maturational processing. In particular, we demonstrate that changes to the lasso are associated with a range of disease phenotypes, variable CFTR modulator response, and changes in protein domains far downstream of the amino terminus. Molecular dynamic analyses and limited proteolysis data were used to evaluate global impact of P67L and other variants within lasso helix 2 (Lh2). We also identified a severe diseaseassociated mutation within Lh2 that is completely resistant to small molecule rescue and second-site suppression, despite proximity to the robust lumacaftor responder, P67L. Strategies defined by this project can be applied to clinical findings and other rare CFTR alleles, as a way to help identify patient populations most likely to benefit from emerging modulator treatment. Moreover, our studies provide an approach by which CFTR mutations can be better characterized mechanistically, and assigned with greater precision to “theratype”, ie. which variants respond best to specific modulator compounds.

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