CFTR's complex folding scheme leads to near-complete ER-associated degradation (ERAD) of the common CF disease mutant, F508del CFTR, and of a significant fraction of the WT protein. Defects in protein folding and aggregation underlie a diverse set of ~100 diseases, and their treatment requires an understanding of the checkpoints that determine protein fate. We have identified novel physical and functional interactions of CFTR with the small heat shock protein (sHsp), Hsp27, which leads to mutant CFTR conjugation with the small ubiquitin-like modifier, SUMO. As opposed to WT CFTR, F508del is selectively degraded by this Hsp27/SUMO pathway, which includes the SUMO E2 enzyme, Ubc9, and a SUMO-targeted ubiquitin ligase, RNF4. RNF4 ubiquitylates CFTR after its conjugation by poly-chains derived from SUMO-2/3 paralogs. Whereas most studies of SUMO modification have focused on nuclear regulatory events, we have implicated the first non- nuclear pathway for SUMO modification and degradation of an integral membrane protein. Data emerging from our array analysis for SUMO binding proteins identified the SUMO E3 enzyme, PIAS 4, which modifies CFTR by SUMO-1, a paralog that cannot form poly-chains, and is therefore not a target of RNF4. PIAS4 co- expression stabilized WT CFTR, and elicited the maturation and trafficking (rescue) of the F508del mutant. Accordingly, this project will evaluate the hypothesis that SUMO-mediated CFTR biogenesis and degradation are competing processes, supported by different SUMO paralogs, SUMO-1 and SUMO-2/3, and that these modifications are carried out by different components of the SUMOylation pathway. To approach these goals, Aim 1 asks what structures of WT and F508del CFTR lead to its SUMOylation, and if CFTR modification by different paralogs and by ubiquitin are competing or complementary processes. We will assess also whether a SUMO site at the C-terminus impacts CFTR stability at the plasma membrane.
Aim 2 uses biophysical measurements to ask about the properties of WT and F508del NBD1 that lead to its SUMOylation, and in turn, how this influences the physical properties of the protein (solubility, structure, stability) that impact CFR biogenesis/degradation.
Aim 3 examines mono-and poly-SUMO binding proteins identified in the Protoarray for their impact on CFTR biogenesis/degradation, including potential SUMO-targeted deubiquitylating proteases (DUBs). It is critical that these studies of CFTR fate are performed in airway cells wherever possible, especially differentiated primary cultures of human bronchial epithelia (HBE), whose phenotype is predictive of small molecule efficacy in clinical studies, because our data indicates that the Hsp27/SUMO pathway is highly significant in airway cells. Finally, we will evaluate the potential for the Hsp27/SUMO pathway to determine the fate of misfolded proteins in which mutations lead to neurodegenerative diseases: amyotropic lateral sclerosis (ALS) and Huntington's disease. This project will provide a mechanistic understanding of a new cytoplasmic triage checkpoint and its impact in the clinically important conflict between protein folding and degradation.

Public Health Relevance

This project capitalizes on the discovery of a new quality control pathway that determines the fate of CFTR, the protein mutated in the genetic disease, Cystic Fibrosis (CF). One limb of this pathway improves the biogenesis of the common CFTR mutant, and this provides therapeutic targets for correcting CF and potentially other diseases of protein folding/aggregation, e.g. neurodegenerative diseases.

National Institute of Health (NIH)
Research Project (R01)
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Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
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Eggerman, Thomas L
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University of Pittsburgh
Anatomy/Cell Biology
Schools of Medicine
United States
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Ahner, Annette; Gong, Xiaoyan; Schmidt, Bela Z et al. (2013) Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier-dependent pathway. Mol Biol Cell 24:74-84
Ahner, Annette; Gong, Xiaoyan; Frizzell, Raymond A (2013) Cystic fibrosis transmembrane conductance regulator degradation: cross-talk between the ubiquitylation and SUMOylation pathways. FEBS J 280:4430-8
Liang, Xiubin; Da Paula, Ana Carina; Bozoky, Zoltan et al. (2012) Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis. Mol Biol Cell 23:996-1009
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Schmidt, Bela Z; Watts, Rebecca J; Aridor, Meir et al. (2009) Cysteine string protein promotes proteasomal degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) by increasing its interaction with the C terminus of Hsp70-interacting protein and promoting CFTR ubiquitylation. J Biol Chem 284:4168-78
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Bertrand, Carol A; Zhang, Ruilin; Pilewski, Joseph M et al. (2009) SLC26A9 is a constitutively active, CFTR-regulated anion conductance in human bronchial epithelia. J Gen Physiol 133:421-38
Silvis, Mark R; Bertrand, Carol A; Ameen, Nadia et al. (2009) Rab11b regulates the apical recycling of the cystic fibrosis transmembrane conductance regulator in polarized intestinal epithelial cells. Mol Biol Cell 20:2337-50

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