title: Non-native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation.
abstract: A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50-60% by Hsp27; it interacted preferentially with the mutant and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, Lys(447), obviated Hsp27-mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 versus WT and for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: (a) its modification decreased as [ATP] increased, reflecting stabilization of the nucleotide-binding domain by ligand binding; (b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitional NBD1 conformation, was followed by its SUMO modification; and (c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system.
authors.affiliation: From the Departments of Cell Biology and.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Gong
authors.affiliation: From the Departments of Cell Biology and.
authors.forename: Annette
authors.initials: A
authors.lastname: Ahner
authors.affiliation: the Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada.
authors.forename: Ariel
authors.initials: A
authors.lastname: Roldan
authors.affiliation: the Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada.
authors.forename: Gergely L
authors.initials: GL
authors.lastname: Lukacs
authors.affiliation: Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 and.
authors.forename: Patrick H
authors.initials: PH
authors.lastname: Thibodeau
authors.affiliation: From the Departments of Cell Biology and. Electronic address: frizzell@pitt.edu.
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Frizzell
mesh_terms: D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation
chemical_list: D055551:HSP27 Heat-Shock Proteins; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: ABC transporter; Prot; SUMO; SUMOylation; chloride channel; cystic fibrosis; cystic fibrosis transmembrane conductance regulator (CFTR); post-translational modification (PTM); protein conformation; protein degradation