MSACL 2016 US Abstract

Using Mass Spectrometry to Understand Cystic Fibrosis as a Protein Misfolding Disease

John Yates (Presenter)
The Scripps Research Institute

Bio: John R. Yates is the Ernest W. Hahn Professor in the Department of Chemical Physiology and Molecular and Cellular Neurobiology at The Scripps Research Institute. His research interests include development of integrated methods for tandem mass spectrometry analysis of protein mixtures, bioinformatics using mass spectrometry data, and biological studies involving proteomics. He is the lead inventor of the SEQUEST software for correlating tandem mass spectrometry data to sequences in the database and developer of the shotgun proteomics technique for the analysis of protein mixtures. His laboratory has developed the use of proteomic techniques to analyze protein complexes, posttranslational modifications, organelles and quantitative analysis of protein expression for the discovery of new biology. Many proteomic approaches developed by Yates have become a national and international resource to many investigators in the scientific community. He has received the American Society for Mass Spectrometry research award, the Pehr Edman Award in Protein Chemistry, the American Society for Mass Spectrometry Biemann Medal, the HUPO Distinguished Achievement Award in Proteomics, Herbert Sober Award from the ASBMB, and the Christian Anfinsen Award from The Protein Society, the 2015 ACS’s Analytical Chemistry award and 2015 The Ralph N. Adams Award in Bioanalytical Chemistry. He was ranked by Citation Impact, Science Watch as one of the Top 100 Chemists for the decade, 2000-2010. He was #1 on a List of Most Influential in Analytical Chemistry compiled by The Analytical Scientist 10/30/2013 and is on the List Of Most Highly Influential Biomedical Researchers, 1996-2011, European J. Clinical Investigation 2013, 43, 1339-1365 and the 2015 List of Highly Cited Scientists. He has published over 750 scientific articles with ~70,000 citations, and an H index 130.

Authorship: John R. Yates, Sandra Pankow, Casimir Bamberger, Diego Calzolari, Salvador Martínez-Bartolomé, Mathieu Lavallée-Adam
Department of Chemical Physiology, The Scripps Research Institute, LaJolla, CA, USA

Short Abstract Recent studies on the loss of function mutant form of the Cystic Fibrosis Transport Regulator (F508) as it progresses through the folding pathway will be presented. Through the study of protein-protein interactions and modifications that regulate maturation of CFTR, we are beginning to understand the critical interactions regulating pathways for export or destruction.

Long Abstract

Deletion of phenylalanine 508 (∆F508) in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is the major cause of Cystic Fibrosis (CF), one of the most common inherited childhood diseases for which no cure exists.  The mutated anion channel is not fully glycosylated or integrated into the plasma membrane and has little to no activity in lung epithelial cells of CF patients 1-3.  The defects in cellular processing of mutant CFTR can be rescued, in part, by low temperature (26-30˚C) or by inhibition of histone deacetylases (HDACs) 4,5.  Proposed rescue mechanisms include a favorable change in protein interactions for ∆F508 at low temperature and upon treatment with HDAC inhibitors, which under normal conditions prevent proper processing and function of the mutant chloride channel.  However, only 38 interactors have been identified for both wt and ∆F508 CFTR in lung cells 6 and the interactome dynamics that occur during temperature shift and HDAC inhibition (HDACi) are unknown.  Here, we report the first systematic and comprehensive analysis of the normal and ∆F508 CFTR interactome in patient-derived lung epithelial cells and its dynamics during temperature shift and HDACi.  By using a novel deep proteomic analysis method based on co-immunoprecipitation-mass spectrometry (CoPIT), we were able to achieve comprehensive interactome coverage.  We identified more than 21,000 spectra for CFTR alone and found a total of 638 individual high-confidence interactors with 208 specific for ∆F508 CFTR.  These constitute a mutation-specific interactome, which is extensively remodeled upon rescue by temperature-shift, HDACi or RNAi of HDAC7.  Network-analysis identified a number of key novel interactors that influence translation and degradation of ∆F508 CFTR in particular, as well as its ER-dependent folding and trafficking upon rescue.  52 of these were functionally tested and 48 altered ∆F508 CFTR maturation, among them the ER-resident protein tyrosine kinase-like protein PTPLAD1 and the protein disulfide isomerase PDIA4, the translation regulating protein YBX1 and the ubiquitin E3-ligase TRIM21.  These proteins were four of 31 novel interactors that prevent proper CFTR maturation and stability in mutant cells.  In contrast, 17 proteins are critical for normal CFTR biogenesis and include the lectin binding protein LGALS3BP whose loss leads to elimination of CFTR protein.  Our results demonstrate that global remodeling of protein interactions involved in CFTR biogenesis and degradation is crucial for ∆F508 CFTR rescue, and identify individual key interactors like PTPLAD1 or SURF4 whose loss enhanced ∆F508 CFTR channel function. This study provides comprehensive insight into the molecular disease mechanisms of CF caused by deletion of F508.

References & Acknowledgements:

Remodeling of a ∆F508 CFTR-mutation specific interactome promotes rescue of Cystic Fibrosis, Pankow et al, Nature (in press)

This work is supported by NIH grants 5R01HL079442-08 (J.R.Y. and W.E.B.), P01AG031097 (J.R.Y and W.E.B.), P41 RR011823 (J.R.Y), HHSN268201000035C (J.R.Y), and a CFF mass spectrometry fellowship BALCH050X6 (S.P. and J.R.Y).

Financial Disclosure

IP Royalty: yes

IP Desc:University of Washington, The Scripps Research Institute

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