A Chaperone Trap Contributes to the Onset of Cystic Fibrosis

Judith A Coppinger,Atanas V Koulov,William E Balch,Darren M Hutt,John R Yates,Abbas Razvi,Sandra Pankow,Harm Kampinga

doi:10.1371/journal.pone.0037682

Judith A Coppinger, Atanas V Koulov + Show 6 more

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https://doi.org/10.1371/journal.pone.0037682

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Journal: PloS one	Publication Date: May 31, 2012
Citations: 81	License type: CC BY 4.0

Affiliation: Scripps Research Institute

Abstract

Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a ‘chaperone trap’. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF.

Highlights

The proteostasis network (PN) is composed of numerous components that direct the highly variable and adaptable folding and degradative capacity of the cell [1,2,3]
DF508-CFTR provides a unique opportunity to quantitatively determine the properties of folding intermediate(s) that impact disease etiology
In order to understand CFTR folding by the PN components differentially associated with WT- and DF508-CFTR [33], we used 15N-labeled protein standards expressed in E. coli and single ion reaction monitoring mass spectrometry (SRMMS) to provide a quantitative analysis of the complexes generated during CFTR biogenesis (Fig. 1 & Fig. S1)

Summary

Introduction

The proteostasis network (PN) is composed of numerous components that direct the highly variable and adaptable folding and degradative capacity of the cell [1,2,3]. Substantial evidence demonstrates that failure to deliver DF508-CFTR to the cell surface reflects its inability to achieve sufficient thermodynamic stability of the cytoplasmic oriented NBD1 domain missing the Phe508 residue [20,21,22,23,24,25,26,27,28,29,30,31,32] This defect limits the ability of additional domains, including the TMD, to interact with NBD1 to form a structure recognized by the ER-associated COPII export machinery for delivery to the plasma membrane [3,17,18,19,33,34]. The loss of CFTR at the cell surface results in dehydration of the epithelial lining of the lung and other tissues through a disruption of Na+ and Cl2 balance, triggering the progressive pathology characteristic of the disease [3,35]

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