Summary Deletion of phenylalanine 508 of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is the major cause of Cystic Fibrosis (CF), one of the most common inherited childhood diseases. The mutated CFTR anion channel is not fully glycosylated and shows minimal activity in bronchial epithelial cells of CF patients. Low temperature or inhibition of histone deacetylases (HDACi) can partially rescue ΔF508 CFTR cellular processing defects and function. A favorable change of ΔF508 CFTR protein-protein interactions was proposed as mechanism of rescue, however CFTR interactome dynamics during temperature-shift and HDACi rescue are unknown. Here, we report the first comprehensive analysis of the wt and ΔF508 CFTR interactome and its dynamics during temperature shift and HDACi. By using a novel deep proteomic analysis method (CoPIT), we identified 638 individual high-confidence CFTR interactors and discovered a mutation-specific interactome, which is extensively remodeled upon rescue. Detailed analysis of the interactome remodeling identified key novel interactors, whose loss promoted enhanced CFTR channel function in primary CF epithelia or which were critical for normal CFTR biogenesis. Our results demonstrate that global remodeling of ΔF508 CFTR interactions is crucial for rescue, and provide comprehensive insight into the molecular disease mechanisms of CF caused by deletion of F508.

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.

Here we report the identification and molecular function of the p53 tumor suppressor-like protein nvp63 in a non-bilaterian animal, the starlet sea anemone Nematostella vectensis. So far, p53-like proteins had been found in bilaterians only. The evolutionary origin of p53-like proteins is highly disputed and primordial p53-like proteins are variably thought to protect somatic cells from genotoxic stress. Here we show that ultraviolet (UV) irradiation at low levels selectively induces programmed cell death in early gametes but not somatic cells of adult N. vectensis polyps. We demonstrate with RNA interference that nvp63 mediates this cell death in vivo. Nvp63 is the most archaic member of three p53-like proteins found in N. vectensis and in congruence with all known p53-like proteins, nvp63 binds to the vertebrate p53 DNA recognition sequence and activates target gene transcription in vitro. A transactivation inhibitory domain at its C-terminus with high homology to the vertebrate p63 may regulate nvp63 on a molecular level. The genotoxic stress induced and nvp63 mediated apoptosis in N. vectensis gametes reveals an evolutionary ancient germ cell protective pathway which relies on p63-like proteins and is conserved from cnidarians to vertebrates.

Misfolding and aggregation of amyloid-β peptide and hyperphosphorylated tau are molecular markers of Alzheimer's disease (AD), and although the 3D structures of these aberrantly folded proteins have been visualized in exquisite detail, no method has been able to survey protein folding across the proteome in AD. Here, we present covalent protein painting (CPP), a mass spectrometry-based protein footprinting approach to quantify the accessibility of lysine ε-amines for covalent modification at the surface of natively folded proteins. We used CPP to survey the reactivity of 2645 lysine residues and therewith the structural proteome of HEK293T cells and found that reactivity increased upon mild heat shock. CPP revealed that the accessibility of lysine residues for covalent modification in tubulin-β (TUBB), in succinate dehydrogenase (SHDB), and in amyloid-β peptide (Aβ) is altered in human postmortem brain samples of patients with neurodegenerative diseases. The structural alterations of TUBB and SHDB in patients with AD, dementia with Lewy bodies (DLB), or both point to broader perturbations of the 3D proteome beyond Aβ and hyperphosphorylated tau.

SUMMARY Gaucher’s disease (GD) is caused by mutations that compromise β-glucocerebrosidase (GCase) folding in the endoplasmic reticulum (ER), leading to excessive degradation instead of trafficking, which results in insufficient lysosomal function. We hypothesized that ER GCase interacting proteins play critical roles in making quality control decisions, i.e., facilitating ER-associated degradation (ERAD) instead of folding and trafficking. Utilizing GCase immunoprecipitation followed by mass spectrometry-based proteomics, we identified endogenous HeLa cell GCase protein interactors, including ERdj3, an ER resident Hsp40 not previously established to interact with GCase. Depleting ERdj3 reduced the rate of mutant GCase degradation in patient-derived fibroblasts, while increasing folding, trafficking and function by directing GCase to the pro-folding ER calnexin pathway. Inhibiting ERdj3-mediated mutant GCase degradation while simultaneously enhancing calnexin-associated folding, by way of a diltiazem-mediated increase in ER Ca2+ levels, yields a synergistic rescue of L444P GCase lysosomal function. Our findings suggest a combination therapeutic strategy for ameliorating GD.