COPII cage assembly factor Sec13 integrates information flow regulating endomembrane function in response to human variation.

How information flow is coordinated for managing transit of 1/3 of the genome through endomembrane pathways by the coat complex II (COPII) system in response to human variation remains an enigma. By examining the interactome of the COPII cage-assembly component Sec13, we show that it is simultaneously associated with multiple protein complexes that facilitate different features of a continuous program of chromatin organization, transcription, translation, trafficking, and degradation steps that are differentially sensitive to Sec13 levels. For the trafficking step, and unlike other COPII components, reduction of Sec13 expression decreased the ubiquitination and degradation of wild-type (WT) and F508del variant cargo protein cystic fibrosis transmembrane conductance regulator (CFTR) leading to a striking increase in fold stability suggesting that the events differentiating export from degradation are critically dependent on COPII cage assembly at the ER Golgi intermediate compartment (ERGIC) associated recycling and degradation step linked to COPI exchange. Given Sec13's multiple roles in protein complex assemblies that change in response to its expression, we suggest that Sec13 serves as an unanticipated master regulator coordinating information flow from the genome to the proteome to facilitate spatial covariant features initiating and maintaining design and function of membrane architecture in response to human variation.

Triangulating variation in the population to define mechanisms for precision management of genetic disease.

To understand mechanistically how the protein fold is shaped by therapeutics to inform precision management of disease, we developed variation-capture (VarC) mapping. VarC triangulates sparse sequence variation information found in the population using Gaussian process regression (GPR)-based machine learning to define the combined pairwise-residue interactions contributing to dynamic protein function in the individual in response to therapeutics. Using VarC mapping, we now reveal the pairwise-residue covariant relationships across the entire protein fold of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) to define the molecular mechanisms of clinically approved CF chemical modulators. We discover an energetically destabilized covariant core containing a di-acidic YKDAD endoplasmic reticulum (ER) exit code that is only weakly corrected by current therapeutics. Our results illustrate that VarC provides a generalizable tool to triangulate information from genetic variation in the population to mechanistically discover therapeutic strategies that guide precision management of the individual.

Spatial covariance analysis reveals the residue-by-residue thermodynamic contribution of variation to the CFTR fold.

Although the impact of genome variation on the thermodynamic properties of function on the protein fold has been studied in vitro, it remains a challenge to assign these relationships across the entire polypeptide sequence in vivo. Using the Gaussian process regression based principle of Spatial CoVariance, we globally assign on a residue-by-residue basis the biological thermodynamic properties that contribute to the functional fold of CFTR in the cell. We demonstrate the existence of a thermodynamically sensitive region of the CFTR fold involving the interface between NBD1 and ICL4 that contributes to its export from endoplasmic reticulum. At the cell surface a new set of residues contribute uniquely to the management of channel function. These results support a general 'quality assurance' view of global protein fold management as an SCV principle describing the differential pre- and post-ER residue interactions contributing to compartmentalization of the energetics of the protein fold for function. Our results set the stage for future analyses of the quality systems managing protein sequence-to-function-to-structure broadly encompassing genome design leading to protein function in complex cellular relationships responsible for diversity and fitness in biology in response to the environment.

HDAC inhibitors rescue multiple disease-causing CFTR variants.

Understanding the role of the epigenome in protein-misfolding diseases remains a challenge in light of genetic diversity found in the world-wide population revealed by human genome sequencing efforts and the highly variable response of the disease population to therapeutics. An ever-growing body of evidence has shown that histone deacetylase (HDAC) inhibitors (HDACi) can have significant benefit in correcting protein-misfolding diseases that occur in response to both familial and somatic mutation. Cystic fibrosis (CF) is a familial autosomal recessive disease, caused by genetic diversity in the CF transmembrane conductance regulator (CFTR) gene, a cyclic Adenosine MonoPhosphate (cAMP)-dependent chloride channel expressed at the apical plasma membrane of epithelial cells in multiple tissues. The potential utility of HDACi in correcting the phenylalanine 508 deletion (F508del) CFTR variant as well as the over 2000 CF-associated variants remains controversial. To address this concern, we examined the impact of US Food and Drug Administration-approved HDACi on the trafficking and function of a panel of CFTR variants. Our data reveal that panobinostat (LBH-589) and romidepsin (FK-228) provide functional correction of Class II and III CFTR variants, restoring cell surface chloride channel activity in primary human bronchial epithelial cells. We further demonstrate a synergistic effect of these HDACi with Vx809, which can significantly restore channel activity for multiple CFTR variants. These data suggest that HDACi can serve to level the cellular playing field for correcting CF-causing mutations, a leveling effect that might also extend to other protein-misfolding diseases.

Silencing of the Hsp70-specific nucleotide-exchange factor BAG3 corrects the F508del-CFTR variant by restoring autophagy.

The protein chaperones heat shock protein 70 (Hsp70) and Hsp90 are required for de novo folding of proteins and protect against misfolding-related cellular stresses by directing misfolded or slowly folding proteins to the ubiquitin/proteasome system (UPS) or autophagy/lysosomal degradation pathways. Here, we examined the role of the Bcl2-associated athanogene (BAG) family of Hsp70-specific nucleotide-exchange factors in the biogenesis and functional correction of genetic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) whose mutations cause cystic fibrosis (CF). We show that siRNA-mediated silencing of BAG1 and -3, two BAG members linked to the clearance of misfolded proteins via the UPS and autophagy pathways, respectively, leads to functional correction of F508del-CFTR and other disease-associated CFTR variants. BAG3 silencing was the most effective, leading to improved F508del-CFTR stability, trafficking, and restoration of cell-surface function, both alone and in combination with the FDA-approved CFTR corrector, VX-809. We also found that the BAG3 silencing-mediated correction of F508del-CFTR restores the autophagy pathway, which is defective in F508del-CFTR-expressing cells, likely because of the maladaptive stress response in CF pathophysiology. These results highlight the potential therapeutic benefits of targeting the cellular chaperone system to improve the functional folding of CFTR variants contributing to CF and possibly other protein-misfolding-associated diseases.

Multilevel interaction of the DnaK/DnaJ(HSP70/HSP40) stress-responsive chaperone machine with the central metabolism.

Networks of molecular chaperones maintain cellular protein homeostasis by acting at nearly every step in the biogenesis of proteins and protein complexes. Herein, we demonstrate that the major chaperone DnaK/HSP70 of the model bacterium Escherichia coli is critical for the proper functioning of the central metabolism and for the cellular response to carbon nutrition changes, either directly or indirectly via the control of the heat-shock response. We identified carbon sources whose utilization was positively or negatively affected by DnaK and isolated several central metabolism genes (among other genes identified in this work) that compensate for the lack of DnaK and/or DnaK/Trigger Factor chaperone functions in vivo. Using carbon sources with specific entry points coupled to NMR analyses of real-time carbon assimilation, metabolic coproducts production and flux rearrangements, we demonstrate that DnaK significantly impacts the hierarchical order of carbon sources utilization, the excretion of main coproducts and the distribution of metabolic fluxes, thus revealing a multilevel interaction of DnaK with the central metabolism.