Jeanne Matteson

Reduced histone deacetylase 7 activity restores function to misfolded CFTR in cystic fibrosis

Chemical modulation of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACi) is an increasingly important approach for modifying the etiology of human disease. Loss-of-function diseases arise as a consequence of protein misfolding and degradation, which lead to system failures. The DeltaF508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) results in the absence of the cell surface chloride channel and a loss of airway hydration, leading to the premature lung failure and reduced lifespan responsible for cystic fibrosis. We now show that the HDACi suberoylanilide hydroxamic acid (SAHA) restores surface channel activity in human primary airway epithelia to levels that are 28% of those of wild-type CFTR. Biological silencing of all known class I and II HDACs reveals that HDAC7 plays a central role in restoration of DeltaF508 function. We suggest that the tunable capacity of HDACs can be manipulated by chemical biology to counter the onset of cystic fibrosis and other human misfolding disorders.

COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code

Cystic fibrosis (CF) is a childhood hereditary disease in which the most common mutant form of the CF transmembrane conductance regulator (CFTR) ΔF508 fails to exit the endoplasmic reticulum (ER). Export of wild-type CFTR from the ER requires the coat complex II (COPII) machinery, as it is sensitive to Sar1 mutants that disrupt normal coat assembly and disassembly. In contrast, COPII is not used to deliver CFTR to ER-associated degradation. We find that exit of wild-type CFTR from the ER is blocked by mutation of a consensus di-acidic ER exit motif present in the first nucleotide binding domain. Mutation of the code disrupts interaction with the COPII coat selection complex Sec23/Sec24. We propose that the di-acidic exit code plays a key role in linking CFTR to the COPII coat machinery and is the primary defect responsible for CF in ΔF508-expressing patients.

A Di-acidic (DXE) Code Directs Concentration of Cargo during Export from the Endoplasmic Reticulum

Efficient export of vesicular stomatitis virus glycoprotein (VSV-G), a type I transmembrane protein, from the endoplasmic reticulum requires a di-acidic code (DXE) located in the cytosolic carboxyl-terminal tail (Nishimura, N., and Balch, W. E. (1997) Science 277, 556–558). Mutation of the DXE code by mutation to AXA did not prevent VSV-G recruitment to pre-budding complexes formed in the presence of the activated form of the Sar1 and the Sec23/24 complex, components of the COPII budding machinery. However, the signal was required at a subsequent concentration step preceding vesicle fission. By using green fluorescence protein-tagged VSV-G to image movement in a single cell, we found that VSV-G lacking the DXE code fails to be concentrated into COPII vesicles. As a result, the normal 5–10-fold increase in the steady-state concentration of VSV-G in downstream pre-Golgi intermediates and Golgi compartments was lost. These results demonstrate for the first time that inactivation of the DXE signal uncouples early cargo selection steps from concentration into COPII vesicles. We propose that two sequential steps are required for efficient export from the endoplasmic reticulum.

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

We propose a general model for the role of the Hsp90 ATPase cycle in proteostasis in which Aha1 regulates the dwell time of Hsp90 with client by integrating chaperone function and client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions.

Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca2+ stabilization

Hereditary familial amyloidosis of Finnish type (FAF) leading to amyloid in the peripheral and central nervous systems stems from deposition of a 71 residue fragment generated from the D187N/Y variants of plasma gelsolin by two sequential endoproteolytic events. We identify the protease accomplishing the first cleavage as furin, a proprotein convertase. Endoproteolysis of plasma gelsolin occurs in the trans‐Golgi network due to the inability of the FAF variants to bind and be stabilized by Ca2+. Secretion and processing of the FAF variants by furin can be uncoupled by blocking the convergence of the exocytic pathway transporting plasma gelsolin and the endocytic recycling of furin. We propose that coincidence of membrane trafficking pathways contributes to the development of proteolysis‐initiated amyloid disease.

Rab-αGDI activity is regulated by a Hsp90 chaperone complex

The Rab‐specific αGDP‐dissociation inhibitor (αGDI) regulates the recycling of Rab GTPases. We have now identified a novel αGDI complex from synaptic membranes that contains three chaperone components: Hsp90, Hsc70 and cysteine string protein (CSP). We find that the αGDI–chaperone complex is dissociated in response to Ca2+‐induced neurotransmitter release, that chaperone complex dissociation is sensitive to the Hsp90 inhibitor geldanamycin (GA) and that GA inhibits the ability of αGDI to recycle Rab3A during neurotransmitter release. We propose that αGDI interacts with a specialized membrane‐associated Rab recycling Hsp90 chaperone system on the vesicle membrane to coordinate the Ca2+‐dependent events triggering Rab‐GTP hydrolysis with retrieval of Rab‐GDP to the cytosol.

Geranylgeranyl Switching Regulates

Rab GTPases, key regulators of membrane targeting and fusion, require the covalent attachment of geranylgeranyl lipids to their C terminus for function. To elucidate the role of lipid in Rab recycling, we have determined the crystal structure of Rab guanine nucleotide dissociation inhibitor (alphaGDI) in complex with a geranylgeranyl (GG) ligand (H(2)N-Cys-(S-GG)-OMe). The lipid is bound beneath the Rab binding platform in a shallow hydrophobic groove. Mutation of the binding pocket in the brain-specific alphaGDI leads to mental retardation. Strikingly, lipid binding acts through a conserved allosteric switching mechanism to promote release of the GDI-Rab[GDP] complex from the membrane.

Molecular Role for the Rab Binding Platform of Guanine Nucleotide Dissociation Inhibitor in Endoplasmic Reticulum to Golgi Transport

Guanine nucleotide dissociation inhibitor (GDI) regulates the recycling of Rab GTPases involved in vesicle targeting and fusion. We have analyzed the requirement for conserved amino acid residues in the binding of Rab1A and the function of GDI in transport of cargo between the endoplasmic reticulum (ER) and the Golgi apparatus. Using a new approach to monitor GDI-Rab interactions based on the change in fluorescence associated with the release of methylanthraniloyl guanosine di(tri)phosphate-GDP (mGDP) from Rab, we show that residues previously implicated in the binding of the synapse-specific Rab3A, including Gln-236, Arg-240, and Thr-248, are essential for the binding of Rab1A. Mutation of each of these residues has potent effects on the ability of GDI to remove Rab1A from membranes and inhibit ER to Golgi transport in vitro. Given the sequence divergence between Rab1A and 3A (35% identity), these residues are proposed to play a general role in GDI function in the cell. In contrast, several other residues found within or flanking the Rab-binding region were found to have differential effects in the recognition and recycling of Rab1A and 3A, and therefore direct selective interaction of GDI with individual Rab proteins. Intriguingly, mutation of one residue, Arg-70, led to a reduction of Rab1A binding, failed to extract Rab1A from membranes in vitro, yet bound membranes tightly and potently inhibited ER to Golgi transport. These results provide evidence that novel membrane-associated factor(s) mediate Rab-independent GDI interaction with membranes.

The role of ARF1 and rab GTPases in polarization of the Golgi stack.

The organization and sorting of proteins within the Golgi stack to establish and maintain its cis to trans polarization remains an enigma. The function of Golgi compartments involves coat assemblages that facilitate vesicle traffic, Rab‐tether‐SNAP receptor (SNARE) machineries that dictate membrane identity, as well as matrix components that maintain structure. We have investigated how the Golgi complex achieves compartmentalization in response to a key component of the coat complex I (COPI) coat assembly pathway, the ARF1 GTPase, in relationship to GTPases‐regulating endoplasmic reticulum (ER) exit (Sar1) and targeting fusion (Rab1). Following collapse of the Golgi into the ER in response to inhibition of activation of ARF1 by Brefeldin A, we found that Sar1‐ and Rab1‐dependent Golgi reformation took place at multiple peripheral and perinuclear ER exit sites. These rapidly converged into immature Golgi that appeared as onion‐like structures composed of multiple concentrically arrayed cisternae of mixed enzyme composition. During clustering to the perinuclear region, Golgi enzymes were sorted to achieve the degree of polarization within the stack found in mature Golgi. Surprisingly, we found that sorting of Golgi enzymes into their subcompartments was insensitive to the dominant negative GTP‐restricted ARF1 mutant, a potent inhibitor of COPI coat disassembly and vesicular traffic. We suggest that a COPI‐independent, Rab‐dependent mechanism is involved in the rapid reorganization of resident enzymes within the Golgi stack following synchronized release from the ER, suggesting an important role for Rab hubs in directing Golgi polarization.

FK506 Binding Protein 8 Peptidylprolyl Isomerase Activity Manages a Late Stage of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Folding and Stability

Background: The cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel critical for ionic homeostasis in epithelial cells, is mutated in cystic fibrosis. Results: FKBP8 stabilizes WT and ΔF508 CFTR in the ER and appears to act downstream of Hsp90. Conclusion: FKBP8 is critical for the biogenesis of WT and ΔF508 CFTR. Significance: Our findings suggest that FKBP8 is a late acting chaperone for WT and ΔF508 CFTR. Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.