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.
The pathways that distinguish transport of folded and misfolded cargo through the exocytic (secretory) pathway of eukaryotic cells remain unknown. Using proteomics to assess global cystic fibrosis ...(CF) transmembrane conductance regulator (CFTR) protein interactions (the CFTR interactome), we show that Hsp90 cochaperones modulate Hsp90-dependent stability of CFTR protein folding in the endoplasmic reticulum (ER). Cell-surface rescue of the most common disease variant that is restricted to the ER, ΔF508, can be initiated by partial siRNA silencing of the Hsp90 cochaperone ATPase regulator Aha1. We propose that failure of ΔF508 to achieve an energetically favorable fold in response to the steady-state dynamics of the chaperone folding environment (the “chaperome”) is responsible for the pathophysiology of CF. The activity of cargo-associated chaperome components may be a common mechanism regulating folding for ER exit, providing a general framework for correction of misfolding disease.
α1-Antitrypsin (α1AT) deficiency (α1ATD) is a consequence of defective folding, trafficking, and secretion of α1AT in response to a defect in its interaction with the endoplasmic reticulum ...proteostasis machineries. The most common and severe form of α1ATD is caused by the Z-variant and is characterized by the accumulation of α1AT polymers in the endoplasmic reticulum of the liver leading to a severe reduction (>85%) of α1AT in the serum and its anti-protease activity in the lung. In this organ α1AT is critical for ensuring tissue integrity by inhibiting neutrophil elastase, a protease that degrades elastin. Given the limited therapeutic options in α1ATD, a more detailed understanding of the folding and trafficking biology governing α1AT biogenesis and its response to small molecule regulators is required. Herein we report the correction of Z-α1AT secretion in response to treatment with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), acting in part through HDAC7 silencing and involving a calnexin-sensitive mechanism. SAHA-mediated correction restores Z-α1AT secretion and serpin activity to a level 50% that observed for wild-type α1AT. These data suggest that HDAC activity can influence Z-α1AT protein traffic and that SAHA may represent a potential therapeutic approach for α1ATD and other protein misfolding diseases.
Background: α1-Antitrypsin (α1AT) deficiency (α1ATD) is a consequence of defective folding, trafficking, and secretion of α1AT.
Results: SAHA restores the secretion of an active form of Z-α1AT in part through a calnexin- and HDAC7-sensitive dependent mechanism(s).
Conclusion: SAHA may represent a potential therapeutic approach for α1ATD.
Significance: SAHA is a regulator of the proteostasis biology of Z-α1AT, favoring export of a functional form to serum.
Niemann-Pick disease type C1 (NPC1) is a lysosomal disorder due to impaired intracellular cholesterol transport out of the endolysosomal compartment.. Marked heterogeneity has been observed in ...individuals with the same
genotype, thus suggesting a significant effect of modifier genes. Prior work demonstrated that decreased SOAT1 activity decreased disease severity in an NPC1 mouse model. Thus, we hypothesized that a polymorphism associated with decreased SOAT1 expression might influence the NPC1 phenotype. Phenotyping and genomic sequencing of 117 individuals with NPC1 was performed as part of a Natural History trial. Phenotyping included determination of disease severity and disease burden. Significant clinical heterogeneity is present in individuals homozygous for the
variant and in siblings. Analysis of the
polymorphism, rs1044925 (A>C), showed a significant association of the C-allele with earlier age of neurological onset. The C-allele may be associated with a higher Annualized Severity Index Score as well as increased frequency of liver disease and seizures. A polymorphism associated with decreased expression of
appears to be a genetic modifier of the NPC1 phenotype. This finding is consistent with prior data showing decreased phenotypic severity in
mice and supports efforts to investigate the potential of SOAT1 inhibitors as a potential therapy for NPC1.
Protein function is generated and maintained by the proteostasis network (PN) (Balch et al. (2008) Science, 319:916). The PN is a modular, yet integrated system unique to each cell type that is ...sensitive to signaling pathways that direct development and aging, and respond to folding stress. Mismanagement of protein folding and function triggered by genetic, epigenetic and environmental causes poses a major challenge to human health and lifespan. Herein, we address the impact of proteostasis defined by the FoldFx model on our understanding of protein folding and function in biology. FoldFx describes how general proteostasis control (GPC) enables the polypeptide chain sequence to achieve functional balance in the context of the cellular proteome. By linking together the chemical and energetic properties of the protein fold with the composition of the PN we discuss the principle of the proteostasis boundary (PB) as a key component of GPC. The curved surface of the PB observed in 3-dimensional space suggests that the polypeptide chain sequence and the PN operate as an evolutionarily conserved functional unit to generate and sustain protein dynamics required for biology. Modeling general proteostasis provides a rational basis for tackling some of the most challenging diseases facing mankind in the 21st century.
Niemann-Pick type C (NPC) disease is primarily caused by mutations in the NPC1 gene and is characterized by the accumulation of unesterified cholesterol and lipids in the late endosomal (LE) and ...lysosomal (Ly) compartments. The most prevalent disease-linked mutation is the I1061T variant of NPC1, which exhibits defective folding and trafficking from the endoplasmic reticulum to the LE/Ly compartments. We now show that the FDA-approved histone deacetylase inhibitor (HDACi) valproic acid (VPA) corrects the folding and trafficking defect associated with I1061T-NPC1 leading to restoration of cholesterol homeostasis, an effect that is largely driven by a reduction in HDAC7 expression. The VPA-mediated trafficking correction is in part associated with an increase in the acetylation of lysine residues in the cysteine-rich domain of NPC1. The HDACi-mediated correction is synergistically improved by combining it with the FDA-approved anti-malarial, chloroquine, a known lysosomotropic compound, which improved the stability of the LE/Ly-localized fraction of the I1061T variant. We posit that combining the activity of VPA, to modulate epigenetically the cellular acetylome, with chloroquine, to alter the lysosomal environment to favor stability of the trafficked I1061T variant protein can have a significant therapeutic benefit in patients carrying at least one copy of the I1061T variant of NPC1, the most common disease-associated mutation leading to NPC disease. Given its ability to cross the blood-brain barrier, we posit VPA provides a potential mechanism to improve the response to 2-hydroxypropyl-β-cyclodextrin, by restoring a functional NPC1 to the cholesterol managing compartment as an adjunct therapy.
Transport of membrane proteins between intracellular compartments requires specific sequences in the protein cytoplasmic domain to direct packaging into vesicle shuttles. A sequence that mediates ...export from the endoplasmic reticulum (ER) has proved elusive. A di-acidic signal (Asp-X-Glu, where X represents any amino acid) on the cytoplasmic tail of vesicular stomatitis virus glycoprotein (VSV-G) and other cargo molecules was required for efficient recruitment to vesicles mediating export from the ER in baby hamster kidney cells. The existence of such a signal provides evidence that export from the ER occurs through a selective mechanism.
Loss-of-function diseases are often caused by a mutation in a protein traversing the secretory pathway that compromises the normal balance between protein folding, trafficking, and degradation. We ...demonstrate that the innate cellular protein homeostasis, or proteostasis, capacity can be enhanced to fold mutated enzymes that would otherwise misfold and be degraded, using small molecule proteostasis regulators. Two proteostasis regulators are reported that alter the composition of the proteostasis network in the endoplasmic reticulum through the unfolded protein response, increasing the mutant folded protein concentration that can engage the trafficking machinery, restoring function to two nonhomologous mutant enzymes associated with distinct lysosomal storage diseases. Coapplication of a pharmacologic chaperone and a proteostasis regulator exhibits synergy because of the former's ability to further increase the concentration of trafficking-competent mutant folded enzymes. It may be possible to ameliorate loss-of-function diseases by using proteostasis regulators alone or in combination with a pharmacologic chaperone.
Gaucher disease is a lysosomal storage disorder caused by deficient lysosomal β-glucosidase (β-Glu) activity. A marked decrease in enzyme activity results in progressive accumulation of the substrate ...(glucosylceramide) in macrophages, leading to hepatosplenomegaly, anemia, skeletal lesions, and sometimes CNS involvement. Enzyme replacement therapy for Gaucher disease is costly and relatively ineffective for CNS involvement. Chemical chaperones have been shown to stabilize various proteins against misfolding, increasing proper trafficking from the endoplasmic reticulum. We report herein that the addition of subinhibitory concentrations (10 µM) of N-(n-nonyl)deoxynojirimycin (NN-DNJ) to a fibroblast culture medium for 9 days leads to a 2-fold increase in the activity of N370S β-Glu, the most common mutation causing Gaucher disease. Moreover, the increased activity persists for at least 6 days after the withdrawal of the putative chaperone. The NN-DNJ chaperone also increases WT β-Glu activity, but not that of L444P, a less prevalent Gaucher disease variant. Incubation of isolated soluble WT enzyme with NN-DNJ reveals that β-Glu is stabilized against heat denaturation in a dose-dependent fashion. We propose that NN-DNJ chaperones β-Glu folding at neutral pH, thus allowing the stabilized enzyme to transit from the endoplasmic reticulum to the Golgi, enabling proper trafficking to the lysosome. Clinical data suggest that a modest increase in β-Glu activity may be sufficient to achieve a therapeutic effect.
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