HSP90 inhibitors reduce cholesterol storage in Niemann-Pick type C1 mutant fibroblasts.

Niemann-Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder caused by mutations of the NPC1 gene. More than 300 disease-associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids, and other lipids in late endosomes and lysosomes (LE/Ly) of many cell types. Previously, we showed that treatment of many different NPC1 mutant fibroblasts with histone deacetylase inhibitors resulted in reduction of cholesterol storage, and we found that this was associated with enhanced exit of the NPC1 protein from the endoplasmic reticulum and delivery to LE/Ly. This suggested that histone deacetylase inhibitors may work through changes in protein chaperones to enhance the folding of NPC1 mutants, allowing them to be delivered to LE/Ly. In this study, we evaluated the effect of several HSP90 inhibitors on NPC1I1061T skin fibroblasts. We found that HSP90 inhibition resulted in clearance of cholesterol from LE/Ly, and this was associated with enhanced delivery of the mutant NPC1I1061T protein to LE/Ly. We also observed that inhibition of HSP90 increased the expression of HSP70, and overexpression of HSP70 also reduced cholesterol storage in NPC1I1061T fibroblasts. However, we did not see correction of cholesterol storage by arimoclomol, a drug that is reported to increase HSP70 expression, at doses up to 0.5 mM. The increase in other chaperones as a consequence of HSP90 improves folding of NPC1 protein and relieves cholesterol accumulation in NPC1 mutant fibroblasts.

Correction of Niemann-Pick type C1 trafficking and activity with the histone deacetylase inhibitor valproic acid.

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-ß-cyclodextrin, by restoring a functional NPC1 to the cholesterol managing compartment as an adjunct therapy.

Quantitative Analysis of the Proteome Response to the Histone Deacetylase Inhibitor (HDACi) Vorinostat in Niemann-Pick Type C1 disease.

Niemann-Pick type C (NPC) disease is an inherited, progressive neurodegenerative disorder principally caused by mutations in the NPC1 gene. NPC disease is characterized by the accumulation of unesterified cholesterol in the late endosomes (LE) and lysosomes (Ly) (LE/Ly). Vorinostat, a histone deacetylase inhibitor (HDACi), restores cholesterol homeostasis in fibroblasts derived from NPC patients; however, the exact mechanism by which Vorinostat restores cholesterol level is not known yet. In this study, we performed comparative proteomic profiling of the response of NPC1I1061T fibroblasts to Vorinostat. After stringent statistical criteria to filter identified proteins, we observed 202 proteins that are differentially expressed in Vorinostat-treated fibroblasts. These proteins are members of diverse cellular pathways including the endomembrane dependent protein folding-stability-degradation-trafficking axis, energy metabolism, and lipid metabolism. Our study shows that treatment of NPC1I1061T fibroblasts with Vorinostat not only enhances pathways promoting the folding, stabilization and trafficking of NPC1 (I1061T) mutant to the LE/Ly, but alters the expression of lysosomal proteins, specifically the lysosomal acid lipase (LIPA) involved in the LIPA->NPC2->NPC1 based flow of cholesterol from the LE/Ly lumen to the LE/Ly membrane. We posit that the Vorinostat may modulate numerous pathways that operate in an integrated fashion through epigenetic and post-translational modifications reflecting acetylation/deacetylation balance to help manage the defective NPC1 fold, the function of the LE/Ly system and/or additional cholesterol metabolism/distribution pathways, that could globally contribute to improved mitigation of NPC1 disease in the clinic based on as yet uncharacterized principles of cellular metabolism dictating cholesterol homeostasis.

Histone deacetylase inhibitors correct the cholesterol storage defect in most Niemann-Pick C1 mutant cells.

Niemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. The large majority of NPC disease is caused by mutations in NPC1, a large polytopic membrane protein that functions in late endosomes. There are many disease-associated mutations in NPC1, and most patients are compound heterozygotes. The most common mutation, NPC1I1061T, has been shown to cause endoplasmic reticulum-associated degradation of the NPC1 protein. Treatment of patient-derived NPC1I1061T fibroblasts with histone deacetylase inhibitors (HDACis) vorinostat or panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Here, we show that several other human NPC1 mutant fibroblast cell lines can also be corrected by vorinostat or panobinostat and that treatment with vorinostat extends the lifetime of the NPC1I1061T protein. To test effects of HDACi on a large number of NPC1 mutants, we engineered a U2OS cell line to suppress NPC1 expression by shRNA and then transiently transfected these cells with 60 different NPC1 mutant constructs. The mutant NPC1 did not significantly reduce cholesterol accumulation, but approximately 85% of the mutants showed reduced cholesterol accumulation when treated with vorinostat or panobinostat.

Quantitative Proteomics of Human Fibroblasts with I1061T Mutation in Niemann-Pick C1 (NPC1) Protein Provides Insights into the Disease Pathogenesis.

Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of unesterified cholesterol in the late endosomal/lysosomal compartments. Mutations in the NPC1 protein are implicated in 95% of patients with NPC disease. The most prevalent mutation is the missense mutation I1061T that occurs in ∼ 15-20% of the disease alleles. In our study, an isobaric labeling-based quantitative analysis of proteome of NPC1(I1061T) primary fibroblasts when compared with wild-type cells identified 281 differentially expressed proteins based on stringent data analysis criteria. Gene ontology enrichment analysis revealed that these proteins play important roles in diverse cellular processes such as protein maturation, energy metabolism, metabolism of reactive oxygen species, antioxidant activity, steroid metabolism, lipid localization, and apoptosis. The relative expression level of a subset of differentially expressed proteins (TOR4A, DHCR24, CLGN, SOD2, CHORDC1, HSPB7, and GAA) was independently and successfully substantiated by Western blotting. We observed that treating NPC1(I1061T) cells with four classes of seven different compounds that are potential NPC drugs increased the expression level of SOD2 and DHCR24. We have also shown an abnormal accumulation of glycogen in NPC1(I1061T) fibroblasts possibly triggered by defective processing of lysosomal alpha-glucosidase. Our study provides a starting point for future more focused investigations to better understand the mechanisms by which the reported dysregulated proteins triggers the pathological cascade in NPC, and furthermore, their effect upon therapeutic interventions.

Adding hydrophobicity or positive charges to the cytosolic half of the α-synuclein 3-11 helix increases membrane association and S129 phosphorylation.

The neuronal protein α-synuclein is centrally involved in the neurodegeneration occurring in Parkinson's disease and related synucleinopathies. α-Synuclein's membrane-induced 3-11 helix conformation has a hydrophobic membrane-embedded half and a hydrophilic cytosolic half. Here, we studied the significance of (a) the surprising hydrophobicity of amino-acids at cytosol-exposed helix position 8; (b) the absence of positively charged lysine/arginine from all cytosol-exposed positions (1-5-8-9). We found that (a) further increasing hydrophobicity or adding lysine, but not glutamate, at position 8 augments both membrane interaction and S129 phosphorylation; (b) adding lysines at cytosol-exposed positions 1, 5, 8, or 9 has similar effects. Variants abundantly present in membranes by biochemical fractionation markedly colocalized with transferrin-receptor (an endosomal marker) in immunofluorescence-microscopy, indicating accumulation at vesicle membranes. Thus, we observed a striking correlation between membrane attraction and S129 phosphorylation, relevant for understanding α-synuclein biology in health and disease.

Analysis of DHHC acyltransferases implies overlapping substrate specificity and a two-step reaction mechanism.

Asp-His-His-Cys (DHHC) cysteine-rich domain (CRD) acyltransferases are polytopic transmembrane proteins that are found along the endomembrane system of eukaryotic cells and mediate palmitoylation of peripheral and integral membrane proteins. Here, we address the in vivo substrate specificity of five of the seven DHHC acyltransferases for peripheral membrane proteins by an overexpression approach. For all analysed DHHC proteins we detect strongly overlapping substrate specificity. In addition, we now show acyltransferase activity for Pfa5. More importantly, the DHHC protein Pfa3 is able to trap several substrates at the vacuole. For Pfa3 and its substrate Vac8, we can distinguish two consecutive steps in the acylation reaction: an initial binding that occurs independently of its central cysteine in the DHHC box, but requires myristoylation of its substrate Vac8, and a DHHC-motif dependent acylation. Our data also suggest that proteins can be palmitoylated on several organelles. Thus, the intracellular distribution of DHHC proteins provides an acyltransferase network, which may promote dynamic membrane association of substrate proteins.

Quantitating the epigenetic transformation contributing to cholesterol homeostasis using Gaussian process.

To understand the impact of epigenetics on human misfolding disease, we apply Gaussian-process regression (GPR) based machine learning (ML) (GPR-ML) through variation spatial profiling (VSP). VSP generates population-based matrices describing the spatial covariance (SCV) relationships that link genetic diversity to fitness of the individual in response to histone deacetylases inhibitors (HDACi). Niemann-Pick C1 (NPC1) is a Mendelian disorder caused by >300 variants in the NPC1 gene that disrupt cholesterol homeostasis leading to the rapid onset and progression of neurodegenerative disease. We determine the sequence-to-function-to-structure relationships of the NPC1 polypeptide fold required for membrane trafficking and generation of a tunnel that mediates cholesterol flux in late endosomal/lysosomal (LE/Ly) compartments. HDACi treatment reveals unanticipated epigenomic plasticity in SCV relationships that restore NPC1 functionality. GPR-ML based matrices capture the epigenetic processes impacting information flow through central dogma, providing a framework for quantifying the effect of the environment on the healthspan of the individual.

2-Hydroxypropyl-ß-cyclodextrin is the active component in a triple combination formulation for treatment of Niemann-Pick C1 disease.

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPßCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPßCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPßCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.

Palmitoylation determines the function of Vac8 at the yeast vacuole.

Palmitoylation stably anchors specific proteins to membranes, but may also have a direct effect on the function of a protein. The yeast protein Vac8 is required for efficient vacuole fusion, inheritance and cytosol-to-vacuole trafficking. It is anchored to vacuoles by an N-terminal myristoylation site and three palmitoylation sites, also known as the SH4 domain. Here, we address the role of Vac8 palmitoylation and show that the position and number of substrate cysteines within the SH4 domain determine the vacuole localization of Vac8: stable vacuole binding of Vac8 requires two cysteines within the N-terminus, regardless of the combination. Importantly, our data suggest that palmitoylation adds functionality to Vac8 beyond simple localization. A mutant Vac8 protein, in which the palmitoylation sites were replaced by a stretch of basic residues, still localizes to vacuole membranes and functions in cytosol-to-vacuole transport, but can only complement the function of Vac8 in morphology and inheritance if it also contains a single cysteine within the SH4 domain. Our data suggest that palmitoylation is not a mere hydrophobic anchor required solely for localization, but influences the protein function(s).

The DHHC protein Pfa3 affects vacuole-associated palmitoylation of the fusion factor Vac8.

Vacuole biogenesis depends on specific targeting and retention of peripheral membrane proteins. At least three palmitoylated proteins are found exclusively on yeast vacuoles: the fusion factor Vac8, the kinase Yck3, and a novel adaptor protein implicated in microautophagy, Meh1. Here, we analyze the role that putative acyltransferases of the DHHC family play in their localization and function. We find that Pfa3/Ynl326c is required for efficient localization of Vac8 to vacuoles in vivo, while Yck3 or Meh1 localization is not impaired in any of the seven DHHC deletions. Vacuole-associated Vac8 appears to be palmitoylated in a pfa3 mutant, but this population is refractive to further palmitoylation on isolated vacuoles. Vacuole morphology and inheritance, which both depend on Vac8 palmitoylation, appear normal, although there is a reduction in vacuole fusion. Interestingly, Pfa3 is required for the vacuolar localization of not only an SH4 domain that is targeted by myristate/palmitate (as in Vac8) but also one that is targeted by a myristate/basic stretch (as in Src). Our data indicate that Pfa3 has an important but not exclusive function for Vac8 localization to the vacuole.