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 NPC

Cholesterol Pathways Affected by Small Molecules That Decrease Sterol Levels in Niemann-Pick Type C Mutant Cells

Niemann-Pick type C (NPC) disease is a genetically inherited multi-lipid storage disorder with impaired efflux of cholesterol from lysosomal storage organelles.The effect of screen-selected cholesterol lowering compounds on the major sterol pathways was studied in CT60 mutant CHO cells lacking NPC1 protein. Each of the selected chemicals decreases cholesterol in the lysosomal storage organelles of NPC1 mutant cells through one or more of the following mechanisms: increased cholesterol efflux from the cell, decreased uptake of low-density lipoproteins, and/or increased levels of cholesteryl esters. Several chemicals promote efflux of cholesterol to extracellular acceptors in both non-NPC and NPC1 mutant cells. The uptake of low-density lipoprotein-derived cholesterol is inhibited by some of the studied compounds.Results herein provide the information for prioritized further studies in identifying molecular targets of the chemicals. This approach proved successful in the identification of seven chemicals as novel inhibitors of lysosomal acid lipase (Rosenbaum et al, Biochim. Biophys. Acta. 2009, 1791:1155-1165)

Role of STARD4 and NPC1 in intracellular sterol transport

Cholesterol plays an important role in determining the biophysical properties of membranes in mammalian cells, and the concentration of cholesterol in membranes is tightly regulated. Cholesterol moves among membrane organelles by a combination of vesicular and nonvesicular transport pathways, but the details of these transport pathways are not well understood. In this review, we discuss the mechanisms for nonvesicular sterol transport with an emphasis on the role of STARD4, a small, soluble, cytoplasmic sterol transport protein. STARD4 can rapidly equilibrate sterol between membranes, especially membranes with anionic lipid headgroups. We also discuss the sterol transport in late endosomes and lysosomes, which is mediated by a soluble protein, NPC2, and a membrane protein NPC1. Homozygous mutations in these proteins lead to a lysosomal lipid storage disorder, Niemann-Pick disease type C. Many of the disease causing mutations in NPC1 are associated with degradation of the mutant NPC1 proteins in the endoplasmic reticulum. Several histone deacetylase inhibitors have been found to rescue the premature degradation of the mutant NPC1 proteins, and one of these is now in a small clinical trial.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Efficiency of immunotoxin cytotoxicity is modulated by the intracellular itinerary.

Pseudomonas exotoxin-based immunotoxins, including LMB-2 (antiTac(F(v))-PE38), are proposed to traffic to the trans-Golgi network (TGN) and move by a retrograde pathway to the endoplasmic reticulum, where they undergo translocation to the cytoplasm, a step that is essential for cytotoxicity. The retrograde transport pathways used by LMB-2 are not completely understood, so it is unclear if transit through specific organelles is critical for maximal cytotoxic activity. In this study, we used Chinese hamster ovary (CHO) cell lines that express chimeric constructs of CD25, the Tac antigen, attached to the cytoplasmic domain of the TGN-targeted transmembrane proteins, TGN38 and furin. These chimeras are both targeted to the TGN, but the itineraries they follow are quite different. LMB-2 was incubated with the two cell lines, and the efficiency of cell killing was determined using cell viability and cytotoxicity assays. LMB-2 that is targeted through the endocytic recycling compartment to the TGN via Tac-TGN38 kills the cells more efficiently than immunotoxins delivered through the late endosomes by Tac-furin. Although the processing to the 37 kDa active fragment was more efficient in Tac-furin cells than in Tac-TGN38 cells, this was not associated with enhanced cytotoxicity - presumably because the toxin was also degraded more rapidly in these cells. These data indicate that trafficking through specific organelles is an important factor modulating toxicity by LMB-2

Comparison of cytotoxic effects and immunotoxin specificity in Tac-receptor expressing cells.

<p>Tac-TGN38 and Tac-furin cells were treated with up to 10 ng/ml of LMB-2. After a 24 hour incubation with immunotoxin, cells were washed and the cells remaining in the well were used for <b>A</b>) Hoechst-based cell count assay or <b>B</b>) LDH assay as described in Methods. Panels A and B show % cytotoxicity plotted against the concentration of LMB-2. The data points represent the cytotoxicity values averaged over several experiments, cell count assay (n = 9), LDH assay (n = 8). The standard error of the mean is shown. <b>C</b>) Tac-TGN38 and Tac-furin cells were seeded and then treated with up to 10 ng/ml of LMB-2 or Erb38 one day after plating. After a 24 hour incubation with immunotoxin, cytotoxicity was assessed by a cell count assay. The number of cells remaining after toxin treatment is shown as a fraction of cells incubated without toxin (n = 5). The standard error of the mean is shown.</p

Effect of lysosomal protease inhibitors on LMB-2 cytotoxicity.

<p>Tac-furin cells were pretreated with 100 µM Leupeptin (Leu), E-64, Chymostatin (Chy), a combination of all three inhibitors or DMSO control for 24 hours. Cells were subsequently treated with 1 ng/ml LMB-2 in the continued presence of inhibitors. After a 24 hour incubation with immunotoxin, cells were counted using the Hoechst-based assay. The number of cells remaining after toxin treatment is shown as a fraction of cells incubated without toxin normalized to the DMSO control (n = 6 wells). The standard error of the mean is shown. Similar experiments were performed twice and one representative data set is shown.</p

t_1/2 for degradation were based on exponential decay curve fitting equation (see figure 5 legend for equation).

<p>The EC₅₀'s were also calculated after correcting for Tac expression and fitted in SigmaPlot using a four parameter logistic model (Hill-Slope model) with the equation y = y₀+(a/(1+(x/x₀)^b)) where b = slope, y = concentration, x₀ = EC₅₀ and a = maximum response. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047320#pone.0047320-Dudley1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047320#pone.0047320-Khinkis1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047320#pone.0047320-LaurenceMLevasseur1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047320#pone.0047320-NIH1" target="_blank">[38]</a>. The p-values for Tac-TGN38: Tac-furin were 0.03.</p