A molecular target for viral killer toxin: TOK1 potassium channels.

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes

Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking

Background Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. Methodology/Principal Findings Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. Conclusions/Significance These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation

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)

Clues to Neuro-Degeneration in Niemann-Pick Type C Disease from Global Gene Expression Profiling

BACKGROUND: Niemann-Pick Type C (NPC) disease is a neurodegenerative disease that is characterized by the accumulation of cholesterol and glycosphingolipids in the late endocytic pathway. The majority of NPC cases are due to mutations in the NPC1 gene. The precise function of this gene is not yet known. METHODOLOGY/PRINCIPAL FINDINGS: Using cDNA microarrays, we analyzed the genome-wide expression patterns of human fibroblasts homozygous for the I1061T NPC1 mutation that is characterized by a severe defect in the intracellular processing of low density lipoprotein-derived cholesterol. A distinct gene expression profile was identified in NPC fibroblasts from different individuals when compared with fibroblasts isolated from normal subjects. As expected, NPC1 mutant cells displayed an inappropriate homeostatic response to accumulated intracellular cholesterol. In addition, a number of striking parallels were observed between NPC disease and Alzheimer's disease. CONCLUSIONS/SIGNIFICANCE: Many genes involved in the trafficking and processing of amyloid precursor protein and the microtubule binding protein, tau, were more highly expressed. Numerous genes important for membrane traffic and the cellular regulation of calcium, metals and other ions were upregulated. Finally, NPC fibroblasts exhibited a gene expression profile indicative of oxidative stress. These changes are likely contributors to the pathophysiology of Niemann-Pick Type C disease

HacA-Independent Functions of the ER Stress Sensor IreA Synergize with the Canonical UPR to Influence Virulence Traits in Aspergillus fumigatus

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacAi, or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireAΔ10. Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus

A molecular target for viral killer toxin: TOK1 potassium channels.

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes

Human Apolipoprotein-b Signal Sequence Variants Confer a Secretion-defective Phenotype When Expressed in Yeast

Hyperlipidemia arises from a disturbance in the balance between production and degradation of Lipoprotein particles. Variation in the secretion of human apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins, directly affects this homeostasis. Naturally occurring apoB signal peptide variants (associated with hypertriglyceridemia, altered postprandial lipid metabolism, or atherosclerosis) were investigated for their ability to direct transit through the secretion pathway. Three apoB signal peptide iso forms were fused to the secretory protein, invertase, and expressed in yeast. A deletion or insertion in the hydrophobic core of the signal peptide mediated inefficient translocation into the endoplasmic reticulum and was secretion-defective, relative to the common 27-residue isoform. Additionally, the insertion apoB isoform was observed in yeast to confer a defect in export from the endoplasmic reticulum. Secretion of the apoB signal peptide-invertase fusions responded positively to an inhibitor of calpain type I proteases. These observations suggest that the apoB signal peptide plays a role in determining the levels of apoB degradation and secretion and, thus, hyperlipidemia