Fannie W. Chen

Ribosomal proteins in cell proliferation and apoptosis.

Ribosomal proteins have the complex task of coordinating protein biosynthesis to maintain cell homeostasis and survival. Recent evidence suggests that a number of ribosomal proteins have secondary functions independent of their involvement in protein biosynthesis. A number of these proteins function as cell proliferation regulators and in some instances as inducers of cell death. Specifically, expression of human ribosomal protein L13a has been shown to induce apoptosis, presumably by arresting cell growth in the G2/M phase of the cell cycle. In addition, inhibition of expression of L13a induces apoptosis in target cells, suggesting that this protein is necessary for cell survival. Similar results have been obtained in the yeast Saccharomyces cerevisiae, where inactivation of the yeast homologues of L13a, rp22 and rp23, by homologous recombination results in severe growth retardation and death. In addition, a closely related ribosomal protein, L7, arrests cells in G1 and also induces apoptosis. Thus, it appears that a group of ribosomal proteins may function as cell cycle checkpoints and compose a new family of cell proliferation regulators.

Cyclodextrin Induces Calcium-Dependent Lysosomal Exocytosis

Cyclodextrins (CDs) have long been used to manipulate cellular cholesterol levels both in vitro and in vivo, but their direct effects at a cellular level are not well characterized. Recently, CDs have garnered much interest because of their ability to clear stored cholesterol from Niemann Pick Type C (NPC) cells and markedly prolong the life of NPC1 disease mice. Here, we investigate the hypothesis that treatment with 2-hydroxypropyl- β-cyclodextrin (HPB-CD) stimulates lysosomal exocytosis in a calcium-enhanced manner. We propose that this exocytosis is the mechanism by which HPB-CD ameliorates the endolysosomal cholesterol storage phenotype in NPC cells. These findings have significant implications for the use of HPB-CD in biochemical assays and data interpretation as well as for their use for the treatment for NPC and other disorders.

An"exacerbate-reverse" strategy in yeast identifies histone deacetylase inhibition as a correction for cholesterol and sphingolipid transport defects in human niemann-pick type C disease

Niemann-Pick type C (NP-C) disease is a fatal lysosomal lipid storage disorder for which no effective therapy exists. A genome-wide, conditional synthetic lethality screen was performed using the yeast model of NP-C disease during anaerobiosis, an auxotrophic condition that requires yeast to utilize exogenous sterol. We identified 12 pathways and 13 genes as modifiers of the absence of the yeast NPC1 ortholog (NCR1) and quantified the impact of loss of these genes on sterol metabolism in ncr1Δ strains grown under viable aerobic conditions. Deletion of components of the yeast NuA4 histone acetyltransferase complex in ncr1Δ strains conferred anaerobic inviability and accumulation of multiple sterol intermediates. Thus, we hypothesize an imbalance in histone acetylation in human NP-C disease. Accordingly, we show that the majority of the 11 histone deacetylase (HDAC) genes are transcriptionally up-regulated in three genetically distinct fibroblast lines derived from patients with NP-C disease. A clinically approved HDAC inhibitor (suberoylanilide hydroxamic acid) reverses the dysregulation of the majority of the HDAC genes. Consequently, three key cellular diagnostic criteria of NP-C disease are dramatically ameliorated as follows: lysosomal accumulation of both cholesterol and sphingolipids and defective esterification of LDL-derived cholesterol. These data suggest HDAC inhibition as a candidate therapy for NP-C disease. We conclude that pathways that exacerbate lethality in a model organism can be reversed in human cells as a novel therapeutic strategy. This “exacerbate-reverse” approach can potentially be utilized in any model organism for any disease.

MLN64 Transport to the Late Endosome Is Regulated by Binding to 14-3-3 via a Non-canonical Binding Site

MLN64 is an integral membrane protein localized to the late endosome and plasma membrane that is thought to function as a mediator of cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria. The protein consists of two distinct domains: an N-terminal membrane-spanning domain that shares homology with the MENTHO protein and a C-terminal steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain that binds cholesterol. To further characterize the MLN64 protein, full-length and truncated proteins were overexpressed in cells and the effects on MLN64 trafficking and endosomal morphology were observed. To gain insight into MLN64 function, affinity chromatography and mass spectrometric techniques were used to identify potential MLN64 interacting partners. Of the 15 candidate proteins identified, 14-3-3 was chosen for further characterization. We show that MLN64 interacts with 14-3-3 in vitro as well as in vivo and that the strength of the interaction is dependent on the 14-3-3 isoform. Furthermore, blocking the interaction through the use of a 14-3-3 antagonist or MLN64 mutagenesis delays the trafficking of MLN64 to the late endosome and also results in the dispersal of endocytic vesicles to the cell periphery. Taken together, these studies have determined that MLN64 is a novel 14-3-3 binding protein and indicate that 14-3-3 plays a role in the endosomal trafficking of MLN64. Furthermore, these studies suggest that 14-3-3 may be the link by which MLN64 exerts its effects on the actin-mediated endosome dynamics.

PKC activation in Niemann pick C1 cells restores subcellular cholesterol transport.

Activation of protein kinase C (PKC) has previously been shown to ameliorate the cholesterol transport defect in Niemann Pick Type C1 (NPC1) cells, presumably by increasing the soluble levels of one of its substrates, vimentin. This activity would then restore the vimentin cycle in these cells and allow vimentin-dependent retrograde transport to proceed. Here, we further investigate the effects of PKC activation in NPC1 cells by evaluating different isoforms for their ability to solubilize vimentin and correct the NPC1 cholesterol storage phenotype. We also examine the effects of PKC activators, including free fatty acids and the PKC-specific activator diazoxide, on the NPC1 disease phenotype. Our results indicate that PKC isoforms α, βII, and ε have the greatest effects on vimentin solubilization. Furthermore, expression or activation of PKCε in NPC1 cells dramatically reduces the amount of stored cholesterol and restores cholesterol transport out of endocytic vesicles. These results provide further support for the contribution of PKCs in NPC1 disease pathogenesis and suggest that PKCs may be targeted in future efforts to develop therapeutics for NPC1 disease.

Normalization of hepatic homeostasis in the Npc1nmf164 mouse model of Niemann-Pick type C disease treated with the histone deacetylase inhibitor vorinostat

Niemann-Pick type C (NP-C) disease is a fatal genetic lipidosis for which there is no Food and Drug Administration (FDA)-approved therapy. Vorinostat, an FDA-approved inhibitor of histone deacetylases, ameliorates lysosomal lipid accumulation in cultured NP-C patient fibroblasts. To assess the therapeutic potential of histone deacetylase inhibition, we pursued these in vitro observations in two murine models of NP-C disease. Npc1nmf164 mice, which express a missense mutation in the Npc1 gene, were treated intraperitoneally, from weaning, with the maximum tolerated dose of vorinostat (150 mg/kg, 5 days/week). Disease progression was measured via gene expression, liver function and pathology, serum and tissue lipid levels, body weight, and life span. Transcriptome analyses of treated livers indicated multiple changes consistent with reversal of liver dysfunction that typifies NP-C disease. Significant improvements in liver pathology and function were achieved by this treatment regimen; however, NPC1 protein maturation and levels, disease progression, weight loss, and animal morbidity were not detectably altered. Vorinostat concentrations were >200 μm in the plasma compartment of treated animals but were almost 100-fold lower in brain tissue. Apolipoprotein B metabolism and the expression of key components of lipid homeostasis in primary hepatocytes from null (Npc1−/−) and missense (Npc1nmf164) mutant mice were altered by vorinostat treatment, consistent with a response by these cells independent of the status of the Npc1 locus. These results suggest that HDAC inhibitors have utility to treat visceral NP-C disease. However, it is clear that improved blood-brain barrier penetration will be required to alleviate the neurological symptoms of human NP-C disease.