No abstract

Lipid rafts have been identified in the membranes of mammalian cells, the yeast Saccharomyces cerevisiae, and the pathogenic fungus Candida albicans. Formed by a lateral association of sphingolipids and sterols, rafts concentrate proteins carrying a glycosylphosphatidylinositol (GPI) anchor. We report the isolation of membranes with the characteristics of rafts from the fungal pathogen Cryptococcus neoformans. These characteristics include insolubility in Triton X-100 (TX100) at 4°C, more-buoyant density within a sucrose gradient than the remaining membranes, and threefold enrichment with sterols. The virulence determinant phospholipase B1 (PLB1), a GPI-anchored protein, was highly concentrated in raft membranes and could be displaced from them by treatment with the sterol-sequestering agent methyl-␤-cyclodextrin (M␤CD). Phospholipase B enzyme activity was inhibited in the raft environment and increased 15-fold following disruption of rafts with TX100 at 37°C. Treatment of viable cryptococcal cells in suspension with M␤CD also released PLB1 protein and enzyme activity, consistent with localization of PLB1 in plasma membrane rafts prior to secretion. The antioxidant virulence factor Cu/Zn superoxide dismutase (SOD1) was concentrated six-to ninefold in raft membrane fractions compared with nonraft membranes, whereas the cell wall-associated virulence factor laccase was not detected in membranes. We hypothesize that raft membranes function to cluster certain virulence factors at the cell surface to allow efficient access to enzyme substrate and/or to provide rapid release to the external environment.

Phospholipase B (Plb1) is secreted by pathogenic fungi and is a proven virulence determinant in Cryptococcus neoformans. Cell-associated Plb1 is presumptively involved in fungal membrane biogenesis and remodelling. We have also identified it in cryptococcal cell walls. Motif scanning programs predict that Plb1 is attached to cryptococcal membranes via a glycosylphosphatidylinositol (GPI) anchor, which could regulate Plb1 export and secretion. A functional GPI anchor was identified in cellassociated Plb1 by (G)PI-specific phospholipase C (PLC)-induced release of Plb1 from strain H99 membrane rafts and inhibition of GPI anchor synthesis by YW3548, which prevented Plb1 secretion and transport to membranes and cell walls. Plb1 containing ␤-1,6-linked glucan was released from H99 (wildtype strain) cell walls by ␤-1,3 glucanase, consistent with covalent attachment of Plb1 via ␤-1,6-linked glucans to ␤-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained ␤-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, ⌬PLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of ⌬PLB1 was unaffected by caffeine, excluding an effect of Plb1 on cell wall biogenesis-related signaling pathways. Environmental (heat) stress caused Plb1 accumulation in cell walls, with loss from membranes and reduced secretion, further supporting the importance of Plb1 in cell wall integrity. This is the first demonstration that Plb1 contributes to fungal survival by maintaining cell wall integrity and that the cell wall is a source of secreted enzyme.

No abstract

SummaryPhospholipase B1 (Plb1) is secreted after release from its glycosylphosphatidylinositol anchor and is implicated in initiation and dissemination of infection of the pathogenic fungus, Cryptococcus neoformans. To investigate the role of phosphatidylinositolspecific phospholipase C (PI-PLC) in Plb1 secretion, we identified two putative PI-PLC-encoding genes in C. neoformans var. grubii (PLC1 and PLC2), and created Dplc1 and Dplc2 deletion mutants. In Dplc1, which expressed less PI-PLC activity than wild type (WT), three major cryptococcal virulence traits, Plb1 secretion, melanin production and growth at host temperature (37°C) were abolished and absence of Plb1 secretion coincided with Plb1 accumulation in plasma membranes. In addition, Dplc1 cell walls were defective, as indicated by cell clumping and irregular morphology, slower growth and an inability to activate mitogen-activated protein kinase (MAPK) in the presence of cell wall-perturbing agents. In contrast to Dplc2, which was as virulent as WT, Dplc1 was avirulent in mice and exhibited attenuated killing of Caenorhabditis elegans at 25°C, demonstrating that mechanism(s) independent of the 37°C growth defect contribute to the virulence composite. We conclude that Plc1 is a central regulator of cryptococcal virulence, acting through the protein kinase C/MAPK pathway, that it regulates release of Plb1 from the plasma membrane and is a candidate antifungal drug target.

Iron plays a crucial part in proliferation while iron deficiency results in G 1 /S arrest, DNA damage, and apoptosis. However, the precise role of iron in cell cycle control remains unclear. We showed that iron depletion using the iron chelators, desferrioxamine (DFO), or 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311), increased the mRNA levels of the growth arrest and DNA damage 45␣ gene, GADD45␣ (Darnell, G. and Richardson, D. R. (1999) Blood 94, 781-792). In this study, we examined the effect of iron depletion on up-regulating GADD family members involved in growth control, including cell cycle arrest, apoptosis, and DNA repair, making them therapeutic targets for tumor suppression. We showed the GADD family members were up-regulated by cellular iron depletion. Further, upregulation of GADD45␣ after iron deprivation was independent of hypoxia-inducible factor-1␣ (HIF-1␣), octamer-1 (Oct-1), p53 and early growth response 1 (Egr1). We then analyzed the regulatory elements responsible for iron depletion-mediated regulation of GADD45␣ and identified the specific transcription factor/s involved. This region was within ؊117 bp and ؊81 bp relative to the start codon where the consensus sequences of three transcription factors are located: the CCAAT-binding factor/nuclear factor-Y (NF-Y), the stabilizing molecule v-MYB and the enhancer, CCAAT enhancer-binding protein (CEBP␣). Mutation analysis, shRNA studies, Western blotting, and electrophoretic mobility shift assays led to the identification of NF-Y in the transcriptional up-regulation of GADD45␣ after iron depletion. Furthermore, like GADD45␣, NF-YA was upregulated after iron chelation and down-regulated by iron supplementation. These results are important for understanding the mechanisms of iron depletion-mediated cell cycle arrest, DNA damage repair, and apoptosis.Iron is vital for proliferation and multiple mechanisms are involved in the anti-tumor activity of iron chelators whose effect induces cell cycle arrest and apoptosis (1, 2). Iron depletion results in: 1) inhibition of the iron-containing enzyme, ribonucleotide reductase, which is critical for DNA synthesis (1); 2) increased expression of the growth and metastasis suppressor, N-myc downstream-regulated gene 1 (3); 3) up-regulation of p53 (4); 4) decreased cyclin D1 expression (5) that is required for G 1 /S progression; 5) down-regulation of p21 CIP1/WAF1 protein in some cell types (5) that may induce apoptosis; 6) down-regulation of Bcl-2 and up-regulation of pro-apoptotic Bax (6, 7), and 7) up-regulation of cell division cycle 14A, which acts on substrates important for cell cycle progression (e.g. p27 kip1 and cyclin E) (8). The current investigation focuses on five members of the growth arrest and DNA damage (GADD) 2 family of genes, namely: GADD34, GADD45␣, GADD45␤, GADD45␥, and GADD153. The expression of these genes is often increased when cells are subjected to stress such as nutrient deprivation (9 -11) or exposure to DNA-damaging agents (12), which may cause cell cycle arrest and/or...

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the "reprogramming" of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.

Organic chemistry Z 0200Thiosemicarbazones from the Old to New: Iron Chelators that Are More than Just Ribonucleotide Reductase Inhibitors -[183 refs.]. -(YU, Y.; KALINOWSKI, D. S.; KOVACEVIC, Z.; SIAFAKAS, A. R.; JANSSON, P. J.; STEFANI, C.; LOVEJOY, D. B.; SHARPE, P. C.; BERNHARDT, P. V.; RICHARDSON*, D. R.; J.