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Sphingosine kinase A is a pleiotropic and essential enzyme for Leishmania survival and virulence.

ABSTRACT: Sphingosine kinase is a key enzyme in sphingolipid metabolism, catalysing the conversion of sphingosine or dihydrosphingosine into sphingosine-1-phosphate or dihydrosphingosine-1-phosphate respectively. In mammals, sphingosine-1-phosphate is a powerful signalling molecule regulating cell growth, differentiation, apoptosis and immunity. Functions of sphingosine kinase or sphingosine-1-phosphate in pathogenic protozoans are virtually unknown. While most organisms possess two closely related sphingosine kinases, only one sphingosine kinase homologue (SKa) can be identified in Leishmania, which are vector-borne protozoan parasites responsible for leishmaniasis. Leishmania?SKa is a large, cytoplasmic enzyme capable of phosphorylating both sphingosine and dihydrosphingosine. Remarkably, deletion of SKa leads to catastrophic defects in both the insect stage and mammalian stage of Leishmania parasites. Genetic and biochemical analyses demonstrate that proper expression of SKa is essential for Leishmania parasites to remove toxic metabolites, to survive stressful conditions, and to cause disease in mice. Therefore, SKa is a pleiotropic enzyme with vital roles throughout the life cycle of Leishmania. The essentiality of SKa and its apparent divergence from mammalian counterparts suggests that this enzyme can be selectively targeted to reduce Leishmania infection.

SUBMITTER: Zhang O

PROVIDER: S-EPMC3938578 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Sphingosine kinase A is a pleiotropic and essential enzyme for Leishmania survival and virulence.

Zhang Ou O Hsu Fong-Fu FF Xu Wei W Pawlowic Mattie M Zhang Kai K

Molecular microbiology 20130916 3

Sphingosine kinase is a key enzyme in sphingolipid metabolism, catalysing the conversion of sphingosine or dihydrosphingosine into sphingosine-1-phosphate or dihydrosphingosine-1-phosphate respectively. In mammals, sphingosine-1-phosphate is a powerful signalling molecule regulating cell growth, differentiation, apoptosis and immunity. Functions of sphingosine kinase or sphingosine-1-phosphate in pathogenic protozoans are virtually unknown. While most organisms possess two closely related sphing ...[more]

PMID: 23980754

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Project description:Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.

Project description:BackgroundCytochrome P450s (CYP450s) are hemoproteins catalysing diverse biochemical reactions important for metabolism of xenobiotics and synthesis of physiologically important compounds such as sterols. Therefore, they are functionally important for survival of invading pathogens. One such opportunistic pathogen Leishmania donovani causes visceral leishmaniasis worldwide, which is an important public health problem due to significant disease burden. The parasite genome database, Gene DB, annotates 3 CYP450s in Leishmania, however, the functional role of cytochrome P450 enzymes in Leishmania spp. remains elusive.Methodology/principal findingsA CYP450-like gene cloned from Leishmania donovani was identified as a novel CYP450, the CYP5122A1. Upon co-localization with organelle specific markers, CYP5122A1 distribution was shown to be localized in the promastigote ER, mitochondria and the glycosomes. Replacement of one allele of CYP5122A1 with either neomycin or hygromycin gene by homologous recombination in Leishmania promastigotes induced substantial reduction of CYP5122A1 expression. These parasites showed impaired growth, lower mitochondrial Ca(2+) and membrane potential resulting in low ATP generation. Also, these parasites were less infective in vitro and in vivo than their wild-type counterparts as assessed by incubation of Leishmania promastigotes with macrophages in vitro as well as through administration of parasites into hamsters. The HKOs were more susceptible to drugs like miltefosine and antimony, but showed reduced sensitivity to amphotericin B. Removal of two alleles of CYP5122A1 did not allow the parasites to survive. The mutant parasites showed 3.5 times lower ergosterol level as compared to the wild-type parasites when estimated by Gas chromatography/mass spectrometry. Complementation of CYP5122A1 through episomal expression of protein by using pXG-GFP+2 vector partially rescued CYP5122A1 expression and restored ergosterol levels by 1.8 times. Phenotype reversal included restored growth pattern and lesser drug susceptibility.Conclusions/significanceIn summary, this study establishes CYP5122A1 as an important molecule linked to processes like cell growth, infection and ergosterol biosynthesis in Leishmania donovani.

Project description:BackgroundSphingosine kinase-1 (SphK1) is an oncogenic lipid kinase notably involved in response to anticancer therapies in prostate cancer. Androgens regulate prostate cancer cell proliferation, and androgen deprivation therapy is the standard of care in the management of patients with advanced disease. Here, we explored the role of SphK1 in the regulation of androgen-dependent prostate cancer cell growth and survival.Methodology/principal findingsShort-term androgen removal induced a rapid and transient SphK1 inhibition associated with a reduced cell growth in vitro and in vivo, an event that was not observed in the hormono-insensitive PC-3 cells. Supporting the critical role of SphK1 inhibition in the rapid effect of androgen depletion, its overexpression could impair the cell growth decrease. Similarly, the addition of dihydrotestosterone (DHT) to androgen-deprived LNCaP cells re-established cell proliferation, through an androgen receptor/PI3K/Akt dependent stimulation of SphK1, and inhibition of SphK1 could markedly impede the effects of DHT. Conversely, long-term removal of androgen support in LNCaP and C4-2B cells resulted in a progressive increase in SphK1 expression and activity throughout the progression to androgen-independence state, which was characterized by the acquisition of a neuroendocrine (NE)-like cell phenotype. Importantly, inhibition of the PI3K/Akt pathway--by negatively impacting SphK1 activity--could prevent NE differentiation in both cell models, an event that could be mimicked by SphK1 inhibitors. Fascinatingly, the reversability of the NE phenotype by exposure to normal medium was linked with a pronounced inhibition of SphK1 activity.Conclusions/significanceWe report the first evidence that androgen deprivation induces a differential effect on SphK1 activity in hormone-sensitive prostate cancer cell models. These results also suggest that SphK1 activation upon chronic androgen deprivation may serve as a compensatory mechanism allowing prostate cancer cells to survive in androgen-depleted environment, giving support to its inhibition as a potential therapeutic strategy to delay/prevent the transition to androgen-independent prostate cancer.

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Sphingosine kinase A is a pleiotropic and essential enzyme for Leishmania survival and virulence.

Publications

Sphingosine kinase A is a pleiotropic and essential enzyme for Leishmania survival and virulence.

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