The Biosynthetic Incorporation of the Intact Leucine Skeleton into Sterol by the Trypanosomatid Leishmania mexicana

Ginger, Michael L.; Chance, Michaël La; Sadler, Ian H.; Goad, L. John

doi:10.1074/jbc.m006850200

Cited by 68 publications

(59 citation statements)

References 42 publications

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“…At least some species of Leishmania seem to be able to overcome their effect, possibly by using the host's steroids. On the other hand, besides the synthesis of ergosterol instead of cholesterol, there are other peculiar steps in the isoprenoid pathway in Leishmania: leucine is the main precursor of mevalonate, as opposed to acetyl coenzyme A in mammals (14); dolichols containing 11 to 12 isoprene units are synthesized instead of 19 to 20 observed in human dolichol. These differences could be explored in the identification of new targets for chemotherapy.…”

Section: Discussionmentioning

confidence: 99%

Antileishmanial Activity of the Terpene Nerolidol

Arruda

D’Alexandri

Katzin

et al. 2005

Antimicrob Agents Chemother

169

104

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The activity of nerolidol, a sesquiterpene used as a food-flavoring agent and currently under testing as a skin penetration enhancer for the transdermal delivery of therapeutic drugs, was evaluated against Leishmania species. Nerolidol inhibited the growth of Leishmania amazonensis, L. braziliensis, and L. chagasi promastigotes and L. amazonensis amastigotes with in vitro 50% inhibitory concentrations of 85, 74, 75, and 67 M, respectively. The treatment of L. amazonensis-infected macrophages with 100 M nerolidol resulted in 95% reduction in infection rates. Inhibition of isoprenoid biosynthesis, as shown by reduced incorporation of [2-14 C]mevalonic acid (MVA) or [1-14 C]acetic acid precursors into dolichol, ergosterol, and ubiquinone, was observed in nerolidol-treated promastigotes. This drug effect can be attributed to the blockage of an early step in the mevalonate pathway, since incorporation of the precursor [1(n)-3 H]farnesyl pyrophosphate in polyisoprenoids is not inhibited by nerolidol. L. amazonensis-infected BALB/c mice were treated with intraperitoneal doses of 100 mg/kg/day for 12 days or topically with 5 or 10% ointments for 4 weeks. Significant reduction of lesion sizes in nerolidol treated mice was observed for both treatment routes. However, long-term follow up indicated that the disease was not cured in this highly susceptible animal model. Nonetheless, the in vitro activity of nerolidol against these parasites may prove a useful tool for the development of new drugs for the treatment of leishmaniasis. In addition, biosynthesis of dolichols with 11 and 12 isoprene units was identified in Leishmania, as described for other trypanosomatids and Apicomplexa.

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Section: Discussionmentioning

confidence: 99%

Antileishmanial Activity of the Terpene Nerolidol

Arruda

D’Alexandri

Katzin

et al. 2005

Antimicrob Agents Chemother

169

104

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“…Little is known for trypanosomatids about the localization of other proteins of the mevalonate pathway. The description of HMG-CoA reductase as a mitochondrial enzyme (40) has interest in relation to studies performed with Leishmania that established leucine as a major substrate for the generation of sterols (18). The latter would occur through degradation of the amino acid to HMG-CoA, a process that has been described to be mitochondrial in different cell types, although this issue remains to be established for the Trypanosomatidae.…”

Section: Discussionmentioning

confidence: 99%

Farnesyl Diphosphate Synthase Is a Cytosolic Enzyme inLeishmania majorPromastigotes and Its Overexpression Confers Resistance to Risedronate

et al. 2006

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Farnesyl diphosphate synthase is the most likely molecular target of aminobisphosphonates (e.g., risedronate), a set of compounds that have been shown to have antiprotozoal activity both in vitro and in vivo. This protein, together with other enzymes involved in isoprenoid biosynthesis, is an attractive drug target, yet little is known about the compartmentalization of the biosynthetic pathway. Here we show the intracellular localization of the enzyme in wild-type Leishmania major promastigote cells and in transfectants overexpressing farnesyl diphosphate synthase by using purified antibodies generated towards a homogenous recombinant Leishmania major farnesyl diphosphate synthase protein. Indirect immunofluorescence, together with immunoelectron microscopy, indicated that the enzyme is mainly located in the cytoplasm of both wild-type cells and transfectants. Digitonin titration experiments also confirmed this observation. Hence, while the initial step of isoprenoid biosynthesis catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase is located in the mitochondrion, synthesis of farnesyl diphosphate by farnesyl diphosphate synthase is a cytosolic process. Leishmania major promastigote transfectants overexpressing farnesyl diphosphate synthase were highly resistant to risedronate, and the degree of resistance correlated with the increase in enzyme activity. Likewise, when resistance was induced by stepwise selection with the drug, the resulting resistant promastigotes exhibited increased levels of farnesyl diphosphate synthase. The overproduction of protein under different conditions of exposure to risedronate further supports the hypothesis that this enzyme is the main target of aminobisphosphonates in Leishmania cells.

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“…For isolation of amastigote stages, BALB/c mice were subcutaneously infected with 5 ϫ 10 6 stationary phase promastigotes, and lesions were harvested after 3-5 wk. Partial inhibition of sphingolipid and sterol biosynthesis was achieved by incubating L. major promastigotes in medium containing 1 g/ml myriosin (Sigma, St. Louis, MO) or 2 g/ml ketoconazole (Sigma), respectively (Ginger et al, 2001;Ralton et al, 2002).…”

Section: Cell Culturementioning

confidence: 99%

“…To investi- gate whether the flagellar localization of SMP-1 is dependent on the lipid composition of the flagellar DRM, L. major promastigotes were treated with myriocin, an inhibitor of serine: palmitoyl-CoA transferase, the first committed enzyme in sphingolipid biosynthesis (Miyake et al, 1995) and ketoconazole, an inhibitor of sterol 14Ј-demethylase (Beach et al, 1988). At the concentrations used, these inhibitors cause a reduction of 70 and 50% in inositolphosphoceramide and sterol levels, respectively (Ginger et al, 2001;Ralton et al, 2002;our unpublished results). Myriocin-treatment alone had no effect on the growth rate of L. major promastigotes (our unpublished results), the solubility of SMP-1 and GIPLs in cold Triton X-100 ( Figure 7A) or the flagellar localization of SMP-1 ( Figure 7B).…”

Section: Flagellar Localization Of Smp-1 Is Not Affected By Changes Imentioning

confidence: 99%

SMP-1, a Member of a New Family of Small Myristoylated Proteins in Kinetoplastid Parasites, Is Targeted to the Flagellum Membrane inLeishmania

Tull

Vince

Callaghan

et al. 2004

MBoC

101

118

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The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.

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The Biosynthetic Incorporation of the Intact Leucine Skeleton into Sterol by the Trypanosomatid Leishmania mexicana

Cited by 68 publications

References 42 publications

Antileishmanial Activity of the Terpene Nerolidol

Antileishmanial Activity of the Terpene Nerolidol

Farnesyl Diphosphate Synthase Is a Cytosolic Enzyme inLeishmania majorPromastigotes and Its Overexpression Confers Resistance to Risedronate

SMP-1, a Member of a New Family of Small Myristoylated Proteins in Kinetoplastid Parasites, Is Targeted to the Flagellum Membrane inLeishmania

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