RESUMO
The isoprenoid metabolic pathway in protozoa of the Leishmania genus exhibits distinctive characteristics. These parasites, as well as other members of the Trypanosomatidae family, synthesize ergosterol, instead of cholesterol, as the main membrane sterol lipid. Leishmania has been shown to utilize leucine, instead of acetate as the main precursor for sterol biosynthesis. While mammalian dolichols are molecules containing 15-23 isoprene units, Leishmania amazonensis promastigotes synthesize dolichol of 11 and 12 units. In this paper, we show that the intracellular stages of L. amazonensis, amastigotes, synthesize mainly polyprenols of 9 isoprene units, instead of dolichol.
Assuntos
Leishmania mexicana/metabolismo , Terpenos/química , Animais , Butadienos/química , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Dolicóis/biossíntese , Dolicóis/química , Hemiterpenos/química , Pentanos/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We performed reverse-phase thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of polyisoprenoids released by sulfonium-salt cleavage with methyl iodide from Plasmodium falciparum proteins labeled with [3H]FPP or [3H]GGPP and showed that a dolichol of 11 isoprene units is bound to 21-28-kDa protein clusters from trophozoite and schizont stages. The dolichol structure was confirmed by electrospray-ionization mass spectrometry analysis. Treatment with protein synthesis inhibitors and RP-HPLC analysis of the proteolytic digestion products from parasite proteins labeled with [35S]cysteine and [3H]FPP showed that the attachment of dolichol to protein is a post-translational event and probably occurs via a covalent bond to cysteine residues.
Assuntos
Dolicóis/metabolismo , Plasmodium falciparum/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas de Protozoários/metabolismo , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Camada Fina/métodos , Cisteína/química , Cisteína/metabolismo , Dolicóis/química , Plasmodium falciparum/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Proteínas de Protozoários/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Direct analysis of polyisoprenoids by electrospray ionization mass spectrometry (ESI-MS) often produces poor results requiring off-line time and sample-consuming derivatization techniques. We describe a simple ESI-MS approach for the direct analysis of polyisoprenoids using several dolichols and polyprenols with different chain sizes as proof-of-principle cases. Lithium iodide is used to promote cationization by intense formation of [M+Li]+ adducts. Thus, detection of polyisoprenoids with mass determination can be performed with high sensitivity (limit of detection [LOD] approximately 100 rhoM), whereas characteristic collision-induced dissociations observed for both dolichols and polyprenols permit investigation of their structure. Using ESI(Li+)-MS and ESI(Li+)-MS/MS analysis, we screened for polyprenol products of an octaprenyl pyrophosphate synthase of Plasmodium falciparum and dolichols in a complex mixture of compounds produced by Leishmania amazonensis and P. falciparum.
Assuntos
Álcoois/análise , Iodetos/química , Lítio/química , Polímeros/análise , Terpenos/análise , Álcoois/química , Alquil e Aril Transferases/metabolismo , Animais , Cátions/química , Cromatografia Líquida de Alta Pressão/métodos , Dolicóis/química , Dolicóis/metabolismo , Leishmania/química , Leishmania/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Polímeros/química , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/métodos , Terpenos/químicaRESUMO
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 microM, respectively. The treatment of L. amazonensis-infected macrophages with 100 microM 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.