Prefractionation by Digitonin Extraction Increases Representation of the Cytosolic and Intracellular Proteome of Leishmania infantum

Foucher, Aude L.; Papadopoulou, Barbara; Ouellette, Marc

doi:10.1021/pr060081j

Cited by 49 publications

(47 citation statements)

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“…Western Blot Analysis-Total Leishmania proteins (30 g) were run on 12% polyacrylamide gels and transferred onto nitrocellulose membranes as described previously (33). The blots were blocked overnight in 5% skimmed milk in 1ϫ phosphate-buffered saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family

Dridi¹,

Ouameur²,

Ouellette

2010

Journal of Biological Chemistry

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S-Adenosylmethionine (AdoMet) is an important methyl group donor that plays a central role in many essential biochemical processes. The parasite Leishmania can both synthesize and transport AdoMet. Leishmania cells resistant to the antifolate methotrexate due to a rearrangement in folate biopterin transporter (FBT) genes were cross-resistant to sinefungin, an AdoMet analogue. FBT gene rearrangements were also observed in Leishmania major cells selected for sinefungin resistance. One of the rearranged FBT genes corresponded to the main AdoMet transporter (AdoMetT1) of Leishmania as determined by gene transfection and gene inactivation experiments. AdoMetT1 was determined to be a high affinity plasma membrane transporter expressed constitutively throughout the growth phases of the parasite. Leishmania cells selected for resistance or naturally insensitive to sinefungin had lower expression of AdoMetT1. A new function in one carbon metabolism, also a pathway of interest for chemotherapeutic interventions, is described for a novel class of membrane proteins found in diverse organisms.The parasite Leishmania is distributed globally and causes a variety of clinical symptoms ranging from self-healing cutaneous lesions to visceral infections that are usually fatal if left untreated (1, 2). Current first-line chemotherapy against leishmaniasis relies on a rather limited arsenal of drugs, including pentavalent antimonials, liposomal amphotericin B, or miltefosine (2). These drugs are associated with side effects, high costs, and drug resistance, and therefore, the search for new drugs and targets to control this parasite is warranted (3, 4).S-Adenosylmethionine (AdoMet or SAM) 5 is an important biological sulfonium compound recognized as the universal methyl donor for methylation of lipids, proteins, nucleic acids, and xenobiotics in living cells (5). AdoMet is also a donor of propylamine groups for the synthesis of polyamines and participates in the reverse trans-sulfuration pathway where AdoMet is converted into cysteine and glutathione and in Leishmania in the spermidine-glutathione conjugate trypanothione (6, 7). AdoMet is also used as a source of methylene groups, amino groups, and ribosyl groups in the synthesis of fatty acids, biotin, and the modified nucleoside epoxyqueusine of tRNAs (reviewed in Ref. 8).Most cells are capable of synthesizing AdoMet from L-methionine and ATP, in a process requiring the enzyme methionine adenosyltransferase (MAT). The gene coding for this enzyme is present in most sequenced organisms (reviewed in Ref. 9). However, in some Rickettsia strains, the MAT gene is inactivated by a mutation (10), and in the fungus Pneumocystis carinii, no MAT activity has been detected (11). These organisms have the rare distinction of meeting their AdoMet needs by importing it (12). The Rickettsia transporter is part of the drug/ metabolite transporter superfamily (10), and the identity of the Pneumocystis transporter is not known. Transport of AdoMet across the plasma membrane does not occur to a signifi...

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Section: Methodsmentioning

confidence: 99%

High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family

Dridi¹,

Ouameur²,

Ouellette

2010

Journal of Biological Chemistry

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“…The capacity of digitonin to bind to the membrane and to form pores depends on sterol composition that is variable between intracellular organella and the cytoplasmic membrane. Such differential lysis capacity of digitonin was used to increase the resolution and the depth of total cytosolic and intracellular proteome via sequential lysis of Leishmania parasites (Foucher et al 2006). In this study, we improved the method for the isolation of an enriched mitochondrial fraction previously described by Castro et al (2004).…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we improved the method for the isolation of an enriched mitochondrial fraction previously described by Castro et al (2004). Purity fractions were estimated using luciferase activity as cytosolic marker and LimTXNPx as mitochondrial marker protein (Castro et al 2004;Foucher et al 2006). …”

Section: Introductionmentioning

confidence: 99%

Leishmania infantum: tuning digitonin fractionation for comparative proteomic of the mitochondrial protein content

Hide¹,

Ritleng²,

Brizard³

et al. 2008

Parasitol Res

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Leishmania infantum belongs to the Kinetoplastidae that is characterized by a specific mitochondrial DNA, the kinetoplast. This parasite is responsible for both benign cutaneous leishmaniasis and severe visceral leishmaniasis in humans. Molecular determinants of such differences in pathogenesis are not well understood, and the parasites as well as their hosts may contribute to the disease phenotype. Factors that help parasite to adapt its metabolism to nutritional conditions encountered in different location might play pivotal roles in controlling parasite development in these various host environments. Thus, we have decided to initiate studies aimed to compare the mitochondrial protein content of L. infantum. To avoid the drawback caused by the most abundant proteins such as tubulin and proteins of the cytoskeleton present in whole cell extract, we have decided to fractionate the subcellular components of the cells. Using both cytosolic and mitochondrial markers, we have improved a protein pre-fractionation protocol using digitonin that allowed us to generate an enriched mitochondrial fraction.

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“…To reduce complexity and enable identification of subproteomes, Foucher and colleagues used differential solubilization of membranes by digitonin for pre-fractionation 70 . Digitonin is a non-ionic detergent which solubilizes sterols and at lower concentrations forms pores in the membrane.…”

Section: Prefractionation Of Parasite Lysates To Increase Proteome Comentioning

confidence: 99%

“…This differential lysis protocol of Leishmania spp. and its membranes thus may yield directly information about the likely subcellular localization of proteins 70 .…”

Section: Prefractionation Of Parasite Lysates To Increase Proteome Comentioning

confidence: 99%

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

Paape

Aebischer

2011

Journal of Proteomics

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:Leishmania spp., protozoan parasites with a digenetic life cycle, cause a spectrum of diseases in humans. Recently several Leishmania spp. have been sequenced which significantly boosted the number and quality of proteomic studies conducted. Here a historic review will summarize work of the pre-genomic era and then focus on studies after genome information became available. Firstly works comparing the different life cycle stages, in order to identify stage specific proteins, will be discussed. Identifying of post-translational modifications by proteomics especially phosphorylation events will be discussed. Further the contribution of proteomics to the understanding of the molecular mechanism of drug resistance and the investigation of immunogenic proteins for the identification of vaccine candidates will be summarized. Approaches of how potentially secreted proteins were identified are discussed. So far 30 -35% of the total predicted proteome of Leishmania spp. has been identified. This comprises mainly the abundant proteins, therefore the last section will look into technological approaches how this coverage may be increased and what the gel-free and gel-based proteomics have to offer will be compared.

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Prefractionation by Digitonin Extraction Increases Representation of the Cytosolic and Intracellular Proteome of Leishmania infantum

Cited by 49 publications

References 38 publications

High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family

High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family

Leishmania infantum: tuning digitonin fractionation for comparative proteomic of the mitochondrial protein content

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

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