Cloning Expression and Characterization of Methionine Adenosyltransferase in Leishmania infantumPromastigotes

Reguera, Rosa M.; Balaña‐Fouce, Rafael; Pérez-Pertejo, Yolanda; Fernandez, F; Garcı́a-Estrada, Carlos; Cubría, J.C.; Ordóñez, César Alberto Collazos; Ordóñez, David

doi:10.1074/jbc.m105512200

Cited by 39 publications

(54 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In L. major, two copies of this gene are present within a 7-kilobase region of chromosome 30 and are separated by a hypothetical open reading frame (www.genedb.org). A similar organization is seen in Leishmania infantum (29).…”

Section: Analysis Of the Initial Responses Of L Major To Mtx By Two-supporting

confidence: 65%

Differential Protein Expression Analysis of Leishmania major Reveals Novel Roles for Methionine Adenosyltransferase and S-Adenosylmethionine in Methotrexate Resistance

Drummelsmith

Girard

Trudel

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Leishmania is a trypanosomatid parasite causing serious disease and displaying resistance to various drugs. Here, we present comparative proteomic analyses of Leishmania major parasites that have been either shocked with or selected in vitro for high level resistance to the model antifolate drug methotrexate. Numerous differentially expressed proteins were identified by these experiments. Some were associated with the stress response, whereas others were found to be overexpressed due to genetic linkage to primary resistance mediators present on DNA amplicons. Several proteins not previously associated with resistance were also identified. The role of one of these, methionine adenosyltransferase, was confirmed by gene transfection and metabolite analysis. After a single exposure to low levels of methotrexate, L. major methionine adenosyltransferase transfectants could grow at high concentrations of the drug. Methotrexate resistance was also correlated to increased cellular S-adenosylmethionine levels. The folate and S-adenosylmethionine regeneration pathways are intimately connected, which may provide a basis for this novel resistance phenotype. This thorough comparative proteomic analysis highlights the variety of responses required for drug resistance to be achieved.

show abstract

Section: Analysis Of the Initial Responses Of L Major To Mtx By Two-supporting

confidence: 65%

Differential Protein Expression Analysis of Leishmania major Reveals Novel Roles for Methionine Adenosyltransferase and S-Adenosylmethionine in Methotrexate Resistance

Drummelsmith

Girard

Trudel

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The blots were washed as above, incubated with ECL Plus chemiluminescent substrate (Amersham Biosciences), and exposed to x-ray films. The polyclonal rabbit MAT antibody was kindly provided by Prof. Balana-Fouce (University of Leon, Spain) and was used as described previously (34).…”

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

View full text Add to dashboard Cite

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...

show abstract

“…When they adjusted the enzyme activity of transaldolase (TAL1), transketolase (TKL1), and pyruvate kinase (PYK1) by tuning the promoter strength, the enzyme on May 11, 2018 by guest http://aem.asm.org/ activity of PYK1 was significantly lower than those of the other two genes. In the present study, the extraordinarily low MAT activity may suggest that dimerization of the protein, which is important for MAT activity (9), maintenance of the cellular redox potential (26), and SAM inhibition (32), plays important roles in the modulation of MAT activity. In summary, the expression of these three reporters was regulated at several stages, and the huge variance in mRNA level exerted by the different promoters was attenuated at the protein/enzyme activity level by various degrees for each reporter.…”

Section: Vol 77 2011mentioning

confidence: 70%

GAP Promoter Library for Fine-Tuning of Gene Expression in Pichia pastoris

Qin

Qian

Yao

et al. 2011

Appl Environ Microbiol

127

View full text Add to dashboard Cite

A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris, we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ϳ0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (␤-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.The creation of a library of engineered promoters of various strengths has enabled the precise control of gene expression in a broad range of activities required for detailed genotypephenotype characterization and the identification of an optimal gene expression level for the desired product yield (1,4,12,20,22,29,35). For example, a library of promoters has been used to assess the influence of phosphoenolpyruvate carboxylase levels on growth yield and deoxyxylulose-P synthase levels on lycopene production and to demonstrate that the optimal expression levels of deoxyxylulose-P synthase are different in a strain preengineered to produce lycopene and in the parental strain (1). In another example, random mutagenesis of the Pm promoter has been applied to improve production of the medically important granulocyte-macrophage colony-stimulating factor in Escherichia coli at an industrial level (4). Furthermore, promoter libraries coupled with in silico modeling have been used to guide predictable gene network construction in Saccharomyces cerevisiae (13). Therefore, the promoter library constitutes a valuable addition to the genetic toolbox for the study of metabolic engineering, functional genomics, and synthetic biology, as well as for various biotechnology applications. However, this powerful tool is implemented mainly in bacteria (2,4,12,20,35,36...

show abstract

Cloning Expression and Characterization of Methionine Adenosyltransferase in Leishmania infantumPromastigotes

Cited by 39 publications

References 41 publications

Differential Protein Expression Analysis of Leishmania major Reveals Novel Roles for Methionine Adenosyltransferase and S-Adenosylmethionine in Methotrexate Resistance

Differential Protein Expression Analysis of Leishmania major Reveals Novel Roles for Methionine Adenosyltransferase and S-Adenosylmethionine in Methotrexate Resistance

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

GAP Promoter Library for Fine-Tuning of Gene Expression in Pichia pastoris

Contact Info

Product

Resources

About