LmaPA2G4, a Homolog of Human Ebp1, Is an Essential Gene and Inhibits Cell Proliferation in L. major

We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in aphosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidasemember of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. Anull mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene inLeishmaniamajor. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigotestages. Interestingly, the over-expression affected cell proliferation and virulence in mouse footpad analysis. LmaPA2G4 binds asynthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] as shown in an electrophoretic mobility shiftassay (EMSA). Quantitative proteomics revealed that the over-expression of LmaPA2G4 led to accumulation of factors involved intranslation initiation and elongation. Significantly, we found a strong reduction ofde novoprotein biosynthesis in transgenicparasites using a non-radioactive metabolic labeling assay. In conclusion, LmaPA2G4 is an essential gene and is potentiallyimplicated in fundamental biological mechanisms, such as translation, making it an attractive target for therapeutic intervention.Fil: Norris Mullins, Brianna. University Of Notre Dame-Indiana; Estados UnidosFil: VanderKolk, Kaitlin. University Of Notre Dame-Indiana; Estados UnidosFil: Vacchina, Paola. University Of Notre Dame-Indiana; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Joyce, Michelle V.. University Of Notre Dame-Indiana; Estados UnidosFil: Morales, Miguel A.. University Of Notre Dame-Indiana; Estados Unido

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Catalytic activity of a novel serine/threonine protein phosphatase PP5 from Leishmania major

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs) and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5) in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention

Catalytic activity of a novel serine/threonine protein phosphatase PP5 from

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs) and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5) in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention

RESEARCH ARTICLE OPEN ACCESS Catalytic activity of a novel serine/threonine protein

Abstract – Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs) and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5) in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed autoinhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention. Key words: signaling, phosphatases, mutagenesis, activity, drug target. Résumé – Activité catalytique d’une nouvelle phosphatase de protéine sérine/thréonine PP5 de Leishmania major. La leishmaniose est une maladie transmise par un vecteur, due à des parasites protozoaires appartenant au genre Leishmania. Notre connaissance des phosphatases de protéines (PPs) et leur implication dans la signalisation est très limitée. Nous rapportons ici l’expression, la caractérisation et les analyses de mutagenèse d’une nouvelle PP5 chez Leishmania major. La PP5 recombinante est une phosphatase authentique, enzymatiquement active. La mutagenèse dirigée a identifié les rôles auto-inhibiteurs de la région N-terminale de la PP5. Il s’agit d’une premièr

2D-DIGE quantitative phosphoproteomics analysis of GFPK7.

<p>An enlarged region of the 2D-DIGE gels showing Cy3-labeled WT stationary promastigotes and Cy5-labeled stationary GFPK7 promastigotes is presented. Spot ID 207 (white arrow) was over-represented in GFPK7. In the lower panel, a graphical representation of the BVA (Biological Variation Analysis) module of Decyder software (GE Healthcare) with statistics for spot ID 207 (2.97 fold and p = 0.00056). The spot was analyzed by mass spectrometry and identified as LmjF19.0160, a putative aminopeptidase.</p

Bioinformatics analysis of <i>L. major</i> LmjF19.0160.

<p>Relationship between human and trypanosomatid members of the M24A subfamily of metallopeptidases was analyzed by multiple alignment and cluster analysis using Clustal X. This subfamily comprises homologs of methionyl aminopeptidases 1 and 2 (METAP1 and 2), and non-peptidases. Alignment was fed into MEGA5.2 software and a Neighbor-Joining tree was computed with 500 bootstrap replicates. Numbers on nodes indicate bootstrap support.</p

Attenuated virulence in GFP-PA2G4 transgenic parasites.

<p>(A) 10⁵<i>L. major</i> metacyclic mock and transgenic parasites expressing GFP-PA2G4 and cured GFP-PA2G4 grown in the absence of G418 were inoculated into the footpad of female BALB/c mice. Lesion formation was followed by measuring the increase in footpad size with a Vernier caliper. Groups of five mice were analyzed and standard deviation is indicated by the bars. Two independent experiments were performed and one representative experiment is shown. (B) <i>L. donovani</i> transgenic (GFP-PA2G4) lines were established. 2×10⁵ promastigotes were inoculated in low pH medium and 37°C to trigger differentiation to axenic amastigotes. Cells were lysed 24 and 48 h after differentiation, and western blot performed with anti-GFP, amastigote-specific A2 antibody and anti-tubulin as a loading control. The molecular weight of standard proteins is indicated in kDa.</p

Electrophoretic Mobility Shift Assay.

<p>Poly (I∶C), a synthetic double stranded RNA was labeled with Cy5 and incubated with 20 ng GFP-PA2G4 fusion protein at room temperature for 45 min. The reaction was resolved in 10% non-denaturing polyacrylamide gels. Lane 1: 40 ng poly (I∶C); lane 2: 20 ng GFP-PA2G4; lane 3: 20 ng GFP-PA2G4 incubated with 40 ng poly (I∶C). Gel was scanned on a Typhoon scanner (GE) using 633/670 nm for Cy5 and 489/508 nm for GFP. Arrows indicate mobility shift of GFP-PA2G4.</p

Establishment of L. major PA2G4 conditional null-mutant parasites.

<p>(A) Schematic representation of the null-mutant strategy. Two alleles of LmaPA2G4 were replaced by homologous recombination with hygromycin B (HYG B) and puromycin (PAC) resistance markers. Replacement was performed in the presence of an ectopic copy of PA2G4 (pXNG- PA2G4), carrying a nourseothricin (SAT) marker, a fluorescent protein (GFP) and a <i>Herpes simplex</i> virus thymidine kinase (TK). (B) PCR analysis of total DNA from wild-type (WT) parasites or one independent clone (PA2G4 −/− [pXNG-PA2G4]). F1,R1 primers (expected size 338 bp) confirm the presence of the endogenous copy of PA2G4, while F2,R1 primers (expected size 513 bp) confirm of the episomal PA2G4 ORF. F1, R2 primer pair (expected size 413 bp) and F1, R3 (expected size 440 bp) show the integration of hygromycin b and puromycin genes, respectively. Molecular weight marker (M) is shown. (C) (Upper panel). WT parasites carrying a copy of pXNG-PA2G4 were selected and grown in SAT and the GFP intensity was analyzed by flow cytometry (GCV−). After negative selection with the addition of 50 µg/mL GCV (GCV+) to the culture (3 passages) a reduction in GFP fluorescence is observed (lower panel). In contrast, conditional PA2G4 null-mutant parasites retained the ectopic copy of pXNG-PA2G4 as shown by the minimal reduction in GFP intensity. Histograms plots of one representative analysis are shown. Dotted lines correspond to fluorescence background levels of control parasites.</p