title: LmaPA2G4, a homolog of human Ebp1, is an essential gene and inhibits cell proliferation in L. major.
abstract: We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in a phosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidase member of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. A null mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene in Leishmania major. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigote stages. Interestingly, the over-expression affected cell proliferation and virulence in mouse footpad analysis. LmaPA2G4 binds a synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I∶C)] as shown in an electrophoretic mobility shift assay (EMSA). Quantitative proteomics revealed that the over-expression of LmaPA2G4 led to accumulation of factors involved in translation initiation and elongation. Significantly, we found a strong reduction of de novo protein biosynthesis in transgenic parasites using a non-radioactive metabolic labeling assay. In conclusion, LmaPA2G4 is an essential gene and is potentially implicated in fundamental biological mechanisms, such as translation, making it an attractive target for therapeutic intervention.
authors.affiliation: Eck Institute for Global Health. Department of Biological Sciences. University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: Brianna
authors.initials: B
authors.lastname: Norris-Mullins
authors.affiliation: Eck Institute for Global Health. Department of Biological Sciences. University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: Kaitlin
authors.initials: K
authors.lastname: VanderKolk
authors.affiliation: Eck Institute for Global Health. Department of Biological Sciences. University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: Paola
authors.initials: P
authors.lastname: Vacchina
authors.affiliation: Mass Spectrometry and Proteomics Facility, University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: Michelle V
authors.initials: MV
authors.lastname: Joyce
authors.affiliation: Eck Institute for Global Health. Department of Biological Sciences. University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Morales
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000818:Animals; D049109:Cell Proliferation; D004195:Disease Models, Animal; D005260:Female; D020043:Genes, Essential; D006801:Humans; D018320:Leishmania major / Q000235:genetics*; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D014176:Protein Biosynthesis; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C101633:PA2G4 protein, human; D015800:Protozoan Proteins; D016601:RNA-Binding Proteins; D037521:Virulence Factors