title: Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.
abstract: Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Juliano S
authors.initials: JS
authors.lastname: Toledo
authors.forename: Tiago R
authors.initials: TR
authors.lastname: Ferreira
authors.forename: Tânia P A
authors.initials: TP
authors.lastname: Defina
authors.forename: Fernando de M
authors.initials: Fde M
authors.lastname: Dossin
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Beattie
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Lamont
authors.forename: Serge
authors.initials: S
authors.lastname: Cloutier
authors.forename: Barbara
authors.initials: B
authors.lastname: Papadopoulou
authors.forename: Sergio
authors.initials: S
authors.lastname: Schenkman
authors.forename: Angela K
authors.initials: AK
authors.lastname: Cruz
mesh_terms: D002478:Cells, Cultured; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D020869:Gene Expression Profiling; D006706:Homeostasis / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D018320:Leishmania major / Q000235:genetics* / Q000472:pathogenicity; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D011132:Polyribosomes / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D020038:RNA, Spliced Leader / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D014774:Virulence / Q000235:genetics
chemical_list: D020543:Proteome; D015800:Protozoan Proteins; D020038:RNA, Spliced Leader; D025801:Ubiquitin; D057056:Cysteine Proteases