title: Leishmania major phosphoglycerate kinase transcript and protein stability contributes to differences in isoform expression levels.
abstract: Leishmania contains two phosphoglycerate kinase (PGK) genes, PGKB and PGKC, which code for the cytosolic and glycosomal isoforms of the enzyme, respectively. Although differences in PGKB and PGKC transcript and protein levels and isoform activities have been well documented, the mechanisms of control of both transcript and protein abundance have not been described to date. To better understand the regulation of Leishmania PGK expression, we investigated the stabilities of both PGK transcripts using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in combination with transcription and trans-splicing inhibitors. Cells were treated with sinefungin and actinomycin D, and RNA decay kinetics were assessed. In addition, immunoblotting and protein synthesis inhibition by cycloheximide were employed to evaluate protein steady states and degradation. We observed increased stabilities of both PGKB mRNA and protein compared with the glycosomal isoform (PGKC). Our results indicate that both post-transcriptional and post-translational events contribute to the distinct expression levels of the PGKB and PGKC isoforms in Leishmania major.
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Avenida Bandeirantes, 3900, Monte Alegre, CEP: 14049-900, Ribeirão Preto-São Paulo, Brazil.
authors.forename: Aline
authors.initials: A
authors.lastname: Azevedo
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Avenida Bandeirantes, 3900, Monte Alegre, CEP: 14049-900, Ribeirão Preto-São Paulo, Brazil; Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Brazil.
authors.forename: Juliano S
authors.initials: JS
authors.lastname: Toledo
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Avenida Bandeirantes, 3900, Monte Alegre, CEP: 14049-900, Ribeirão Preto-São Paulo, Brazil.
authors.forename: Tânia
authors.initials: T
authors.lastname: Defina
authors.affiliation: Departamento de Bioquímica, Farmacologia e Fisiologia, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil.
authors.forename: André L
authors.initials: AL
authors.lastname: Pedrosa
authors.affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Avenida Bandeirantes, 3900, Monte Alegre, CEP: 14049-900, Ribeirão Preto-São Paulo, Brazil. Electronic address: akcruz@fmrp.usp.br.
authors.forename: Angela K
authors.initials: AK
authors.lastname: Cruz
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000981:Antiprotozoal Agents / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D003609:Dactinomycin / Q000494:pharmacology; D005786:Gene Expression Regulation; D006207:Half-Life; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018320:Leishmania major / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D008830:Microbodies / Q000201:enzymology; D008970:Molecular Weight; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D016053:RNA, Protozoan / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
chemical_list: D000981:Antiprotozoal Agents; D007527:Isoenzymes; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D016053:RNA, Protozoan; D003609:Dactinomycin; D003513:Cycloheximide; D010735:Phosphoglycerate Kinase; D000241:Adenosine; C006235:sinefungin
keywords: Gene expression; Glycosome turnover; Leishmania; Phosphoglycerate kinase; Untranslated regions