RESUMEN
Trypanosoma brucei is an important human pathogen. In this study, we have focused on the characterization of FtsH protease, ATP-dependent membrane-bound mitochondrial enzyme important for regulation of protein abundance. We have determined localization and orientation of all six putative T.brucei FtsH homologs in the inner mitochondrial membrane by in silico analyses, by immunofluorescence, and with protease assay. The evolutionary origin of these homologs has been tested by comparative phylogenetic analysis. Surprisingly, some kinetoplastid FtsH proteins display inverted orientation in the mitochondrial membrane compared to related proteins of other examined eukaryotes. Moreover, our data strongly suggest that during evolution the orientation of FtsH protease in T. brucei varied due to both loss and acquisition of the transmembrane domain.
Asunto(s)
Evolución Molecular , Proteínas Mitocondriales/química , Péptido Hidrolasas/química , Proteínas Protozoarias/química , Trypanosoma brucei brucei/enzimología , Animales , Arabidopsis/clasificación , Arabidopsis/enzimología , Arabidopsis/genética , Secuencia Conservada , Euglena gracilis/clasificación , Euglena gracilis/enzimología , Euglena gracilis/genética , Euglena longa/clasificación , Euglena longa/enzimología , Euglena longa/genética , Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Leishmania major/clasificación , Leishmania major/enzimología , Leishmania major/genética , Ratones , Mitocondrias/enzimología , Mitocondrias/genética , Membranas Mitocondriales/química , Membranas Mitocondriales/enzimología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Filogenia , Dominios Proteicos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Trypanosoma brucei brucei/clasificación , Trypanosoma brucei brucei/genéticaRESUMEN
Euglena longa, a close relative of the photosynthetic model alga Euglena gracilis, possesses an enigmatic non-photosynthetic plastid. Its genome has retained a gene for the large subunit of the enzyme RuBisCO (rbcL). Here we provide new data illuminating the putative role of RuBisCO in E. longa. We demonstrated that the E. longa RBCL protein sequence is extremely divergent compared to its homologs from the photosynthetic relatives, suggesting a possible functional shift upon the loss of photosynthesis. Similarly to E. gracilis, E. longa harbors a nuclear gene encoding the small subunit of RuBisCO (RBCS) as a precursor polyprotein comprising multiple RBCS repeats, but one of them is highly divergent. Both RBCL and the RBCS proteins are synthesized in E. longa, but their abundance is very low compared to E. gracilis. No RBCS monomers could be detected in E. longa, suggesting that processing of the precursor polyprotein is inefficient in this species. The abundance of RBCS is regulated post-transcriptionally. Indeed, blocking the cytoplasmic translation by cycloheximide has no immediate effect on the RBCS stability in photosynthetically grown E. gracilis, but in E. longa, the protein is rapidly degraded. Altogether, our results revealed signatures of evolutionary degradation (becoming defunct) of RuBisCO in E. longa and suggest that its biological role in this species may be rather unorthodox, if any.
Asunto(s)
Euglena longa , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas Protozoarias , Ribulosa-Bifosfato Carboxilasa , Transcriptoma/fisiología , Euglena longa/enzimología , Euglena longa/genética , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Ribulosa-Bifosfato Carboxilasa/biosíntesis , Ribulosa-Bifosfato Carboxilasa/genéticaRESUMEN
A new aminopeptidase was isolated from the biomass of the flagellate Astasia longa by precipitation with ammonium sulfate, gel filtration, and affinity chromatography on Arginine-Silochrome in 41% yield and with purification degree 490. The enzyme is irreversible inhibited by mercury chloride, EDTA, o-phenanthroline and, partially, bestatin and zinc chloride. It has an optimum pH 8.5 toward the hydrolysis of a synthetic chromogenic substrate Ala-pNA. The enzyme molecular mass is 45 kDa, isoelectric point 5.5, and temperature optimum 45 degrees C. The enzyme most effectively hydrolyzes p-nitroanilides of alanine, arginine, and leucine; it is classified as metalloaminopeptidase.
