RESUMEN
In Trichomonas vaginalis, a Myb1 protein was previously demonstrated to repress transcription of an iron-inducible ap65-1 gene. In this study, a human cyclophilin A homologue, TvCyclophilin 1 (TvCyP1), was identified as a Myb1-binding protein using a bacterial two-hybrid library screening system. The recombinant TvCyP1 exhibited typical peptidyl-prolyl isomerase activity with kcat/Km of â¼7.1 µm(-1) s(-1). In a pulldown assay, the His-tagged Myb1 interacted with a GST-TvCyP1 fusion protein, which had an enzymatic proficiency half that of recombinant TvCyP1. Both the enzymatic proficiency of GST-TvCyP1 and its binding to His-Myb1 were eliminated by mutation of Arg(63) in the catalytic motif or inhibited by cyclosporin A. TvCyP1 was primarily localized to the hydrogenosomes by immunofluorescence assay, but it was also co-purified with Myb1 in certain vesicle fractions from differential and gradient centrifugations. Transgenic cells overexpressing HA-TvCyP1 had a higher level of nuclear Myb1 but a much lower level of Myb1 associated with the vesicles than control and those overexpressing HA-TvCyP1(R63A). Myb1 was detected at a much higher level in the HA-TvCyP1 protein complex than in the HA-TvCyP1(R63A) protein complex immunoprecipitated from P15 and P100, but not S100, fractions of postnuclear lysates. A TvCyP1-binding motif, (105)YGPKWNK(111), was identified in Myb1 in which Gly(106) and Pro(107) were essential for its binding to TvCyP1. Mutation of Gly(106) and Pro(107), respectively, in HA-Myb1 resulted in cytoplasmic retention and elevated nuclear translocation of the overexpressed protein. These results suggest that TvCyP1 may induce the release of Myb1 that is restrained to certain cytoplasmic vesicles prior to its nuclear translocation.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Trichomonas vaginalis/citología , Trichomonas vaginalis/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Factores de Transcripción/químicaRESUMEN
Iron was previously shown to induce rapid nuclear translocation of a Myb3 transcription factor in the protozoan parasite, Trichomonas vaginalis. In the present study, iron was found to induce a transient increase in cellular cAMP, followed by the nuclear influx of Myb3, whereas the latter was also induced by 8-bromo-cyclic AMP. Iron-inducible cAMP production and nuclear influx of Myb3 were inhibited by suramin and SQ22536, respective inhibitors of the Gα subunit of heterotrimeric G proteins and adenylyl cyclases. In contrast, the nuclear influx of Myb3 induced by iron or 8-bromo-cAMP was delayed or inhibited, respectively, by H89, the inhibitor of protein kinase A. Using liquid chromatography-coupled tandem mass spectrometry, Thr(156) and Lys(143) in Myb3 were found to be phosphorylated and ubiquitinated, respectively. These modifications were induced by iron and inhibited by H89, as shown by immunoprecipitation-coupled Western blotting. Iron-inducible ubiquitination and nuclear influx were aborted in T156A and K143R, but T156D was constitutively ubiquitinated and persistently localized to the nucleus. Myb3 was phosphorylated in vitro by the catalytic subunit of a T. vaginalis protein kinase A, TvPKAc. A transient interaction between TvPKAc and Myb3 and the phosphorylation of both proteins were induced in the parasite shortly after iron or 8-bromo-cAMP treatment. Together, these observations suggest that iron may induce production of cAMP and activation of TvPKAc, which then induces the phosphorylation of Myb3 and subsequent ubiquitination for accelerated nuclear influx. It is conceivable that iron probably exerts a much broader impact on the physiology of the parasite than previously thought to encounter environmental changes.
Asunto(s)
Núcleo Celular/metabolismo , Hierro/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Trichomonas vaginalis/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Lisina/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/genética , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Ubiquitina/metabolismoRESUMEN
In Trichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNA-binding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.
Asunto(s)
Núcleo Celular/metabolismo , Hierro/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Trichomonas vaginalis/metabolismo , Transporte Activo de Núcleo Celular , Sitios de Unión , Señales de Localización Nuclear , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Trichomonas vaginalis/genética , Regulación hacia ArribaRESUMEN
Nuclear proteins usually contain specific peptide sequences, referred to as nuclear localization signals (NLSs), for nuclear import. These signals remain unexplored in the protozoan pathogen, Trichomonas vaginalis. The nuclear import of a Myb2 transcription factor was studied here using immunodetection of a hemagglutinin-tagged Myb2 overexpressed in the parasite. The tagged Myb2 was localized to the nucleus as punctate signals. With mutations of its polybasic sequences, 48KKQK51 and 61KR62, Myb2 was localized to the nucleus, but the signal was diffusive. When fused to a C-terminal non-nuclear protein, the Myb2 sequence spanning amino acid (aa) residues 48 to 143, which is embedded within the R2R3 DNA-binding domain (aa 40 to 156), was essential and sufficient for efficient nuclear import of a bacterial tetracycline repressor (TetR), and yet the transport efficiency was reduced with an additional fusion of a firefly luciferase to TetR, while classical NLSs from the simian virus 40 T-antigen had no function in this assay system. Myb2 nuclear import and DNA-binding activity were substantially perturbed with mutation of a conserved isoleucine (I74) in helix 2 to proline that altered secondary structure and ternary folding of the R2R3 domain. Disruption of DNA-binding activity alone by point mutation of a lysine residue, K51, preceding the structural domain had little effect on Myb2 nuclear localization, suggesting that nuclear translocation of Myb2, which requires an ordered structural domain, is independent of its DNA binding activity. These findings provide useful information for testing whether myriad Mybs in the parasite use a common module to regulate nuclear import.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Trichomonas vaginalis/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia Conservada , ADN/química , Componentes del Gen , Luciferasas de Luciérnaga/química , Luciferasas de Luciérnaga/metabolismo , Datos de Secuencia Molecular , Señales de Localización Nuclear , Mapeo Peptídico , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/químicaRESUMEN
A type III Trichomonas vaginalis virus, which may be involved in transcriptional regulation of the major surface protein gene P270 of the protozoan pathogen Trichomonas vaginalis, was purified and characterized in the present study. The complete 4844-base-pair complementary DNA sequence of the viral genome reveals overlapping cap and pol genes with a putative ribosomal frame-shifting signal within the overlap region. The type III virus is related more closely to the type II virus than to the type I virus in the sequence of its ribosomal frameshift signal and in its capsid protein. Phylogenetic analysis revealed that these viruses could be grouped in the same clade as a genus distantly related to other genera in the family Totiviridae. Virus-induced P270 gene expression was only evident in Trichomonas vaginalis cells infected with either a type II or type III virus, but not with a type I virus. These findings suggest that transcription of the P270 gene is likely regulated by viral factors common to type II and type III viruses and thus provides important information for future investigation of virus-host interactions.
Asunto(s)
Totiviridae/clasificación , Totiviridae/genética , Trichomonas vaginalis/virología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de la Cápside/genética , Clonación Molecular , Femenino , Genes Protozoarios , Genoma Viral , Interacciones Huésped-Patógeno/genética , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Proteínas Protozoarias/genética , ARN Viral/química , ARN Viral/genética , Homología de Secuencia de Aminoácido , Totiviridae/aislamiento & purificación , Transcripción Genética , Trichomonas vaginalis/patogenicidadRESUMEN
In Trichomonas vaginalis, the TvCyP1-catalyzed conformational switches of two glycinyl-prolyl imide bonds in Myb3 were previously shown to regulate the trafficking of Myb3 from cytoplasmic membrane compartments towards the nucleus. In this study, TvCyP2 was identified as a second cyclophilin that binds to Myb3 at the same dipeptide motifs. The enzymatic proficiency of TvCyP2, but not its binding to Myb3, was aborted by a mutation of Arg75 in the catalytic domain. TvCyP2 was localized to the endoplasmic reticulum with a weak signal that extensively extends into the cytoplasm as well as to the plasma membrane according to an immunofluorescence assay. Moreover, TvCyP2 was co-enriched with TvCyP1 and Myb3 in various membrane fractions purified by differential and gradient centrifugation. TvCyP2 was found to proficiently enzymatically regulate the distribution of TvCyP1 and Myb3 among purified membrane fractions, and to localize TvCyP1 in hydrogenosomes and on plasma membranes. Protein complexes immunoprecipitated from lysates of cells overexpressing TvCyP1 and TvCyP2 were found to share some common components, like TvCyP1, TvCyP2, TvBip, Myb3, TvHSP72, and the hydrogenosomal heat shock protein 70 (HSP70). Direct interaction between TvCyP1 and TvCyP2 was confirmed by a GST pull-down assay. Fusion of vesicles with hydrogenosomes was observed by transmission electron microscopy, whereas TvCyP1, TvCyP2, and Myb3 were each detected at the fusion junction by immunoelectron microscopy. These observations suggest that T. vaginalis may have evolved a novel protein trafficking pathway to deliver proteins among the endomembrane compartments, hydrogenosomes and plasma membranes.
Asunto(s)
Familia 2 del Citocromo P450/metabolismo , Transporte de Proteínas/fisiología , Trichomonas vaginalis/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Ciclofilinas/metabolismo , Ciclofilinas/fisiología , Familia 2 del Citocromo P450/fisiología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/fisiología , Proteínas de la Membrana/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismoRESUMEN
In Trichomonas vaginalis, a TvCyP1 cyclophilin was previously demonstrated to regulate the nuclear translocation of Myb1 and Myb3, which respectively repress and activate transcription of an adhesion protein ap65-1 gene. In the present study, TvCyP1 was found to bind to Myb3 at sites spanning 54 Gly-Pro55 and 72 Gly-Pro73 with differential affinities. When Gly54 and Gly72 in Myb3 were both mutated, the mutant protein was restrained on outer membranes of hydrogenosomes and some cytoplasmic vesicles. In the purified Myb3 protein complex, a high molecular weight Myb3-interacting protein (Myb3IPhmw ) and a 72-kDa heat shock protein (TvHSP72) were identified and characterized, with direct binding of Myb3 to Myb3IPhmw and TvHSP72 confirmed in vitro. When cell lysates were fractionated by the differential and gradient centrifugations, TvCyP1 and Myb3 were always associated with membrane fractions enriched with Myb3IPhmw and Myb1, as well as hydrogenosomes and VMyb organelle fractions. Mutations of Gly54 and/or Gly72 resulted in membrane redistribution of Myb3 and the aberrant assembly of the Myb3 protein complex. Consistent with these findings, the involvement of TvCyP1 in membrane distribution of Myb3, and dissociation of Myb3 from TvCyP1 protein complex were demonstrated, with direct interactions between TvCyP1 and Myb3IPhmw and that between TvCyP1 and TvHSP72, confirmed in vitro. These observations suggest that TvCyP1 directly binds to Myb3 and some of its interacting proteins to mediate serial conformational switches of Myb3 for its transition from the membrane compartments toward the nucleus.
Asunto(s)
Ciclofilinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Trichomonas vaginalis/metabolismo , Transporte Activo de Núcleo Celular , Centrifugación por Gradiente de Densidad , Citoplasma/metabolismo , Glicina/química , Proteínas de la Membrana/aislamiento & purificación , Prolina/química , Mapeo de Interacción de Proteínas , Proteínas Protozoarias/aislamiento & purificación , Factores de Transcripción/aislamiento & purificación , Trichomonas vaginalis/ultraestructuraRESUMEN
Multifarious transcription of the adhesion protein ap65-1 gene in the human pathogen, Trichomonas vaginalis, is critically regulated by the coordination of two similar but opposite oriented DNA regulatory regions, MRE-1/MRE-2r and MRE-2f, both of which are binding sites for multiple Myb-like proteins. In the present study, MRE-1/MRE-2r was demonstrated to be composed of multiple overlapping promoter elements, among which the entire region is required for growth-related ap65-1 transcription, and the 5'-MRE-1 antagonizes the suppressive activity of the 3'-MRE-2r in iron-inducible transcription. The recombinant Myb2 protein derived from a previously identified myb2 gene was demonstrated to recognize distinct sequence contexts in MRE-2r and MRE-2f, whereas Myb2 in the nuclear lysate preferentially binds to MRE-2f to MRE-2r. Iron repletion resulted in persistent repression of the myb2 gene, and temporal activation/deactivation of Myb2 promoter entry, which was also activated by prolonged iron depletion. The hemagglutinintagged Myb2 when overexpressed during iron-depleted conditions facilitated basal and growth-related ap65-1 transcription to a level that was achieved in iron-replete cells, whereas ironinducible ap65-1 transcription was abolished with knockdown of Myb2. These findings demonstrated that Myb2 is involved in activation of growth-related and iron-inducible transcription of the ap65-1 gene, possibly through differential promoter selection in competition with other Myb proteins.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Transactivadores/fisiología , Activación Transcripcional , Trichomonas vaginalis/genética , Animales , Sitios de Unión , Hierro/metabolismo , Elementos Reguladores de la Transcripción , Trichomonas vaginalis/crecimiento & desarrollo , Trichomonas vaginalis/metabolismoRESUMEN
The transcription efficiency of an adhesion protein gene, ap65-1, in Trichomonas vaginalis varies with changes in the iron supply and with the growth stage. In the present study, two Myb recognition elements, MRE-1/MRE-2r and MRE-2f, were found to play antagonistic roles in regulating the iron-inducible activity of an ap65-1 reporter gene. Intriguingly, either of these elements was shown to be sufficient to repress basal activity, but together they were also shown to activate growth-related activity of the reporter gene in iron-depleted cells. A myb1 gene which encodes a 24-kDa protein containing a Myb-like R2R3 DNA binding domain was identified from Southwestern screening of MRE-2f-binding proteins. The Myb1 protein was detected as a major 35-kDa protein which exhibited variations in nuclear concentration with changes in the iron supply. A recombinant Myb1 protein was shown to differentially interact with MRE-1/MRE-2r and MRE-2f in vitro. Overexpression of hemagglutinin-tagged Myb1 in T. vaginalis resulted in repression or activation of ap65-1 transcription in iron-depleted cells at an early and a late stage of cell growth, respectively, while iron-inducible ap65-1 transcription was constitutively repressed. The hemagglutinin-tagged Myb1 protein was found to constantly occupy the chromosomal ap65-1 promoter at a proximal site, but it also selected two more distal sites only at the late growth stage. Together, these observations suggest that Myb1 critically regulates multifarious ap65-1 transcription, possibly via differential selection of multiple promoter sites upon environmental changes.
Asunto(s)
Moléculas de Adhesión Celular/genética , Regulación de la Expresión Génica , Genes Protozoarios/genética , Parásitos/genética , Proteínas Protozoarias/metabolismo , Transcripción Genética/genética , Trichomonas vaginalis/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Hierro/farmacología , Datos de Secuencia Molecular , Parásitos/citología , Parásitos/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Alineación de Secuencia , Transcripción Genética/efectos de los fármacos , Trichomonas vaginalis/citologíaRESUMEN
Iron has been shown to regulate transcription in the protozoan pathogen Trichomonas vaginalis. In this study, a DNA transfection system was developed to monitor ap65-1 promoter activity in response to changing iron supply. In conjunction with electrophoretic mobility shift assay, iron-induced transcription of the ap65-1 gene was shown to be regulated by multiple closely spaced DNA elements spanning an iron-responsive region (-110/-54), including an iron-responsive DNA element ((-98)AGATAACGA(-90)), which overlaps with a 3'-MYB-like protein binding sequence ((-95)TAACGATAT(-87)), and three nearby T-rich sequences ((-110)ATTTTT(-105), (-78)ATTATT(-73), and (-59)ATTTTT(-54)). 5'- and 3'-flanking sequences of the iron-responsive region were shown to regulate basal transcription. A distal DNA regulatory region was shown to enhance both basal and iron-induced transcription. These findings delineate the DNA regulatory elements and nuclear proteins involving in iron-induced transcription of the ap65-1 gene, which provide useful tools for the future study of transcriptional regulation in T. vaginalis.
Asunto(s)
Moléculas de Adhesión Celular , Hierro/metabolismo , Regiones Promotoras Genéticas , Proteínas Protozoarias/genética , Trichomonas vaginalis/genética , Trichomonas vaginalis/metabolismo , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Clonación Molecular , ADN/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Mutación , Plásmidos/metabolismo , Unión Proteica , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , TransfecciónRESUMEN
A reputed iron-responsive region, which contains multiple nuclear protein-binding DNA sequences, was shown previously to regulate iron-inducible transcription of the ap65-1 gene in the protozoan pathogen, Trichomonas vaginalis. These DNA sequences include two overlapping MYB recognition elements (MRE-1/MRE-2r) and three abutted T-tract elements. Additional nuclear protein-binding DNA sequences flanking the 5' (AGTGAAGTGA) and 3' (MRE-2f) of the iron-responsive region were identified in the present study. A stable promoter assay and primer extension revealed that transcriptional activity of the ap65-1 promoter is iron inducible as well as growth related, being lowest in the early logarithmic phase and highest in the mid-logarithmic phase. Subsequent mutational analysis of individual DNA elements of the ap65-1 promoter suggests that closely spaced T-tract elements together with an intervening GAAGGAAG sequence within the iron-responsive region are most critical for regulation of overall transcriptional activity, whereas an additional AGTGAAGTGA and MRE-2f together with an upstream T-rich region are required for optimal iron-inducible activity, and the MRE-1/MRE-2r overlap is only involved in growth-related activity. These observations suggest that expression of the ap65-1 gene is dynamically regulated under various growth conditions via interactions among multiple DNA regulatory elements.
Asunto(s)
Moléculas de Adhesión Celular/genética , Regulación de la Expresión Génica , Hierro/metabolismo , Proteínas Protozoarias/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Trichomonas vaginalis/genética , Animales , Secuencia de Bases , Sitios de Unión , Análisis Mutacional de ADN , ADN Protozoario/genética , ADN Protozoario/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Trichomonas vaginalis/crecimiento & desarrollo , Trichomonas vaginalis/metabolismoRESUMEN
We showed previously that transcription of the ran gene in Giardia lamblia is regulated by an AT-rich initiator. In the present study, the ran initiator was found to regulate transcription of a neighbouring PHD zinc-finger protein gene. Deletion and scanning mutagenesis of the phd promoter in a firefly luciferase reporter system showed that the promoter activity is determined by multiple single-stranded T-tract DNA elements distributed into a distal domain spanning the ran initiator (-134/-103) and a proximal domain (-88/-48) spanning phd messenger RNA (mRNA) start sites (-74, -55 and -53 relative to the first ATG). The promoter activity is repressed by the single T-tract element on a non-template strand of the ran initiator, and is activated by closely spaced T-tract elements on the opposite strand. The T-tract elements in the phd and ran initiators compete for similar ssDNA binding proteins. Mutation of -47/-42 resulted in dramatic reduction of luciferase activity without changing luciferase mRNA levels, indicating the potential involvement of a regulatory mechanism in PHD protein translation. These findings suggest that G. lamblia uses multiple copies of a T-tract element as both core and distal elements in regulating transcription initiation, and that expression of the phd gene is regulated at multiple levels.