Transporte y funciones de la prolina en Trypanosoma cruzi : su potencial como blanco terapéutico

The amino acid proline has special relevance for the parasite Trypanosoma cruzi,\nthe etiological agent of Chagas disease. This amino acid can be used as an energy and\ncarbon source alternative to glucose, it participates in different stress resistance\nmechanisms, and it is also essential during t...

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Autor principal:	Martínez Sayé, Melisa Soledad
Otros Autores:	Nowicki, Cristina
Formato:	Tesis doctoral acceptedVersion
Lenguaje:	Español
Publicado:	Facultad de Farmacia y Bioquímica 2017
Materias:	Transportador de prolina Trypanosoma cruzi Enfermedad de chagas Ciencia de la vida
Acceso en línea:	http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_1867 http://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_1867.dir/1867.PDF
Aporte de:	Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires

id	I28-R145-HWA_1867
record_format	dspace
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-145
collection	Repositorio Digital de la Universidad de Buenos Aires (UBA)
language	Español
orig_language_str_mv	spa
topic	Transportador de prolina Trypanosoma cruzi Enfermedad de chagas Ciencia de la vida
spellingShingle	Transportador de prolina Trypanosoma cruzi Enfermedad de chagas Ciencia de la vida Martínez Sayé, Melisa Soledad Transporte y funciones de la prolina en Trypanosoma cruzi : su potencial como blanco terapéutico
topic_facet	Transportador de prolina Trypanosoma cruzi Enfermedad de chagas Ciencia de la vida
description	The amino acid proline has special relevance for the parasite Trypanosoma cruzi,\nthe etiological agent of Chagas disease. This amino acid can be used as an energy and\ncarbon source alternative to glucose, it participates in different stress resistance\nmechanisms, and it is also essential during the cell invasion of metacyclic\ntrypomastigotes and for the life cycle progression inside the mammalian cells.\nIn this work the TcAAAP069 gene of T. cruzi was identified as a proline permease,\nfirst through yeast complementation and then by overexpression in epimastigotes\n(Tc069 parasites). Although the transporter TcAAAP069 proved to be mono-specific, the\nstereoisomer D-proline was able to significantly inhibit L-proline uptake, suggesting the\nexistence of D-amino acid transport processes in the parasite.\nUsing an in silico approach, the TcAAAP733 gene was identified as another\nputative proline permease. Its heterologous expression in defective yeasts for the\nproline transporter showed that despite it is not a permease of this amino acid, it\nencodes a functional permease since significant differences when compared to controls\nwere observed when the uptake of an amino acid mixture was assessed.\nThe TcAAAP069 permease overexpression caused an augment not only on\nproline intracellular concentration but also on ATP levels. When the parasites were\nchallenged against oxidative and nitrosative stress situations, or when were treated with\nthe trypanocidal drugs currently used, nifurtimox and benznidazole, it was evidenced\nthat proline itself participates on the response mechanisms to these conditions since\nTc069 parasites were significantly more resistant than control parasites in all the assays.\nThe study of proline transport throughout the culture progress, as an emulating\nsituation of the different nutritional conditions along the parasites life cycle, proved that\nthe permease TcAAAP069 is regulated both in its activity and expression as in its\nsubcellular localization and this produces variations on proline intracellular\nconcentration too. The maximal activity and expression of TcAAAP069 transporter was observed at the beginning of the exponential growth phase, together with the\nlocalization along the plasmatic membrane besides its presence in the flagellar pocket,\nbeing the latter location a common feature to all the members of the TcAAAP family so\nfar characterized. As the culture proceeded, expression and activity of the transporter\ngradually diminished until undetectable levels were reached on the stationary phase.\nThese decreases were accompanied by the loss of the plasma membrane subcellular\nlocalization and finally by the disappearance of the protein, even in the flagellar pocket.\nIn addition, the proline transport was not regulated by substrate availability. Finally, the\nassays involving medium modification or artificially altered cellular density suggest that\nthe observed changes for the proline permease would be directly influenced by an\nunknown density-dependent factor.\nTaking together the biological relevance of proline for the parasite T. cruzi and\nour advances on the knowledge of its transport, synthetic analogues of this amino acid\nwere evaluated in order to study their effect on the permease TcAAAP069 activity. Of\nthe four analogues tested, only the compounds named ITP-1B and ITP-1G significantly\ninhibited the proline transport and also of other amino acid and/or polyamines, but not\nthe uptake of other metabolites. However, exclusively with the analogue ITP-1G the\nability to inhibit the transport was linked with a trypanocidal action. The study with the\nproline analogues confirmed the amino acid and derivatives transporter family TcAAAP\nas a multiple and promising therapeutic target for the development of new treatments\nagainst Chagas disease.
author2	Nowicki, Cristina
author_facet	Nowicki, Cristina Martínez Sayé, Melisa Soledad
format	Tesis doctoral Tesis doctoral acceptedVersion
author	Martínez Sayé, Melisa Soledad
author_sort	Martínez Sayé, Melisa Soledad
title	Transporte y funciones de la prolina en Trypanosoma cruzi : su potencial como blanco terapéutico
title_short	Transporte y funciones de la prolina en Trypanosoma cruzi : su potencial como blanco terapéutico
title_full	Transporte y funciones de la prolina en Trypanosoma cruzi : su potencial como blanco terapéutico
title_fullStr	Transporte y funciones de la prolina en Trypanosoma cruzi : su potencial como blanco terapéutico
title_full_unstemmed	Transporte y funciones de la prolina en Trypanosoma cruzi : su potencial como blanco terapéutico
title_sort	transporte y funciones de la prolina en trypanosoma cruzi : su potencial como blanco terapéutico
publisher	Facultad de Farmacia y Bioquímica
publishDate	2017
url	http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_1867 http://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_1867.dir/1867.PDF
work_keys_str_mv	AT martinezsayemelisasoledad transporteyfuncionesdelaprolinaentrypanosomacruzisupotencialcomoblancoterapeutico
_version_	1766017486204960768
spelling	I28-R145-HWA_18672019-09-25 The amino acid proline has special relevance for the parasite Trypanosoma cruzi,\nthe etiological agent of Chagas disease. This amino acid can be used as an energy and\ncarbon source alternative to glucose, it participates in different stress resistance\nmechanisms, and it is also essential during the cell invasion of metacyclic\ntrypomastigotes and for the life cycle progression inside the mammalian cells.\nIn this work the TcAAAP069 gene of T. cruzi was identified as a proline permease,\nfirst through yeast complementation and then by overexpression in epimastigotes\n(Tc069 parasites). Although the transporter TcAAAP069 proved to be mono-specific, the\nstereoisomer D-proline was able to significantly inhibit L-proline uptake, suggesting the\nexistence of D-amino acid transport processes in the parasite.\nUsing an in silico approach, the TcAAAP733 gene was identified as another\nputative proline permease. Its heterologous expression in defective yeasts for the\nproline transporter showed that despite it is not a permease of this amino acid, it\nencodes a functional permease since significant differences when compared to controls\nwere observed when the uptake of an amino acid mixture was assessed.\nThe TcAAAP069 permease overexpression caused an augment not only on\nproline intracellular concentration but also on ATP levels. When the parasites were\nchallenged against oxidative and nitrosative stress situations, or when were treated with\nthe trypanocidal drugs currently used, nifurtimox and benznidazole, it was evidenced\nthat proline itself participates on the response mechanisms to these conditions since\nTc069 parasites were significantly more resistant than control parasites in all the assays.\nThe study of proline transport throughout the culture progress, as an emulating\nsituation of the different nutritional conditions along the parasites life cycle, proved that\nthe permease TcAAAP069 is regulated both in its activity and expression as in its\nsubcellular localization and this produces variations on proline intracellular\nconcentration too. The maximal activity and expression of TcAAAP069 transporter was observed at the beginning of the exponential growth phase, together with the\nlocalization along the plasmatic membrane besides its presence in the flagellar pocket,\nbeing the latter location a common feature to all the members of the TcAAAP family so\nfar characterized. As the culture proceeded, expression and activity of the transporter\ngradually diminished until undetectable levels were reached on the stationary phase.\nThese decreases were accompanied by the loss of the plasma membrane subcellular\nlocalization and finally by the disappearance of the protein, even in the flagellar pocket.\nIn addition, the proline transport was not regulated by substrate availability. Finally, the\nassays involving medium modification or artificially altered cellular density suggest that\nthe observed changes for the proline permease would be directly influenced by an\nunknown density-dependent factor.\nTaking together the biological relevance of proline for the parasite T. cruzi and\nour advances on the knowledge of its transport, synthetic analogues of this amino acid\nwere evaluated in order to study their effect on the permease TcAAAP069 activity. Of\nthe four analogues tested, only the compounds named ITP-1B and ITP-1G significantly\ninhibited the proline transport and also of other amino acid and/or polyamines, but not\nthe uptake of other metabolites. However, exclusively with the analogue ITP-1G the\nability to inhibit the transport was linked with a trypanocidal action. The study with the\nproline analogues confirmed the amino acid and derivatives transporter family TcAAAP\nas a multiple and promising therapeutic target for the development of new treatments\nagainst Chagas disease. Fil: Martínez Sayé, Melisa Soledad. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Nowicki, Cristina Facultad de Farmacia y Bioquímica Pereira, Claudio A. Martínez Sayé, Melisa Soledad 2017-03-23 La prolina tiene especial relevancia para el parásito Trypanosoma cruzi, agente\ncausal de la enfermedad de Chagas. Este aminoácido puede ser utilizado como fuente\nde carbono y energía alternativa a la glucosa, participa en mecanismos de resistencia a\ndistintos estreses, y es necesario durante la invasión celular de tripomastigotes\nmetacíclicos y para progresar en el ciclo de vida dentro de las células de mamífero.\nEn este trabajo se identificó el gen TcAAAP069 de T. cruzi como una permeasa\nde prolina, primero por complementación en levaduras y luego por sobre-expresión en\nepimastigotes (parásitos Tc069). Si bien la permeasa demostró ser mono-específica el\nestereoisómero D-prolina fue capaz de inhibir significativamente la incorporación de Lprolina,\nsugiriendo la presencia de procesos de transporte de D-aminoácidos en el\nparásito.\nMediante un análisis bioinformático se identificó el gen TcAAAP733 como otra\nposible permeasa de prolina. Su expresión heteróloga en levaduras deficientes para el\ntransportador de prolina demostró que si bien no es una permeasa de este aminoácido,\ncodifica una permeasa funcional ya que se observaron diferencias significativas respecto\nal control cuando se evaluó el transporte de una mezcla de aminoácidos.\nLa sobre-expresión del transportador causó un aumento, no sólo en la\nconcentración intracelular de prolina, sino también en los niveles de ATP. Al enfrentar\nestos parásitos ante situaciones de estrés oxidativo, nitrosativo o con las drogas\ntripanocidas actualmente disponibles, nifurtimox y benznidazol, se evidenció que la\nprolina participa en los mecanismos de respuesta a estas condiciones ya que los\nparásitos Tc069 fueron significativamente más resistentes en todos los casos.\nEl estudio del transporte de prolina durante el progreso del cultivo, como\nsituación que emula las distintas condiciones nutricionales de los parásitos en su ciclo\nde vida, demostró que la permeasa TcAAAP069 está regulada tanto en su actividad y\nexpresión como en su localización subcelular, y esto produce además variaciones en la\nconcentración intracelular de prolina. La máxima actividad y expresión del\ntransportador TcAAAP069 se observó al inicio de la fase exponencial, coincidiendo con la localización de la permeasa a lo largo de la membrana plasmática además de su\npresencia en el bolsillo flagelar, siendo esta última localización una característica común\na todos los miembros de la familia TcAAAP caracterizados hasta el momento. A medida\nque el cultivo progresó, la expresión y la actividad del transportador disminuyeron\ngradualmente hasta alcanzar niveles indetectables en la fase estacionaria. Estas\ndisminuciones fueron acompañadas por la pérdida de la localización subcelular en la\nmembrana y finalmente por la desaparición de la proteína, incluso en el bolsillo flagelar.\nSe observó además que el transporte de prolina no es regulado por la disponibilidad de\nsustrato. Por último, los estudios realizados modificando el medio o alterando\nartificialmente la densidad celular del cultivo sugieren que los cambios observados para\nla permeasa de prolina TcAAAP069 estarían relacionados directamente con un factor\ndensidad-dependiente de naturaleza aún desconocida.\nTeniendo en cuenta la importancia biológica de la prolina en el parásito T. cruzi\ny nuestros avances en el conocimiento de su transporte, se utilizaron análogos sintéticos\ndel aminoácido a fin de estudiar su efecto sobre la actividad de la permeasa TcAAAP069.\nDe los cuatro análogos evaluados, sólo los compuestos denominados ITP-1B e ITP-1G\ninhibieron el transporte de prolina y también de otros aminoácidos y/o poliaminas pero\nno de otros metabolitos. Sin embargo, únicamente con el análogo ITP-1G la capacidad\nde inhibir el transporte estuvo vinculada con una acción tripanocida. El estudio con los\nanálogos de prolina permitió confirmar que la familia de transportadores de\naminoácidos y derivados TcAAAP es un blanco terapéutico múltiple y prometedor para\nel desarrollo de nuevos tratamientos contra la enfermedad de Chagas. application/pdf Fernández Villamil, Silvia Alonso, Guillermo Cazzulo, Juan José Transportador de prolina Trypanosoma cruzi Enfermedad de chagas spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencia de la vida Transporte y funciones de la prolina en Trypanosoma cruzi : su potencial como blanco terapéutico info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_1867 http://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_1867.dir/1867.PDF

Transporte y funciones de la prolina en Trypanosoma cruzi : su potencial como blanco terapéutico

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