Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms

Autores
Iribarren, Paula Ana; Berazategui, María Agustina; Bayona, Julio Cesar; Almeida, Igor; Cazzulo, Juan José; Álvarez, Vanina Eder
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitinlike MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T. brucei. We expressed various tagged variants of SUMO from the endogenous locus. HisHA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a HisHA-TbSUMOT106K version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others.
Materia
Ciencias Biológicas
trypanosoma
SUMO
proteomic
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-nd/4.0/
Repositorio
CIC Digital (CICBA)
Institución
Comisión de Investigaciones Científicas de la Provincia de Buenos Aires
OAI Identificador
oai:digital.cic.gba.gob.ar:11746/7295

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network_acronym_str CICBA
repository_id_str 9441
network_name_str CIC Digital (CICBA)
spelling Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic formsIribarren, Paula AnaBerazategui, María AgustinaBayona, Julio CesarAlmeida, IgorCazzulo, Juan JoséÁlvarez, Vanina EderCiencias BiológicastrypanosomaSUMOproteomicSUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitinlike MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T. brucei. We expressed various tagged variants of SUMO from the endogenous locus. HisHA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a HisHA-TbSUMOT106K version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others.2015-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttps://digital.cic.gba.gob.ar/handle/11746/7295enginfo:eu-repo/semantics/altIdentifier/doi/10.1111/cmi.12467info:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-nd/4.0/reponame:CIC Digital (CICBA)instname:Comisión de Investigaciones Científicas de la Provincia de Buenos Airesinstacron:CICBA2025-09-29T13:39:47Zoai:digital.cic.gba.gob.ar:11746/7295Institucionalhttp://digital.cic.gba.gob.arOrganismo científico-tecnológicoNo correspondehttp://digital.cic.gba.gob.ar/oai/snrdmarisa.degiusti@sedici.unlp.edu.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:94412025-09-29 13:39:48.087CIC Digital (CICBA) - Comisión de Investigaciones Científicas de la Provincia de Buenos Airesfalse
dc.title.none.fl_str_mv Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms
title Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms
spellingShingle Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms
Iribarren, Paula Ana
Ciencias Biológicas
trypanosoma
SUMO
proteomic
title_short Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms
title_full Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms
title_fullStr Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms
title_full_unstemmed Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms
title_sort Different proteomic strategies to identify genuine SUMO targets and their modification sites in Trypanosoma brucei procyclic forms
dc.creator.none.fl_str_mv Iribarren, Paula Ana
Berazategui, María Agustina
Bayona, Julio Cesar
Almeida, Igor
Cazzulo, Juan José
Álvarez, Vanina Eder
author Iribarren, Paula Ana
author_facet Iribarren, Paula Ana
Berazategui, María Agustina
Bayona, Julio Cesar
Almeida, Igor
Cazzulo, Juan José
Álvarez, Vanina Eder
author_role author
author2 Berazategui, María Agustina
Bayona, Julio Cesar
Almeida, Igor
Cazzulo, Juan José
Álvarez, Vanina Eder
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv Ciencias Biológicas
trypanosoma
SUMO
proteomic
topic Ciencias Biológicas
trypanosoma
SUMO
proteomic
dc.description.none.fl_txt_mv SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitinlike MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T. brucei. We expressed various tagged variants of SUMO from the endogenous locus. HisHA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a HisHA-TbSUMOT106K version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others.
description SUMOylation is an important post-translational modification conserved in eukaryotic organisms. In Trypanosoma brucei, SUMO (Small Ubiquitinlike MOdifier) is essential in procyclic and bloodstream forms. Furthermore, SUMO has been linked to the antigenic variation process, as a highly SUMOylated focus was recently identified within chromatin-associated proteins of the active variant surface glycoprotein expression site. We aimed to establish a reliable strategy to identify SUMO conjugates in T. brucei. We expressed various tagged variants of SUMO from the endogenous locus. HisHA-TbSUMO was useful to validate the tag functionality but SUMO conjugates were not enriched enough over contaminants after affinity purification. A Lys-deficient SUMO version, created to reduce contaminants by Lys-C digestion, was able to overcome this issue but did not allow mapping many SUMOylation sites. This cell line was in turn useful to demonstrate that polySUMO chains are not essential for parasite viability. Finally, a HisHA-TbSUMOT106K version allowed the purification of SUMO conjugates and, after digestion with Lys-C, the enrichment for diGly-Lys peptides using specific antibodies. This site-specific proteomic strategy led us to identify 45 SUMOylated proteins and 53 acceptor sites unambiguously. SUMOylated proteins belong mainly to nuclear processes, such as DNA replication and repair, transcription, rRNA biogenesis and chromatin remodelling, among others.
publishDate 2015
dc.date.none.fl_str_mv 2015-10
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
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info:ar-repo/semantics/articulo
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status_str publishedVersion
dc.identifier.none.fl_str_mv https://digital.cic.gba.gob.ar/handle/11746/7295
url https://digital.cic.gba.gob.ar/handle/11746/7295
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1111/cmi.12467
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
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eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-nd/4.0/
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dc.source.none.fl_str_mv reponame:CIC Digital (CICBA)
instname:Comisión de Investigaciones Científicas de la Provincia de Buenos Aires
instacron:CICBA
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collection CIC Digital (CICBA)
instname_str Comisión de Investigaciones Científicas de la Provincia de Buenos Aires
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institution CICBA
repository.name.fl_str_mv CIC Digital (CICBA) - Comisión de Investigaciones Científicas de la Provincia de Buenos Aires
repository.mail.fl_str_mv marisa.degiusti@sedici.unlp.edu.ar
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