Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling

Autores
Bonnet, Laura Vanesa; Flores Martín, Jésica Belén; Palandri, Anabela; Hallak, Marta Elena
Año de publicación
2024
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Background: Arginyltransferase (Ate1) orchestrates posttranslational protein arginylation, a pivotal regulator of cellular proteolytic processes. In eukaryotic cells, two interconnected systems the ubiquitin proteasome system (UPS) and macroautophagy mediate proteolysis and cooperate to maintain quality protein control and cellular homeostasis. Previous studies have shown that N-terminal arginylation facilitates protein degradation through the UPS. Dysregulation of this machinery triggers p62-mediated autophagy to ensure proper substrate processing. Nevertheless,how Ate1 operates through this intricate mechanism remains elusive.Methods: We investigated Ate1 subcellular distribution through confocal microscopy and biochemical assays using cells transiently or stably expressing either endogenous Ate1 or a GFP-tagged Ate1 isoform transfected in CHO-K1 or MEFs, respectively. To assess Ate1 and p62-cargo clustering, we analyzed their colocalization and multimerization status by immunofuorescence and nonreducing immunoblotting, respectively. Additionally, we employed Ate1 KO cells to examine the role of Ate1 in autophagy. Ate1 KO MEFs cells stably expressing GFP-tagged Ate1-1 isoform wereused as a model for phenotype rescue. Autophagy dynamics were evaluated by analyzing LC3B turnover and p62/SQSTM1 levels under both steady-state and serum-starvation conditions, through immunoblotting and immunofuorescence. We determined mTORC1/AMPk activation by assessing mTOR and AMPk phosphorylation through immunoblotting, while mTORC1 lysosomal localization was monitored by confocal microscopy.Results: Here, we report a multifaceted role for Ate1 in the autophagic process, wherein it clusters with p62, facilitates autophagic clearance, and modulates its signaling. Mechanistically, we found that cell-specifc inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic fux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical signifcance was assessed using a two-sided unpaired t test with a signifcance threshold set at P<0.05.Conclusions: Our fndings uncover a critical housekeeping role of Ate1 in mTORC1/AMPk-regulated autophagy, as a potential therapeutic target related to this pathway, that is dysregulated in many neurodegenerative and cancer diseases.
Fil: Bonnet, Laura Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
Fil: Flores Martín, Jésica Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina
Fil: Palandri, Anabela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
Fil: Hallak, Marta Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
Materia
ATE1
P62
AUTOPHAGY
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/256501
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/256501
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signalingBonnet, Laura VanesaFlores Martín, Jésica BelénPalandri, AnabelaHallak, Marta ElenaATE1P62AUTOPHAGYhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Background: Arginyltransferase (Ate1) orchestrates posttranslational protein arginylation, a pivotal regulator of cellular proteolytic processes. In eukaryotic cells, two interconnected systems the ubiquitin proteasome system (UPS) and macroautophagy mediate proteolysis and cooperate to maintain quality protein control and cellular homeostasis. Previous studies have shown that N-terminal arginylation facilitates protein degradation through the UPS. Dysregulation of this machinery triggers p62-mediated autophagy to ensure proper substrate processing. Nevertheless,how Ate1 operates through this intricate mechanism remains elusive.Methods: We investigated Ate1 subcellular distribution through confocal microscopy and biochemical assays using cells transiently or stably expressing either endogenous Ate1 or a GFP-tagged Ate1 isoform transfected in CHO-K1 or MEFs, respectively. To assess Ate1 and p62-cargo clustering, we analyzed their colocalization and multimerization status by immunofuorescence and nonreducing immunoblotting, respectively. Additionally, we employed Ate1 KO cells to examine the role of Ate1 in autophagy. Ate1 KO MEFs cells stably expressing GFP-tagged Ate1-1 isoform wereused as a model for phenotype rescue. Autophagy dynamics were evaluated by analyzing LC3B turnover and p62/SQSTM1 levels under both steady-state and serum-starvation conditions, through immunoblotting and immunofuorescence. We determined mTORC1/AMPk activation by assessing mTOR and AMPk phosphorylation through immunoblotting, while mTORC1 lysosomal localization was monitored by confocal microscopy.Results: Here, we report a multifaceted role for Ate1 in the autophagic process, wherein it clusters with p62, facilitates autophagic clearance, and modulates its signaling. Mechanistically, we found that cell-specifc inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic fux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical signifcance was assessed using a two-sided unpaired t test with a signifcance threshold set at P<0.05.Conclusions: Our fndings uncover a critical housekeeping role of Ate1 in mTORC1/AMPk-regulated autophagy, as a potential therapeutic target related to this pathway, that is dysregulated in many neurodegenerative and cancer diseases.Fil: Bonnet, Laura Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Flores Martín, Jésica Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Palandri, Anabela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Hallak, Marta Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaBioMed Central2024-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/256501Bonnet, Laura Vanesa; Flores Martín, Jésica Belén; Palandri, Anabela; Hallak, Marta Elena; Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling; BioMed Central; Cell Communication and Signaling; 22; 1; 1-2024; 1-171478-811XCONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1186/s12964-024-01499-9info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-10T13:04:20Zoai:ri.conicet.gov.ar:11336/256501instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-10 13:04:20.904CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling
title Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling
spellingShingle Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling
Bonnet, Laura Vanesa
ATE1
P62
AUTOPHAGY
title_short Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling
title_full Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling
title_fullStr Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling
title_full_unstemmed Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling
title_sort Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling
dc.creator.none.fl_str_mv Bonnet, Laura Vanesa
Flores Martín, Jésica Belén
Palandri, Anabela
Hallak, Marta Elena
author Bonnet, Laura Vanesa
author_facet Bonnet, Laura Vanesa
Flores Martín, Jésica Belén
Palandri, Anabela
Hallak, Marta Elena
author_role author
author2 Flores Martín, Jésica Belén
Palandri, Anabela
Hallak, Marta Elena
author2_role author
author
author
dc.subject.none.fl_str_mv ATE1
P62
AUTOPHAGY
topic ATE1
P62
AUTOPHAGY
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Background: Arginyltransferase (Ate1) orchestrates posttranslational protein arginylation, a pivotal regulator of cellular proteolytic processes. In eukaryotic cells, two interconnected systems the ubiquitin proteasome system (UPS) and macroautophagy mediate proteolysis and cooperate to maintain quality protein control and cellular homeostasis. Previous studies have shown that N-terminal arginylation facilitates protein degradation through the UPS. Dysregulation of this machinery triggers p62-mediated autophagy to ensure proper substrate processing. Nevertheless,how Ate1 operates through this intricate mechanism remains elusive.Methods: We investigated Ate1 subcellular distribution through confocal microscopy and biochemical assays using cells transiently or stably expressing either endogenous Ate1 or a GFP-tagged Ate1 isoform transfected in CHO-K1 or MEFs, respectively. To assess Ate1 and p62-cargo clustering, we analyzed their colocalization and multimerization status by immunofuorescence and nonreducing immunoblotting, respectively. Additionally, we employed Ate1 KO cells to examine the role of Ate1 in autophagy. Ate1 KO MEFs cells stably expressing GFP-tagged Ate1-1 isoform wereused as a model for phenotype rescue. Autophagy dynamics were evaluated by analyzing LC3B turnover and p62/SQSTM1 levels under both steady-state and serum-starvation conditions, through immunoblotting and immunofuorescence. We determined mTORC1/AMPk activation by assessing mTOR and AMPk phosphorylation through immunoblotting, while mTORC1 lysosomal localization was monitored by confocal microscopy.Results: Here, we report a multifaceted role for Ate1 in the autophagic process, wherein it clusters with p62, facilitates autophagic clearance, and modulates its signaling. Mechanistically, we found that cell-specifc inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic fux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical signifcance was assessed using a two-sided unpaired t test with a signifcance threshold set at P<0.05.Conclusions: Our fndings uncover a critical housekeeping role of Ate1 in mTORC1/AMPk-regulated autophagy, as a potential therapeutic target related to this pathway, that is dysregulated in many neurodegenerative and cancer diseases.
Fil: Bonnet, Laura Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
Fil: Flores Martín, Jésica Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina
Fil: Palandri, Anabela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
Fil: Hallak, Marta Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
description Background: Arginyltransferase (Ate1) orchestrates posttranslational protein arginylation, a pivotal regulator of cellular proteolytic processes. In eukaryotic cells, two interconnected systems the ubiquitin proteasome system (UPS) and macroautophagy mediate proteolysis and cooperate to maintain quality protein control and cellular homeostasis. Previous studies have shown that N-terminal arginylation facilitates protein degradation through the UPS. Dysregulation of this machinery triggers p62-mediated autophagy to ensure proper substrate processing. Nevertheless,how Ate1 operates through this intricate mechanism remains elusive.Methods: We investigated Ate1 subcellular distribution through confocal microscopy and biochemical assays using cells transiently or stably expressing either endogenous Ate1 or a GFP-tagged Ate1 isoform transfected in CHO-K1 or MEFs, respectively. To assess Ate1 and p62-cargo clustering, we analyzed their colocalization and multimerization status by immunofuorescence and nonreducing immunoblotting, respectively. Additionally, we employed Ate1 KO cells to examine the role of Ate1 in autophagy. Ate1 KO MEFs cells stably expressing GFP-tagged Ate1-1 isoform wereused as a model for phenotype rescue. Autophagy dynamics were evaluated by analyzing LC3B turnover and p62/SQSTM1 levels under both steady-state and serum-starvation conditions, through immunoblotting and immunofuorescence. We determined mTORC1/AMPk activation by assessing mTOR and AMPk phosphorylation through immunoblotting, while mTORC1 lysosomal localization was monitored by confocal microscopy.Results: Here, we report a multifaceted role for Ate1 in the autophagic process, wherein it clusters with p62, facilitates autophagic clearance, and modulates its signaling. Mechanistically, we found that cell-specifc inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic fux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical signifcance was assessed using a two-sided unpaired t test with a signifcance threshold set at P<0.05.Conclusions: Our fndings uncover a critical housekeeping role of Ate1 in mTORC1/AMPk-regulated autophagy, as a potential therapeutic target related to this pathway, that is dysregulated in many neurodegenerative and cancer diseases.
publishDate 2024
dc.date.none.fl_str_mv 2024-01
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/256501
Bonnet, Laura Vanesa; Flores Martín, Jésica Belén; Palandri, Anabela; Hallak, Marta Elena; Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling; BioMed Central; Cell Communication and Signaling; 22; 1; 1-2024; 1-17
1478-811X
CONICET Digital
CONICET
url http://hdl.handle.net/11336/256501
identifier_str_mv Bonnet, Laura Vanesa; Flores Martín, Jésica Belén; Palandri, Anabela; Hallak, Marta Elena; Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling; BioMed Central; Cell Communication and Signaling; 22; 1; 1-2024; 1-17
1478-811X
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1186/s12964-024-01499-9
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv BioMed Central
publisher.none.fl_str_mv BioMed Central
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.993085

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