Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling
Abstract 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 main...
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| Format: | Article |
| Language: | English |
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BMC
2024-01-01
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| Series: | Cell Communication and Signaling |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12964-024-01499-9 |
| _version_ | 1797273819826618368 |
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| author | Laura V. Bonnet Anabela Palandri Jesica B. Flores-Martin Marta E. Hallak |
| author_facet | Laura V. Bonnet Anabela Palandri Jesica B. Flores-Martin Marta E. Hallak |
| author_sort | Laura V. Bonnet |
| collection | DOAJ |
| description | Abstract 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 immunofluorescence 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 were used 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 immunofluorescence. 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-specific inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic flux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical significance was assessed using a two-sided unpaired t test with a significance threshold set at P<0.05. Conclusions Our findings 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. |
| first_indexed | 2024-03-07T14:49:41Z |
| format | Article |
| id | doaj.art-80774d2b7ef044ca81148c1ba202484a |
| institution | Directory Open Access Journal |
| issn | 1478-811X |
| language | English |
| last_indexed | 2024-03-07T14:49:41Z |
| publishDate | 2024-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell Communication and Signaling |
| spelling | doaj.art-80774d2b7ef044ca81148c1ba202484a2024-03-05T19:47:06ZengBMCCell Communication and Signaling1478-811X2024-01-0122111710.1186/s12964-024-01499-9Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signalingLaura V. Bonnet0Anabela Palandri1Jesica B. Flores-Martin2Marta E. Hallak3Departamento de Química Biológica Ranwel Caputto, Universidad Nacional de CórdobaDepartamento de Química Biológica Ranwel Caputto, Universidad Nacional de CórdobaDepartamento de Química Biológica Ranwel Caputto, Universidad Nacional de CórdobaDepartamento de Química Biológica Ranwel Caputto, Universidad Nacional de CórdobaAbstract 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 immunofluorescence 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 were used 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 immunofluorescence. 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-specific inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic flux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical significance was assessed using a two-sided unpaired t test with a significance threshold set at P<0.05. Conclusions Our findings 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.https://doi.org/10.1186/s12964-024-01499-9ArginylationArginyltransferase 1AutophagymTORC1Posttranslational modificationp62/SQSTM1 |
| spellingShingle | Laura V. Bonnet Anabela Palandri Jesica B. Flores-Martin Marta E. Hallak Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling Cell Communication and Signaling Arginylation Arginyltransferase 1 Autophagy mTORC1 Posttranslational modification p62/SQSTM1 |
| title | 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_short | Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling |
| title_sort | arginyltransferase 1 modulates p62 driven autophagy via mtorc1 ampk signaling |
| topic | Arginylation Arginyltransferase 1 Autophagy mTORC1 Posttranslational modification p62/SQSTM1 |
| url | https://doi.org/10.1186/s12964-024-01499-9 |
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