Quantitative Proteome Analysis of <italic toggle="yes">Atg5</italic>-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome

ABSTRACT Basal autophagy is crucial for maintenance of cellular homeostasis. ATG5 is an essential protein for autophagosome formation, and its depletion has been extensively used as a tool to disrupt autophagy. Here, we characterize the impact of Atg5 deficiency on the cellular proteome of mouse emb...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Kiran Bala Sharma, Manish Sharma, Suruchi Aggarwal, Amit Kumar Yadav, Shinjini Bhatnagar, Sudhanshu Vrati, Manjula Kalia
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Atg5 IL-6 JAK-STAT TLR2 TMT mass spectrometry cell adhesion Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ef8a5037fb3d49e8a375a55a264e36a4
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:ef8a5037fb3d49e8a375a55a264e36a4
record_format	dspace
spelling	oai:doaj.org-article:ef8a5037fb3d49e8a375a55a264e36a42021-12-02T19:46:17ZQuantitative Proteome Analysis of <italic toggle="yes">Atg5</italic>-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome10.1128/mSystems.00481-192379-5077https://doaj.org/article/ef8a5037fb3d49e8a375a55a264e36a42019-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00481-19https://doaj.org/toc/2379-5077ABSTRACT Basal autophagy is crucial for maintenance of cellular homeostasis. ATG5 is an essential protein for autophagosome formation, and its depletion has been extensively used as a tool to disrupt autophagy. Here, we characterize the impact of Atg5 deficiency on the cellular proteome of mouse embryonic fibroblasts (MEFs). Using a tandem mass tagging (TMT)-based quantitative proteomics analysis, we observe that 14% of identified proteins show dysregulated levels in atg5−/− MEFs. These proteins were distributed across diverse biological processes, such as cell adhesion, development, differentiation, transport, metabolism, and immune responses. Several of the upregulated proteins were receptors involved in transforming growth factor β (TGF-β) signaling, JAK-STAT signaling, junction adhesion, and interferon/cytokine-receptor interactions and were validated as autophagy substrates. Nearly equal numbers of proteins, including several lysosomal proteins and enzymes, were downregulated, suggesting a complex role of autophagy/ATG5 in regulating their levels. The atg5−/− MEFs had lower levels of key immune sensors and effectors, including Toll-like receptor 2 (TLR2), interferon regulatory factor 3 (IRF3), IRF7, MLKL, and STAT1/3/5/6, which were restored by reexpression of ATG5. While these cells could efficiently mount a type I interferon response to the double-stranded RNA (dsRNA) mimic poly(I·C), they were compromised in their inflammatory response to the bacterial pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and Pam3CSK4. Transcriptional activation and secretion of interleukin-6 (IL-6) in these cells could be recovered by ATG5 expression, supporting the role of autophagy in the TLR2-induced inflammatory response. This study provides a key resource for understanding the effect of autophagy/ATG5 deficiency on the fibroblast proteome. IMPORTANCE Autophagy performs housekeeping functions for cells and maintains a functional mode by degrading damaged proteins and organelles and providing energy under starvation conditions. The process is tightly regulated by the evolutionarily conserved Atg genes, of which Atg5 is one such crucial mediator. Here, we have done a comprehensive quantitative proteome analysis of mouse embryonic fibroblasts that lack a functional autophagy pathway (Atg5 knockout). We observe that 14% of the identified cellular proteome is remodeled, and several proteins distributed across diverse cellular processes with functions in signaling, cell adhesion, development, and immunity show either higher or lower levels under autophagy-deficient conditions. These cells have lower levels of crucial immune proteins that are required to mount a protective inflammatory response. This study will serve as a valuable resource to determine the role of autophagy in modulating specific protein levels in cells. Author Video: An author video summary of this article is available.Kiran Bala SharmaManish SharmaSuruchi AggarwalAmit Kumar YadavShinjini BhatnagarSudhanshu VratiManjula KaliaAmerican Society for MicrobiologyarticleAtg5IL-6JAK-STATTLR2TMT mass spectrometrycell adhesionMicrobiologyQR1-502ENmSystems, Vol 4, Iss 6 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Atg5 IL-6 JAK-STAT TLR2 TMT mass spectrometry cell adhesion Microbiology QR1-502
spellingShingle	Atg5 IL-6 JAK-STAT TLR2 TMT mass spectrometry cell adhesion Microbiology QR1-502 Kiran Bala Sharma Manish Sharma Suruchi Aggarwal Amit Kumar Yadav Shinjini Bhatnagar Sudhanshu Vrati Manjula Kalia Quantitative Proteome Analysis of <italic toggle="yes">Atg5</italic>-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome
description	ABSTRACT Basal autophagy is crucial for maintenance of cellular homeostasis. ATG5 is an essential protein for autophagosome formation, and its depletion has been extensively used as a tool to disrupt autophagy. Here, we characterize the impact of Atg5 deficiency on the cellular proteome of mouse embryonic fibroblasts (MEFs). Using a tandem mass tagging (TMT)-based quantitative proteomics analysis, we observe that 14% of identified proteins show dysregulated levels in atg5−/− MEFs. These proteins were distributed across diverse biological processes, such as cell adhesion, development, differentiation, transport, metabolism, and immune responses. Several of the upregulated proteins were receptors involved in transforming growth factor β (TGF-β) signaling, JAK-STAT signaling, junction adhesion, and interferon/cytokine-receptor interactions and were validated as autophagy substrates. Nearly equal numbers of proteins, including several lysosomal proteins and enzymes, were downregulated, suggesting a complex role of autophagy/ATG5 in regulating their levels. The atg5−/− MEFs had lower levels of key immune sensors and effectors, including Toll-like receptor 2 (TLR2), interferon regulatory factor 3 (IRF3), IRF7, MLKL, and STAT1/3/5/6, which were restored by reexpression of ATG5. While these cells could efficiently mount a type I interferon response to the double-stranded RNA (dsRNA) mimic poly(I·C), they were compromised in their inflammatory response to the bacterial pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and Pam3CSK4. Transcriptional activation and secretion of interleukin-6 (IL-6) in these cells could be recovered by ATG5 expression, supporting the role of autophagy in the TLR2-induced inflammatory response. This study provides a key resource for understanding the effect of autophagy/ATG5 deficiency on the fibroblast proteome. IMPORTANCE Autophagy performs housekeeping functions for cells and maintains a functional mode by degrading damaged proteins and organelles and providing energy under starvation conditions. The process is tightly regulated by the evolutionarily conserved Atg genes, of which Atg5 is one such crucial mediator. Here, we have done a comprehensive quantitative proteome analysis of mouse embryonic fibroblasts that lack a functional autophagy pathway (Atg5 knockout). We observe that 14% of the identified cellular proteome is remodeled, and several proteins distributed across diverse cellular processes with functions in signaling, cell adhesion, development, and immunity show either higher or lower levels under autophagy-deficient conditions. These cells have lower levels of crucial immune proteins that are required to mount a protective inflammatory response. This study will serve as a valuable resource to determine the role of autophagy in modulating specific protein levels in cells. Author Video: An author video summary of this article is available.
format	article
author	Kiran Bala Sharma Manish Sharma Suruchi Aggarwal Amit Kumar Yadav Shinjini Bhatnagar Sudhanshu Vrati Manjula Kalia
author_facet	Kiran Bala Sharma Manish Sharma Suruchi Aggarwal Amit Kumar Yadav Shinjini Bhatnagar Sudhanshu Vrati Manjula Kalia
author_sort	Kiran Bala Sharma
title	Quantitative Proteome Analysis of <italic toggle="yes">Atg5</italic>-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome
title_short	Quantitative Proteome Analysis of <italic toggle="yes">Atg5</italic>-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome
title_full	Quantitative Proteome Analysis of <italic toggle="yes">Atg5</italic>-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome
title_fullStr	Quantitative Proteome Analysis of <italic toggle="yes">Atg5</italic>-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome
title_full_unstemmed	Quantitative Proteome Analysis of <italic toggle="yes">Atg5</italic>-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome
title_sort	quantitative proteome analysis of <italic toggle="yes">atg5</italic>-deficient mouse embryonic fibroblasts reveals the range of the autophagy-modulated basal cellular proteome
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/ef8a5037fb3d49e8a375a55a264e36a4
work_keys_str_mv	AT kiranbalasharma quantitativeproteomeanalysisofitalictoggleyesatg5italicdeficientmouseembryonicfibroblastsrevealstherangeoftheautophagymodulatedbasalcellularproteome AT manishsharma quantitativeproteomeanalysisofitalictoggleyesatg5italicdeficientmouseembryonicfibroblastsrevealstherangeoftheautophagymodulatedbasalcellularproteome AT suruchiaggarwal quantitativeproteomeanalysisofitalictoggleyesatg5italicdeficientmouseembryonicfibroblastsrevealstherangeoftheautophagymodulatedbasalcellularproteome AT amitkumaryadav quantitativeproteomeanalysisofitalictoggleyesatg5italicdeficientmouseembryonicfibroblastsrevealstherangeoftheautophagymodulatedbasalcellularproteome AT shinjinibhatnagar quantitativeproteomeanalysisofitalictoggleyesatg5italicdeficientmouseembryonicfibroblastsrevealstherangeoftheautophagymodulatedbasalcellularproteome AT sudhanshuvrati quantitativeproteomeanalysisofitalictoggleyesatg5italicdeficientmouseembryonicfibroblastsrevealstherangeoftheautophagymodulatedbasalcellularproteome AT manjulakalia quantitativeproteomeanalysisofitalictoggleyesatg5italicdeficientmouseembryonicfibroblastsrevealstherangeoftheautophagymodulatedbasalcellularproteome
_version_	1718376059131396096

Quantitative Proteome Analysis of <italic toggle="yes">Atg5</italic>-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome

Ejemplares similares