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Authors: Juretschke, Thomas
Title: Identification of DNA damage-induced autophagic cargo by mass-spectrometry based proteomics
Online publication date: 10-Feb-2022
Year of first publication: 2022
Language: english
Abstract: Autophagy is a catabolic recycling pathway initiated under periods of starvation with energy-saving and cytoprotective effects. Previously considered a non-targeted process that degrades bulk cytoplasmic material, the discovery of autophagy receptors revealed that autophagy also leads to the targeted turnover of different cargo. Upon its perception, DNA damage triggers tailor-made signaling and repair pathways, collectively designated as the DNA damage response (DDR) that can also result in apoptosis or cellular senescence. Kinases that orchestrate the DDR are ATM, ATR and DNA-PKcs. Autophagy succeeds DNA damage, but the signaling pathways required to transmit the information for autophagy activation from the nucleus to the cytoplasm as well as its purpose remain poorly understood. This study focused on two aspects of DNA damage-induced autophagy. First, I investigated whether the DDR kinases play a role in the activation of autophagy after DNA double strand breaks. Second, I employed mass spectrometry (MS)-based proteomics to probe the cargo of DNA damage-induced autophagy to investigate its relevance in the DDR. To assess the role of the DDR kinases in DNA damage-induced autophagy, the conversion of the autophagy marker protein LC3B into its lipidated form and the degradation of the receptor p62 were monitored in DDR kinase knockdown cells. As a complementary approach, the tandem-LC3B assay was engaged to measure DNA damage-induced autophagy in the presence of DDR kinase inhibitors. Using different read-out systems, we came to indecisive results indicating no, mild or strong involvement of the DDR kinases in DNA damage-induced autophagy. The off-target effects of commonly used DDR kinase inhibitors that might also cause the inhibition of the phosphatidylinositol-3-kinase VPS34 could explain observed inconsistency between the results obtained in knockdown and chemical inhibitor-treated cells. I established a MS-based proximity proteomics approach to identify systematically DNA damage-induced autophagic cargo. Applying this technique, many known and previously undescribed putative cargo proteins were identified. Computational analyses permitted to associate the identified cargo with focal adhesions proteins, ribosomal proteins, the subunits of the TRiQ/CCT chaperonin complex as well as with nuclear-localized proteins. The proteinase K protection assay enabled to validate most of the selected proteins as autophagic cargo. Ultimately, the cargo that was identified indicates that micronuclei might be degraded by autophagy after genotoxic stress. Further, identification of focal adhesions proteins and validation of the metastasis suppressor NME1 could indicate for a metastasis-promoting role of autophagy after treatment with chemotherapeutic agents. This study presents the first approach to investigate DNA damage-induced autophagic cargo using proximity proteomics and reveals a role for autophagy in the degradation of nuclear proteins in human cells.
DDC: 570 Biowissenschaften
570 Life sciences
Institution: Johannes Gutenberg-Universität Mainz
Department: FB 10 Biologie
Place: Mainz
URN: urn:nbn:de:hebis:77-openscience-de724be1-9d09-42d4-973e-90fa778c04484
Version: Original work
Publication type: Dissertation
License: In Copyright
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Extent: V, 107 Seiten, Illustrationen, Diagramme
Appears in collections:JGU-Publikationen

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