Quantitative proteomic analysis of Parkin substrates in Drosophila neurons
Abstract Background Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson’s Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2017-04-01
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| Series: | Molecular Neurodegeneration |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13024-017-0170-3 |
| _version_ | 1819146027347738624 |
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| author | Aitor Martinez Benoit Lectez Juanma Ramirez Oliver Popp James D. Sutherland Sylvie Urbé Gunnar Dittmar Michael J. Clague Ugo Mayor |
| author_facet | Aitor Martinez Benoit Lectez Juanma Ramirez Oliver Popp James D. Sutherland Sylvie Urbé Gunnar Dittmar Michael J. Clague Ugo Mayor |
| author_sort | Aitor Martinez |
| collection | DOAJ |
| description | Abstract Background Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson’s Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings. Methods Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells. Results We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells. Conclusions Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation. |
| first_indexed | 2024-12-22T13:07:24Z |
| format | Article |
| id | doaj.art-27faf3193c6f4049a8c444845e938b87 |
| institution | Directory Open Access Journal |
| issn | 1750-1326 |
| language | English |
| last_indexed | 2024-12-22T13:07:24Z |
| publishDate | 2017-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Molecular Neurodegeneration |
| spelling | doaj.art-27faf3193c6f4049a8c444845e938b872022-12-21T18:24:50ZengBMCMolecular Neurodegeneration1750-13262017-04-0112111910.1186/s13024-017-0170-3Quantitative proteomic analysis of Parkin substrates in Drosophila neuronsAitor Martinez0Benoit Lectez1Juanma Ramirez2Oliver Popp3James D. Sutherland4Sylvie Urbé5Gunnar Dittmar6Michael J. Clague7Ugo Mayor8Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU)Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU)Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU)Max Delbrück Center for Molecular MedicineFunctional Genomics Unit, CIC bioGUNEDepartment of Cellular and Molecular Physiology, Institute of Translational Medicine, University of LiverpoolDepartment of Oncology, Luxembourg Institute of HealthDepartment of Cellular and Molecular Physiology, Institute of Translational Medicine, University of LiverpoolDepartment of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU)Abstract Background Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson’s Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings. Methods Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells. Results We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells. Conclusions Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation.http://link.springer.com/article/10.1186/s13024-017-0170-3Parkin (PARK2)Parkin substratesUbiquitinationVPS35NeurodegenerationIn vivo |
| spellingShingle | Aitor Martinez Benoit Lectez Juanma Ramirez Oliver Popp James D. Sutherland Sylvie Urbé Gunnar Dittmar Michael J. Clague Ugo Mayor Quantitative proteomic analysis of Parkin substrates in Drosophila neurons Molecular Neurodegeneration Parkin (PARK2) Parkin substrates Ubiquitination VPS35 Neurodegeneration In vivo |
| title | Quantitative proteomic analysis of Parkin substrates in Drosophila neurons |
| title_full | Quantitative proteomic analysis of Parkin substrates in Drosophila neurons |
| title_fullStr | Quantitative proteomic analysis of Parkin substrates in Drosophila neurons |
| title_full_unstemmed | Quantitative proteomic analysis of Parkin substrates in Drosophila neurons |
| title_short | Quantitative proteomic analysis of Parkin substrates in Drosophila neurons |
| title_sort | quantitative proteomic analysis of parkin substrates in drosophila neurons |
| topic | Parkin (PARK2) Parkin substrates Ubiquitination VPS35 Neurodegeneration In vivo |
| url | http://link.springer.com/article/10.1186/s13024-017-0170-3 |
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