Dataset Information

Highly Multiplexed Quantitative Mass Spectrometry Analysis of Ubiquitylomes

ABSTRACT: Systems-wide quantitative analysis of ubiquitylomes has proven to be a valuable tool for elucidating targets and mechanisms of the ubiquitin-driven signaling systems, as well as gaining insights in neurodegenerative diseases and cancer. Current mass spectrometry methods for ubiquitylome detection require large amounts of starting material and rely on stochastic data collection to increase replicate analyses. We describe a method compatible with cell line and tissue samples for large-scale quantification of 5,000-9,000 ubiquitylation forms across ten samples simultaneously. Using this method we reveal site-specific ubiquitylation in mammalian brain and liver tissues, as well as in cancer cells undergoing proteasome inhibition. To demonstrate the power of the approach for signal-dependent ubiquitylation, we examined protein and ubiquitylation dynamics for mitochondria undergoing PARKIN and PINK1-dependent mitophagy. This analysis revealed the largest collection of PARKIN and PINK1-dependent ubiquitylation targets to date in a single experiment, and also revealed a subset of proteins recruited to the mitochondria during mitophagy.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Brain, Liver, Cell Culture

SUBMITTER: Marta Isasa

LAB HEAD: Steven P. Gygi

PROVIDER: PXD004628 | Pride | 2016-10-10

REPOSITORIES: Pride

ACCESS DATA

Publications

Highly Multiplexed Quantitative Mass Spectrometry Analysis of Ubiquitylomes.

Rose Christopher M CM Isasa Marta M Ordureau Alban A Prado Miguel A MA Beausoleil Sean A SA Jedrychowski Mark P MP Finley Daniel J DJ Harper J Wade JW Gygi Steven P SP

Cell systems 20160922 4

System-wide quantitative analysis of ubiquitylomes has proven to be a valuable tool for elucidating targets and mechanisms of the ubiquitin-driven signaling systems, as well as gaining insights into neurodegenerative diseases and cancer. Current mass spectrometry methods for ubiquitylome detection require large amounts of starting material and rely on stochastic data collection to increase replicate analyses. We describe a method compatible with cell line and tissue samples for large-scale quant ...[more]

PMID: 27667366

Similar Datasets

Project description:This study aims to investigate the role and mechanism of DEK in asthmatic airway inflammation and in regulating PTEN-induced putative kinase 1 (PINK1)-Parkin mediated mitophagy, NLRP3 (NOD-like receptor family pyrin domain containing 3) inflammasome activation, and apoptosis. We found that recombinant DEK protein (rmDEK) promoted eosinophils recruitment, mitochondrial fragmentation, and outer membrane 20 (TOM20) and LC3 co-localization representing mitophagosomes in bronchoalveolar lavage fluid (BALF) in house dust mite (HDM) induced-asthma. rmDEK also reduced co-localization of mitochondrial fusion protein mitofusin1 (MFN1) and mitochondria, and the protein level of manganese superoxide dismutase (MnSOD), enhanced microtubule-associated protein1 light chain 3 (LC3) and voltage-dependent anion channels (VDAC) co-localization which also represent the mitophagosomes in airway epithelial cells, furthermore, increased dynamin-related protein 1 (DRP1) expression, PINK1-Parkin-mediated mitophagy, NLRP3 inflammasome activation, and apoptosis. In the DEK knockout mice, HDM induced asthmatic airway inflammation, MnSOD, PINK1-Parkin protein level, Parkin mediated mitophagy characterized by LC3 and Parkin co-localization in the airways, ROS generation, NLRP3 inflammation and apoptosis were fully reversed. Similar effects of rmDEK were also observed in the BEAS-2B cells, which were rescued by the autophagy inhibitor 3-MA. Moreover, DEK silencing diminished the Parkin, LC3, DRP1 translocation to mitochondria; as well as mitochondrial ROS; TOM20 and mitochondrial DNA mediated mitochondrial oxidative damage. ChIP-sequence analysis showed that DEK was enriched on the AAA domain-containing protein 3A (ATAD3A) promoter and could positively regulate ATAD3A expression. Additionally, ATAD3A was highly expressed in HDM-induced asthma models. Furthermore, ATAD3A interacted with DRP1, and knockdown of ATAD3A could down-regulate DRP1 and mitochondrial oxidative damage. Conclusively, DEK deficiency alleviates airway inflammation in asthma by down-regulating PINK1-Parkin mitophagy, NLRP3 inflammasome activation, and apoptosis. The mechanism may be through the DEK/ATAD3A/DRP1 signaling axis. Our findings may provide new potential therapeutic targets for asthma treatment.

Dataset Information

Highly Multiplexed Quantitative Mass Spectrometry Analysis of Ubiquitylomes

Publications

Highly Multiplexed Quantitative Mass Spectrometry Analysis of Ubiquitylomes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure