Browse
Submit Data
Databases
API
Help

Dataset Information

471 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Quantitative K-GG site analysis

ABSTRACT: Post-translational modification of proteins by ubiquitin (UQ) and UQ-like modifiers is emerging as a central pathway in DNA replication. We previously showed that chromatin in the vicinity of replisomes is rich in SUMO and depleted in UQ, whereas an opposite pattern is observed in mature chromatin. How this SUMO-rich/UQ-low environment is maintained at replisomes is not known. Here we identify USP7 as a SUMO deubiquitinase that localizes to replication forks and is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 prevents their ubiquitination. Chemical inhibition or genetic deletion of USP7 leads to the accumulation of UQ on SUMOylated proteins, which are displaced from the replisomes. Our findings provide a model to explain the differential accumulation of SUMO and UQ in replication forks versus mature chromatin, and identify an essential role of USP7 that should be taken into account for the use of USP7 inhibitors as anticancer agents.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Colon Cancer

SUBMITTER: Isabel Ruppen

LAB HEAD: Oscar Fernández Capetillo

PROVIDER: PXD002408 | Pride | 2016-01-13

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	20141030_IR_Capetillo_diGly_Control_CID_120m_01.raw	Raw
	20141030_IR_Capetillo_diGly_Control_CID_120m_02.raw	Raw
	20141030_IR_Capetillo_diGly_Control_CID_120m_03.raw	Raw
	20141030_IR_Capetillo_diGly_USP1i_ML_CID_120m_01.raw	Raw
	20141030_IR_Capetillo_diGly_USP1i_ML_CID_120m_02.raw	Raw

Items per page:

1 - 5 of 13

Similar Datasets

Proteome-wide identification of NEDD8 modification sites reveals distinct proteomes for canonical and atypical NEDDylation

Project description:The Ubiquitin-like molecule NEDD8 controls several biological processes and is a promising target for therapeutic intervention. NEDDylation occurs through specific NEDD8 enzymes (canonical) or enzymes of the Ubiquitin system (atypical). Identification of NEDD8 sites on substrates is critical for delineating the processes controlled by NEDDylation. By combining the use of the NEDD8 R74K mutant with anti-diGly antibodies we identified 1101 unique NEDDylation sites in 620 proteins. Bioinformatics analysis reveals that canonical and atypical NEDDylation have distinct proteomes; the spliceosome/mRNA surveillance/DNA replication and ribosome/proteasome respectively. The data also reveal the formation of poly-NEDD8, hybrid NEDD8-Ubiquitin and NEDD8-SUMO-2 chains as potential molecular signals. In particular, NEDD8-SUMO-2 chains are induced upon proteotoxic stress (atypical) through NEDDylation of K11 in SUMO-2 and conjugates accumulate in previously described nucleolus-related inclusions. The study uncovers a diverse proteome for NEDDylation, and is consistent with the concept of extensive cross-talk between Ubiquitin and Ubls under proteotoxic stress conditions.

2021-02-10 | PXD022812 | Pride

USP7 and VCPFAF1 define the SUMO/Ubiquitin landscape at DNA replication forks

Project description:The AAA+ ATPase VCP regulates the extraction of SUMO and/or ubiquitin modified factors from chromatin. We previously showed that active DNA synthesis is associated with a SUMO-high and ubiquitin-low environment that is maintained by the deubiquitylase USP7. Here we unveil a functional cooperation between USP7 and VCP in the control of DNA replication conserved from Caenorhabditis elegans to mammals. The function of VCP during DNA replication is mediated by its cofactor FAF1, which facilitates the extraction of SUMOylated proteins that accumulate on chromatin upon USP7 inhibition. Supporting this cooperative role, the inactivation of the orthologues of USP7 and FAF1 is synthetic lethal in C. elegans, and USP7 and FAF1 inhibitors display synergistic toxicity. Together, these results reveal a coordinated activity of USP7 and VCPFAF1 in limiting SUMOylation at active replication forks and promoting the extraction and degradation of ubiquitylated proteins.

2021-09-12 | PXD018623 | Pride

Deubiquitinase USP9X loss sensitizes renal cancer cells to mTOR inhibition

Project description:mTOR is a central regulator of mammalian metabolism and physiology. Aberrant hyperactivation of this pathway promotes tumor growth and metastasis and drives tumor resistance to chemotherapy and cancer drugs, making mTOR an attractive cancer therapeutic target. mTOR inhibitors have been approved to treat cancer, however, the basis for drug sensitivity remains poorly understood. Here, whole exome sequencing of three chromophobe renal cell carcinoma (chRCC) patients with exceptional mTOR inhibitor sensitivity, uncovered USP9X deubiquitinase as the only mutated gene shared by the tumors. The clonal characteristics of the mutations, revealed by studying multiple patients’ primary and metastatic samples along the years, together with the low USP9X mutation rate in unselected chRCC series, reinforced a causal link between USP9X and mTOR inhibitor sensitivity. Rapamycin treatment of USP9X depleted HeLa and renal cancer 786-O cells and the pharmacological inhibition of USP9X, confirmed a crucial role of this protein in the patients’ sensitivity to mTOR inhibition. As no direct effect of USP9X in mTORC1 was detected, we performed ubiquitylome analyses that identified p62 as a direct USP9X target. Increased p62 ubiquitination and an augmented rapamycin effect upon bortezomib treatment, together with p62 and LC3 immunofluorescence assays, suggested that dysregulated autophagy in USP9X depleted cells can synergize with mTOR inhibitors. In summary, here we show that USP9X constitutes a potential novel marker of sensitivity to mTOR inhibitors in chRCC patients and represents a clinically exploitable strategy that could increase sensitivity to these drugs

2023-06-14 | PXD040496 | Pride

Ubiquitin proteomics reveals critical targets of the Nse1 RING domain in rDNA and genome stability

Project description:Ubiquitination controls most cellular processes, and its deregulation is associated to many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and important functions in genome integrity. However, Nse1-dependent ubiquitin targets remain largely unknown. Here, we use label-free quantitative proteomics to analyse the nuclear ubiquitinome of nse1 RING mutant cells. Our results show that Nse1 impacts on the ubiquitination of several proteins involved in DNA damage tolerance, ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of the Smc5/6 complex in chromosome segregation. In addition, our analysis uncovers an unexpected connection between Nse1 and Pol I ubiquitination. Specifically, Nse1 promotes the ubiquitination of K408 and K410 in Rpa190, the largest subunit of Pol I, to induce its degradation. We propose that this mechanism contributes to Smc5/6-dependent rDNA disjunction in response to transcriptional elongation defects.

2023-05-05 | PXD028669 | Pride

Linkage Reprogramming by Tailor-made E3s Reveals Polyubiquitin Chain Requirements in DNA Damage Bypass

Project description:A polyubiquitin chain can adopt a variety of shapes, depending on how the ubiquitin monomers are joined. However, the relevance of linkage for the signaling functions of polyubiquitin chains is often poorly understood because of our inability to control or manipulate this parameter in vivo. Here we present a strategy for reprogramming polyubiquitin chain linkage by means of tailor-made, linkage- and substrate-selective ubiquitin ligases. Using the polyubiquitylation of the budding yeast replication factor PCNA in response to DNA damage as a model case, we show that altering the features of a polyubiquitin chain in vivo canchange the fate of the modified substrate. We also provide evidence for redundancy betweendistinct, but structurally similar linkages, and we demonstrate by proof-of-principle experiments that the method can be generalized to targets beyond PCNA. Our study illustrates apromising approach towards the in vivo analysis of polyubiquitin signaling.

2022-04-25 | PXD030735 | Pride

SUMOylation proteomic analysis of renal cortical collecting duct cells

Project description:We profiled SUMO1 and SUMO2 modified proteins in cortical collecting duct (mpkCCD) as a precursor to targeted approaches to assess SUMO function. SUMO levels in cells following heat shock and treatment with deSUMOylation inhibitors were assessed.

2019-10-07 | PXD013346 | Pride

SUMOylation proteomic analysis of renal distal convoluted tubule cells

Project description:We profiled SUMO1 and SUMO2 modified proteins in distal convoluted tubule (mpkDCT) as a precursor to targeted approaches to assess SUMO function. SUMO levels in cells following heat shock and treatment with deSUMOylation inhibitors were assessed.

2021-11-25 | PXD028507 | Pride

The ubiquitin landscape of the Influenza A Virus polymerase

Project description:During influenza A virus (IAV) infections, viral proteins are targeted by cellular E3 ligases for modification with ubiquitin. Here, we decipher and functionally explore the ubiquitin landscape of the IAV polymerase during infection of human alveolar epithelial cells by applying mass spectrometry analysis of immuno-purified K-ε-GG- (di-glycyl)-remnant-bearing peptides. We identified 59 modified lysines across all three subunits of the viral polymerase of which 17 distinctively affected mRNA transcription, vRNA replication and the generation of recombinant viruses via non-proteolytic mechanisms. Moreover, our results demonstrate that the ubiquitinated residue K578 in the PB1 thumb domain is crucial for the dynamic structural transitions of the viral polymerase that are required for vRNA replication. Mutations K578A and K578R impeded the steps of cRNA stabilization and vRNA transcription, respectively, and affected NP binding as well as polymerase dimerization. Collectively, our results indicate that ubiquitin-mediated disruption of the charge-dependent interaction between PB1-K578 and PB2-E72 is required to coordinate polymerase dimerization and facilitate vRNA replication, which demonstrates that IAV exploit the cellular ubiquitin system to modulate the activity of the viral polymerase for the regulation of viral replication.

2023-03-11 | PXD030816 | Pride

Proteomic profiling of ubiquitylated proteins in urinary exosomes

Project description:Exosomes, derived from multivesicular bodies (MVBs), contain proteins and genetic materials from their cell of origin and are secreted from various cells types, including kidney epithelial cells. In general, it is thought that protein cargo is ubiquitylated, but that ubiquitin is cleaved by specific deubiquitylases during the process of cargo incorporation into MVBs. Here, we provide direct evidence that in vivo, deubiquitylation is not essential. Ubiquitin was detected within human MVBs and urinary exosomes by electron microscopy. Protein mass spectrometry identified >6000 proteins in human urinary exosomes, 15% of which were ubiquitylated with various topologies (K63>K48> K11>K6>K29>K33>K27). Ubiquitylated proteins involved in transcriptional regulation or solute transport were significantly enriched. Non-biased analysis of over-represented motifs uncovered a significant preference for basic amino acids upstream of the ubiquitylation site. The current studies demonstrate that in human epithelial cells, deubiquitylation of protein cargo is not necessary for MVB formation and exosome secretion and highlight that urinary exosomes are an enriched source for studying ubiquitin modifications in physiological or disease states.

2016-02-22 | PXD002645 | Pride

Dissecting ubiquitylation and DNA damage response pathways in the yeast Saccharomyces cerevisiae using a proteome-wide approach

Project description:The proper function of DNA damage response pathways (DDR) is essential to protect eukaryotic cells from the accumulation of DNA damage and genome instability, a major cause of cancer and other diseases in humans. Ubiquitylation orchestrates the regulation of DDR events.Blaszczak et al. use quantitative proteomics and a reporter system based on the NanoLuc luciferase to study ubiquitylation and protein abundance changes upon genotoxic stress in yeast. The authors point out proteins that may be regulated by ubiquitylation during the DDR.

2023-12-13 | PXD043291 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data