MS/MS analysis of Ub-conjugated UbcH7 ubiquitination sites
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap XL
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
lu z
LAB HEAD: Cuihua Liu
PROVIDER: PXD020982 | Pride | 2021-04-16
REPOSITORIES: Pride
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Project description:Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation. Mol Cell 33:483-495, 2009. Keywords: Comparison of gene expression profiles
2009-03-24 | GSE14088 | GEO
Project description:Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation. Mol Cell 33:483-495, 2009. Keywords: Comparison of gene expression profiles Ube2m and Ube2f are E2 enzymes that direct protein modification by NEDD8. Here we explore the specific functions of Ube2f and Ube2m by comparing gene expression profiles following knockdown of their function in NIH 3T3 cells. We used microarrays to detail the global programme of gene expression changes following knockdown of Ube2m and Ube2f in NIH 3T3 cells. NIH 3T3 cells were transduced with retroviral constructs containing shRNA directed against Ube2m or Ube2f. Three replicates of each condition were analyzed.
2009-03-23 | E-GEOD-14088 | biostudies-arrayexpress
Project description:Post-translational modification of proteins by Ubiquitin (Ub) and Ubiquitin-like proteins (Ubls) can be reversed by deconjugating enzymes, which have been implicated in different pathways and associated with various human diseases. To understand the activity and dynamics of deconjugating enzymes, multiple synthetic and semi-synthetic Ub/Ubl probes have been developed, and some of them have been applied to screen inhibitors of deconjugating enzymes. Since these Ub/Ubl probes are generally not cell-permeable, different strategies have been developed to deliver Ub/Ubl probes to live cells. However, till now no Ub/Ubl probes can be expressed in live cells to directly report on the activities of deconjugating enzymes in the most relevant cellular environment. Here, we genetically encoded cross-linkable Ub/Ubl probes in live E. coli and HEK293T cells. These probes can cross-link with deconjugating enzymes in vitro and in vivo, and the extent of cross-linking is dependent on the activities of deconjugating enzymes.
2022-09-23 | PXD036955 | iProX
Project description:The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1, however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays and recombinant chromatin substrates we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the “backside” of the E2 to stabilise the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL-domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.
2018-08-29 | GSE119120 | GEO
Project description:Proximity-dependent labelling methods are based on promiscuous labeling enzymes that produce reactive molecules that covalently bind neighbor proteins. Labeled proteins can be then purified and identified using affinity-purification coupled to mass spectrometry methods23. Proximity-dependent biotin identification (BioID)24 uses a promiscuously active Escherichia coli biotin ligase (BirA*) generated by a point mutation (R118G) to biotinylate lysines in nearby proteins within an estimated range of 10 nm25. By fusing BirA* to specific proteins, BioID efficiently identifies interactors at physiological levels in living cells26. It has been extensively used in the Ub field, for instance, to identify substrates of E3 ligases27,28. Recently, a more efficient version of BioID, termed TurboID, has been developed29, being this more suitable for transient protein-protein interaction (PPI) detection. Several studies have developed split-versions and applied “protein fragment complementation” to BioID and TurboID, where proximal biotinylation is dependent on the proximity of the fusion partners, opening new opportunities for spatial and temporal identification of complex-dependent interactomes30,31. To study how SUMOylation and SUMO-SIM interactions can lead to other roles and fates for particular substrates poses particular challenges. SUMOylation occurs transiently and often in a small percentage of a given substrate. Modified proteins can be readily deSUMOylated and SUMO can be recycled and passed to other substrates. SUMO-SIM interactions are also difficult to analyze due to their weak affinity (Kd 1-100 μM). To overcome those technical issues, we developed SUMO-ID, a new strategy based on Split-TurboID to identify interactors of specific substrates dependent on SUMO conjugation or interaction. Using PML as a model, we demonstrate that SUMO-ID can enrich for factors that depend on PML-SUMO interaction. Importantly, the identified proteins are represented among proximal interactors of PML identified using full-length TurboID. We also applied SUMO-ID to a less-characterized SUMO substrate, Spalt Like Transcription Factor 1 (SALL1), and identified both known and novel interactors that depend on intact SUMOylation sites in SALL1. SUMO-ID is thus a powerful tool to study transient and dynamic SUMO-dependent interaction events. The developed methodology is generic and therefore widely applicable in the Ub and UbL field to identify readers of these modifications for individual target proteins to improve our insight in non-covalent signal transduction by Ub and UbL.
2021-09-20 | PXD027759 | Pride
Project description:Posttranslational modifications by ubiquitin-like proteins (UBL) are essential for nearly all cellular processes. Ubiquitin related modifier 1 (Urm1) is a non-canonical UBL which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP). While Urm1 has also been observed to conjugate directly to target proteins like other UBLs, the mechanism of attachment as well as how the SCP properties of Urm1 may impact its conjugation are unknown. Here, we reconstitute the covalent attachment of Urm1 to various cellular target proteins in vitro, revealing that, unlike other known UBLs, this process is E2/E3-independent and requires oxidative stress conditions. We determined the crystal structures of the peroxiredoxin Ahp1 before and after covalent attachment of Urm1. Strikingly, we show that this conjugation process results in persulfidation of a cysteine residue in the target protein. Demonstrating that Urm1 actively transfers sulfur atoms to proteins as part of its conjugation reaction. Our results redefine Urm1 as a key evolutionary link between prokaryotic SCPs and the plethora of UBL modifications observed in modern eukaryotes, and demonstrate a critical role for Urm1 in protecting proteins during oxidative stress.
2021-11-16 | MSV000088390 | MassIVE
Project description:To further identify transcriptomic responses to CAIX deficiency in the brain, genome-wide cDNA microarray analyses were performed. Thirty-one and 37 genes were up- and down-regulated in the brain of Car9-/- mice as compared to wild-type mice. Functional annotation revealed that genes with increased expression are mainly involved in alternative splicing, nucleotide binding, RNA binding, and regulation of cellular protein metabolic process; whereas genes with reduced expression are chiefly implicated in cellular cation homeostasis, positive regulation of ion transport, Ubl conjugation, phosphorus metabolic process, and regulation of cell proliferation. Notably, the biological processes behaviour and locomotory behaviour are two prominent over-representation terms among the down-regulated genes, which is consistent with the results obtained from behavioural tests. Brain tissue samples were collected from five wild-type and four CAIX KO mice, respectively, at the age of eight months. Total RNAs were purified and used for cDNA microarray.
2011-12-31 | E-GEOD-24051 | biostudies-arrayexpress
Project description:The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1, however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RINGmediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays and recombinant chromatin substrates we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the “backside” of the E2 to stabilise the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBLdomain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.
2018-11-04 | PXD011510 | Pride
Project description:Post-translational control by ubiquitin regulates various aspects of cellular biology. This chemical modification on proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Among the various types of poly-ubiquitin constructed are untethered species that comprise a series of ubiquitin moieties linked to one another, but free from another protein. The current notion is that these unanchored poly-ubiquitin species are deleterious to the cell and are rapidly deconstructed. We recently examined the toxicity and utilization of untethered poly-ubiquitin in an intact organism by using the fruit fly, Drosophila melanogaster. We found that these ubiquitin species are largely innocuous to flies and that free poly-Ub can be controlled by being degraded by the proteasome or being conjugated onto another protein as a single unit. However, whether the fly is mounting an organismal defense against untethered chains was not explored in detail. Here, we conducted RNA-seq analyses to examine at the transcriptome level the impact of unanchored poly-Ub in the fly. We found ~90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was mostly devoid of ubiquitin-, proteasome- or autophagy-related components. The largest gene ontology category identified, housing ~15 of the altered genes, was proteolysis. The seeming absence of a large and multipronged response to unanchored ubiquitin chains in the fly supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell. We designed two types of poly-Ub chains with six Ub molecules in tandem. They cannot be cleaved by DUBs (there are no "GG" motifs linking the Ub molecules). Ub6-GG can be conjugated in toto onto other proteins; Ub6-Stop cannot. We drove ubiquitous expression using sqh-Gal4, and then performed RNA-Seq on adult flies to determine any cellular response to the presence of our unanchored Ub chains.
2019-05-01 | GSE125090 | GEO
Project description:To further identify transcriptomic responses to CAIX deficiency in the brain, genome-wide cDNA microarray analyses were performed. Thirty-one and 37 genes were up- and down-regulated in the brain of Car9-/- mice as compared to wild-type mice. Functional annotation revealed that genes with increased expression are mainly involved in alternative splicing, nucleotide binding, RNA binding, and regulation of cellular protein metabolic process; whereas genes with reduced expression are chiefly implicated in cellular cation homeostasis, positive regulation of ion transport, Ubl conjugation, phosphorus metabolic process, and regulation of cell proliferation. Notably, the biological processes behaviour and locomotory behaviour are two prominent over-representation terms among the down-regulated genes, which is consistent with the results obtained from behavioural tests.
2011-12-31 | GSE24051 | GEO