Ubiquitin-activated interaction traps (UBAITs) identify E3 ligase binding partners
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Saccharomyces Cerevisiae (ncbitaxon:4932)
SUBMITTER: Jon Huibregtse
PROVIDER: MSV000079265 | MassIVE | Tue Aug 25 08:29:00 BST 2015
SECONDARY ACCESSION(S): PXD002791
REPOSITORIES: MassIVE
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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.
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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:Ubiquitination of proteins is central to protein homeostasis and other cellular processes including DNA repair, vesicular transport, cell-division etc. The process of ubiquitination is conserved from yeast to humans and is carried out by the sequential action of three enzymes: E1, E2 and E3. There are an estimated >600 E3 ligases in humans that execute ubiquitination of specific target proteins in a spatio-temporal manner to elicit desired signaling effects. Here, we developed a simple, proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. Our method exploits the proximity and the relative orientation of the E3-ligase catalytic domain with respect to ubiquitin observed in the enzymatic intermediate-state structures of E3-E2~Ub. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich the bona fide substrates and potential new substrates of the ligase using a one-step Streptavidin pulldown under denaturing conditions. As proof-of-principle, we applied our method, which we named Ub-POD, to the RING E3 ligase Rad18. Rad18 ubiquitinates DNA-sliding clamp PCNA upon UV-induced DNA damage. We identified PCNA and several other critical players in the DNA damage repair pathway in a single Ub-POD experiment. We further discovered an unintended but important application of Ub-POD, where we were able to pin down the cellular localization of Rad18- mediated ubiquitination to the damaged DNA nuclear puncta through Streptavidin immunofluorescence. We also applied our method to TRAF6, another RING ubiquitin ligase involved in TNF signaling, successfully identifying known and potentially new substrates. Finally, we adapted our method to the U-box type E3 ubiquitin ligase CHIP to demonstrate that we can identify substrates of two major classes of mammalian ubiquitin ligases. We anticipate that our method and principle could be widely adapted to all classes of ubiquitin ligases to identify substrates and localize the cellular site(s) of ubiquitination.
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Project description:Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including for RING-family GID/CTLH E3 ubiquitin ligases and their dedicated E2, Ubc8/UBE2H (yeast/human nomenclature). GID/CTLH-Ubc8/UBE2H-mediated ubiquitylation regulates biological processes ranging from yeast metabolic signaling to human development. Here, cryo-EM, biochemistry, and cell biology reveal exquisitely specific GID/CTLH-Ubc8/UBE2H pairing through an unconventional catalytic assembly and auxiliary interactions 70-100 Å away, mediated by E2 phosphorylation. Rather than dynamic polyelectrostatic interactions reported for other ubiquitylation complexes, multiple Ubc8/UBE2H phosphorylation sites within acidic CK2-targeted sequences specifically anchor the E2 C-termini to E3 basic patches. Positions of phospho-dependent interactions relative to the catalytic domains correlate across evolution. Overall, our data illustrate phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, immobilizes the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites.
2023-12-18 | PXD043646 | Pride
Project description:Proctor2007 - Age related decline of proteolysis, ubiquitin-proteome system
This
is a stochastic model of the ubiquitin-proteasome system for a
generic pool of native proteins (NatP), which have a half-life of
about 10 hours under normal conditions. It is assumed that these
proteins are only degraded after they have lost their native
structure due to a damage event. This is represented in the model
by the misfolding reaction which depends on the level of reactive
oxygen species (ROS) in the cell. Misfolded proteins (MisP) are
first bound by an E3 ubiquitin ligase. Ubiquitin (Ub) is
activated by E1 (ubiquitin-activating enzyme) and then passed to
E2 (ubiquitin-conjugating enzyme). The E2 enzyme then passes the
ubiquitin molecule to the E3/MisP complex with the net effect
that the misfolded protein is monoubiquitinated and both E2 and
E3 are released. Further ubiquitin molecules are added in a
step-wise manner. When the chain of ubiquitin molecules is of
length 4 or more, the polyubiquitinated misfolded protein may
bind to the proteasome. The model also includes de-ubiquitinating
enzymes (DUB) which cleave ubiquitin molecules from the chain in
a step-wise manner. They work on chains attached to misfolded
proteins both unbound and bound to the proteasomes. Misfolded
proteins bound to the proteasome may be degraded releasing
ubiquitin. Misfolded proteins including ubiquitinated proteins
may also aggregate. Aggregates (AggP) may be sequestered
(Seq_AggP) which takes them out of harm's way or they may bind to
the proteasome (AggP_Proteasome). Proteasomes bound by aggregates
are no longer available for protein degradation.
Figure
2 and Figure 3 has been simulated using Gillespie2.
This model is described in the article:
An in silico model of the
ubiquitin-proteasome system that incorporates normal
homeostasis and age-related decline.
Proctor CJ, Tsirigotis M, Gray
DA.
BMC Syst Biol 2007; 1: 17
Abstract:
BACKGROUND: The ubiquitin-proteasome system is responsible
for homeostatic degradation of intact protein substrates as
well as the elimination of damaged or misfolded proteins that
might otherwise aggregate. During ageing there is a decline in
proteasome activity and an increase in aggregated proteins.
Many neurodegenerative diseases are characterised by the
presence of distinctive ubiquitin-positive inclusion bodies in
affected regions of the brain. These inclusions consist of
insoluble, unfolded, ubiquitinated polypeptides that fail to be
targeted and degraded by the proteasome. We are using a systems
biology approach to try and determine the primary event in the
decline in proteolytic capacity with age and whether there is
in fact a vicious cycle of inhibition, with accumulating
aggregates further inhibiting proteolysis, prompting
accumulation of aggregates and so on. A stochastic model of the
ubiquitin-proteasome system has been developed using the
Systems Biology Mark-up Language (SBML). Simulations are
carried out on the BASIS (Biology of Ageing e-Science
Integration and Simulation) system and the model output is
compared to experimental data wherein levels of ubiquitin and
ubiquitinated substrates are monitored in cultured cells under
various conditions. The model can be used to predict the
effects of different experimental procedures such as inhibition
of the proteasome or shutting down the enzyme cascade
responsible for ubiquitin conjugation. RESULTS: The model
output shows good agreement with experimental data under a
number of different conditions. However, our model predicts
that monomeric ubiquitin pools are always depleted under
conditions of proteasome inhibition, whereas experimental data
show that monomeric pools were depleted in IMR-90 cells but not
in ts20 cells, suggesting that cell lines vary in their ability
to replenish ubiquitin pools and there is the need to
incorporate ubiquitin turnover into the model. Sensitivity
analysis of the model revealed which parameters have an
important effect on protein turnover and aggregation kinetics.
CONCLUSION: We have developed a model of the
ubiquitin-proteasome system using an iterative approach of
model building and validation against experimental data. Using
SBML to encode the model ensures that it can be easily modified
and extended as more data become available. Important aspects
to be included in subsequent models are details of ubiquitin
turnover, models of autophagy, the inclusion of a pool of
short-lived proteins and further details of the aggregation
process.
This model is hosted on
BioModels Database
and identified by:
BIOMD0000000105.
To cite BioModels Database, please use:
BioModels Database:
An enhanced, curated and annotated resource for published
quantitative kinetic models.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
CC0
Public Domain Dedication for more information.
2024-09-02 | BIOMD0000000105 | BioModels
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2022-10-24 | GSE139633 | GEO
Project description:Rad6 E2 ubiquitin-conjugating enzyme and Bre1 E3 ubiquitin ligase catalyze histone H2B Lysine-123 monoubiquitination (H2Bub1), which stabilizes nucleosomes and regulates the trans-histone H3K4 and K79 methylation during gene transcription and other nuclear processes. The interaction interfaces within the Rad6-Bre1-containing H2B ubiquitin-conjugating complex have remained unknown. By solving the crystal structure of Rad6 along with a non-RING domain N-terminal region of Bre1, we report a beta-turn in Rad6's so-called backside region away from catalytic pocket as a binding site for a homodimer of Bre1 E3 ligase. Using quantitative ChIP-seq or ChIP-Rx, we further demonstrate that Rad6's backside beta-turn residues also govern the chromatin binding dynamics and transcriptional regulatory functions of the Rad6-Bre1 complex.
2022-10-24 | GSE139632 | GEO
Project description:Our method (Ub-POD) exploits the proximity and the relative orientation of the E3-ligase catalytic domain with respect to ubiquitin observed in the enzymatic intermediate-state structures of E3-E2~Ub. By fusing biotin ligase BirA and an Avi tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich the bona fide substrates and potentially new substrates of the ligase using a one-step Streptavidin pulldown under denaturing conditions.
2024-10-17 | PXD052316 | Pride
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