TUBE-based approach coupled with ligase trapping for identification of E3 ubiquitin ligase substrates
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: Shigetsugu Hatakeyama
PROVIDER: PXD020658 | JPOST Repository | Thu Aug 06 00:00:00 BST 2020
REPOSITORIES: jPOST
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Project description:The TRIM37 gene is mutatedin Mulbery nanism, a rare autosomal recessive disorder, and is in the 17q23 chromosomal region that is amplified in up to ~40% of breast cancers. Trim37 contains a RING finger domain, a hallmark of E3 ubiquitin ligases, but the protein substrate(s) of Trim37 is unknown. Mono-ubiquitination of histone H2A is a chromatin modification associated with transcriptional repression and here we report that Trim37 is an H2A ubiquitin ligase. Genome-wide Chip-CHIP experiments indicate that in human breast cancer cells containing amplified 17q23, Trim37 is bound to the promoters of many tumor suppressor genes. RNA interference (RNAi)-mediated knockdown of Trim37 results in loss of ubiquitinated H2A, dissociation of PRC1 and PRC2, and transcriptional reactivation of silenced genes. Knockdown of Trim37 in human breast cancer cells containing amplified 17q23 substantially decreases tumor growth in mouse xenografts. Collectively, our results reveal Trim37 as a new H2A ubiquitin ligase that is overexpressed in a subset of breast cancers and redirects PRC2 to silence tumor suppressors and other genes resulting in oncogenesis. Identification of TRIM37 Binding targets in MCF7 cells from the two replicate experiments
2014-10-06 | E-GEOD-48196 | biostudies-arrayexpress
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
Project description:The TRIM37 gene is mutatedin Mulbery nanism, a rare autosomal recessive disorder, and is in the 17q23 chromosomal region that is amplified in up to ~40% of breast cancers. Trim37 contains a RING finger domain, a hallmark of E3 ubiquitin ligases, but the protein substrate(s) of Trim37 is unknown. Mono-ubiquitination of histone H2A is a chromatin modification associated with transcriptional repression and here we report that Trim37 is an H2A ubiquitin ligase. Genome-wide Chip-CHIP experiments indicate that in human breast cancer cells containing amplified 17q23, Trim37 is bound to the promoters of many tumor suppressor genes. RNA interference (RNAi)-mediated knockdown of Trim37 results in loss of ubiquitinated H2A, dissociation of PRC1 and PRC2, and transcriptional reactivation of silenced genes. Knockdown of Trim37 in human breast cancer cells containing amplified 17q23 substantially decreases tumor growth in mouse xenografts. Collectively, our results reveal Trim37 as a new H2A ubiquitin ligase that is overexpressed in a subset of breast cancers and redirects PRC2 to silence tumor suppressors and other genes resulting in oncogenesis.
2014-10-06 | GSE48196 | GEO
Project description:Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.
2024-03-20 | PXD042687 | Pride
Project description:Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.
2024-03-20 | PXD042706 | Pride
Project description:Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.
2024-03-20 | PXD042700 | Pride
Project description:Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.
2024-03-20 | PXD042674 | Pride
Project description:Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.
2024-03-20 | PXD042657 | Pride
Project description:Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.
2024-03-20 | PXD047455 | Pride
Project description:Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.
2024-03-20 | PXD042673 | Pride