Designed, linked-domain proteins are pan-UBE2D inhibitors and induce proteostress and the unfolded protein response in HeLa cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE298221 | GEO | 2025/09/01
REPOSITORIES: GEO
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Project description:E3 ubiquitin (UB) ligases, the ending module of the E1-E2-E3 cascades, transmit diverse signals in the cell by attaching UB to cellular proteins. Identifying E3 substrates holds the key to elucidate the roles of E3s in cell regulation. We constructed an orthogonal UB transfer (OUT) cascade to identify the substrates of E6AP, a HECT E3 also known as Ube3a that is implicated in neurodevelopmental disorders and cancer. We used yeast cell surface display to engineer E6AP to exclusively transfer an affinity-tagged UB variant (xUB) to its substrate proteins. Proteomic identification of xUB-conjugated proteins in HEK293 cells afforded 131 potential E6AP targets. Among them we verified MAPK1, CDK1 and CDK4, and PRMT5 are directly ubiquitinated by E6AP in vitro and in the cell. Our work establishes OUT as an efficient platform to profile E6AP substrates and would guide the engineering of OUT cascades with other E3s to interrogate their biological functions.
2018-02-07 | PXD005584 | 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: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.
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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
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Public Domain Dedication for more information.
2024-09-02 | BIOMD0000000105 | BioModels
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 has 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 of the Rad6-Bre1 complex.
2022-10-24 | GSE139633 | GEO
Project description:The E3 ubiquitin -protein ligases (E3s) plays a role as regulators of protein trafficking and degradation. We aimed to identify E3s in rat kidney which are associated with dDAVP-induced urine concentration. Kidney inner medulla collected from vehicle treated control (n=2), dDAVP infusion for 5 d (D5d, n=2) and 3 h-withdrawal of dDAVP after 5 d-infusion (D5d-3h, n=2) groups were subjected to a transcriptome analysis.
2011-02-23 | E-GEOD-27449 | biostudies-arrayexpress
Project description:The E3 ubiquitin -protein ligases (E3s) plays a role as regulators of protein trafficking and degradation. We aimed to identify E3s in rat kidney which are associated with dDAVP-induced urine concentration.
2011-02-23 | GSE27449 | 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:We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins, and in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. UBAITs were made for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, substrate trapping occurred in vitro either through an E3 thioester-linked lariat intermediate or an E2 thioester intermediate, and both WT and active site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells.
2015-08-25 | MSV000079265 | MassIVE
Project description:The E2 ubiquitin (Ub)-conjugating enzyme primarily determines Ub conjugation as Ub-isopeptide (lysine), Ub-oxyester (serine/threonine) or Ub-thioester (cysteine). However, E2-specific Ub conjugation profiles within cells remain elusive. Here, we developed the fusion E2–Ub-R74G profiling (FUSEP) strategy to access E2-specific Ub conjugation profiles in cells with amino acid resolution. The probe-specific leucine-glycine-glycine-glycine-modified Ub remnant enables systematic studies of nonlysine Ub conjugation and provides site-specific information. Multiple E2 enzymes were found to be involved in nonlysine ubiquitination. Profiling with UBE2D3–Ub-R74G probes identified the existence of tyrosine ubiquitination in human Cullin-1, a scaffold protein for Cullin-RING E3 Ub ligases. This modification is distinct from lysine ubiquitination. A single-pass membrane-bound E3 ligase, RNF149, was identified to pair with UBE2D3 to regulate pyroptosis by ubiquitinating apoptosis-associated speck-like protein ASC. The availability of this toolbox paves the way for uncovering E2-specific Ub conjugation profiles and identifying previously unknown E3 Ub ligases for potential therapeutic applications.
2024-12-11 | PXD058017 |
Project description:The assembly of a specific polymeric ubiquitin (Ub) chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The ubiquitin-conjugating (E2) enzymes Ubc1 in yeast and Ube2K in mammals exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a Ub binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with Ub chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched Ub molecules. Genetic experiments link the activity of the UBA domain and hence the formation of this unusual Ub chain topology to the maintenance of cellular proteostasis.
2021-03-02 | PXD022621 | Pride