Abstract 6411: Discovery of covalent ligands to novel E3 ligases enables bispecific degraders with highly differentiated protein degradation across a broad range of targets

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

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The UBR-1 Ubiquitin Ligase Regulates Glutamate Metabolism to Generate Coordinated Motor Pattern in C. elegans

10.1101/198994 ◽

2018 ◽

Author(s):

Jyothsna Chitturi ◽

Wesley Hung ◽

Anas M. Abdel Rahman ◽

Min Wu ◽

Maria A. Lim ◽

...

Keyword(s):

Protein Degradation ◽

Motor Neurons ◽

Ubiquitin Ligase ◽

Motor Pattern ◽

Motor Deficit ◽

Neuron Activity ◽

Glutamate Metabolism ◽

E3 Ligases ◽

C Elegans ◽

Glutamate Level

AbstractUBR1 is an E3 ubiquitin ligase best known for its ability to target protein degradation by the N-end rule. The physiological functions of UBR family proteins, however, remain not fully understood. We found that the functional loss of C. elegans UBR-1 leads to a specific motor deficit: when adult animals generate reversal movements, A-class motor neurons exhibit synchronized activation, preventing body bending. This motor deficit is rescued by removing GOT-1, a transaminase that converts aspartate to glutamate. Both UBR-1 and GOT-1 are expressed and critically required in premotor interneurons of the reversal motor circuit to regulate the motor pattern. ubr-1 and got-1 mutants exhibit elevated and decreased glutamate level, respectively. These results raise an intriguing possibility that UBR proteins regulate glutamate metabolism, which is critical for neuronal development and signaling.Author SummaryUbiquitin-mediated protein degradation is central to diverse biological processes. The selection of substrates for degradation is carried out by the E3 ubiquitin ligases, which target specific groups of proteins for ubiquitination. The human genome encodes hundreds of E3 ligases; many exhibit sequence conservation across animal species, including one such ligase called UBR1. Patients carrying mutations in UBR1 exhibit severe systemic defects, but the biology behinds UBR1’s physiological function remains elusive. Here we found that the C. elegans UBR-1 regulates glutamate level. When UBR-1 is defective, C. elegans exhibits increased glutamate; this leads to synchronization of motor neuron activity, hence defective locomotion when animals reach adulthood. UBR1-mediated glutamate metabolism may contribute to the physiological defects of UBR1 mutations.

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Proteolysis Targeting Chimeras With Reduced Off-targets

10.1101/2021.11.18.468552 ◽

2021 ◽

Author(s):

Tuan M Nguyen ◽

Arghya Deb ◽

Praveen Kokkonda ◽

Vedagopuram Sreekanth ◽

Praveen K Tiwari ◽

...

Keyword(s):

Side Effects ◽

Protein Degradation ◽

High Throughput ◽

Normal Development ◽

Nucleophilic Aromatic Substitution ◽

Aromatic Substitution ◽

E3 Ligases ◽

Current Design ◽

Design Paradigm

Proteolysis Targeting Chimeras (PROTACs), a class of heterobifunctional molecules that recruit target proteins to E3 ligases, have gained traction for targeted protein degradation. However, pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other targets, such as zinc-finger (ZF) proteins, that hold key functions in normal development and disease progression. This off-target degradation of pomalidomide-based PROTACs raises concerns about their therapeutic applicability and long-term side effects. Therefore, there is a crucial need to develop rules for PROTAC design that minimize off-target degradation. In this study, we developed a high-throughput platform that interrogates the off-target degradation of ZF domains and discovered, using this platform, that PROTACs with the current design paradigm induce significant degradation of several ZF proteins. To identify new rules for PROTAC design, we generated a rationalized library of pomalidomide analogs with distinct exit vector modifications on the C4 and C5 positions of the phthalimide ring and profiled their propensities for ZF protein degradation. We found that modifications on the C5 position with nucleophilic aromatic substitution (SNAr) reduce off-target ZF degradation. We applied our newfound design principles on a previously developed ALK oncoprotein-targeting PROTAC and generated PROTACs with enhanced potency and minimal off-target degradation. We envision the reported off-target profiling platform and pomalidomide analogs will find utility in the design of specific PROTACs.

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Expanding the Toolbox of E3 Ligases for Protein Degradation: Targeting the “Undruggable” Fbw7 E3 Ligase

Proceedings ◽

10.3390/proceedings2019022101 ◽

2019 ◽

Vol 22 (1) ◽

pp. 101

Author(s):

Carles Galdeano

Keyword(s):

Protein Degradation ◽

E3 Ligase ◽

Intracellular Protein ◽

E3 Ligases ◽

Intracellular Protein Degradation

Proteolysis targeting chimera molecules (PROTACS) are heterobifunctional small moleculesdesigned to induce intracellular protein degradation [...]

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Insulin down-regulates the expression of ubiquitin E3 ligases partially by inhibiting the activity and expression of AMP-activated protein kinase in L6 myotubes

Bioscience Reports ◽

10.1042/bsr20150017 ◽

2015 ◽

Vol 35 (4) ◽

Cited By ~ 8

Author(s):

Hu-Ping Deng ◽

Jia-Ke Chai ◽

Chuan-An Shen ◽

Xi-Bo Zhang ◽

Li Ma ◽

...

Keyword(s):

Protein Kinase ◽

Protein Degradation ◽

Signalling Pathway ◽

Down Regulation ◽

E3 Ligases ◽

Akt Phosphorylation ◽

L6 Myotubes ◽

Amp Activated Protein Kinase

We conclude that insulin inhibits AMPK through Akt phosphorylation in L6 myotubes, which may serve as a possible signalling pathway for the down-regulation of protein degradation. Besides, decreased expression of AMPK α2 may partially participate in inhibiting the activity of AMPK.

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Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16

10.1101/443804 ◽

2018 ◽

Cited By ~ 1

Author(s):

Xiaoyu Zhang ◽

Vincent M. Crowley ◽

Thomas G. Wucherpfennig ◽

Melissa M. Dix ◽

Benjamin F. Cravatt

Keyword(s):

Protein Degradation ◽

Protein Content ◽

Substrate Recognition ◽

Ubiquitin Ligases ◽

Nuclear Proteins ◽

E3 Ubiquitin Ligases ◽

E3 Ligases ◽

Dependent Protein

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy to discover that DCAF16 – a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases – promotes nuclear-restricted protein degradation upon modification by cysteine-directed heterobifunctional electrophilic compounds.

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PhyB induces intron retention and uORF-mediated translational inhibition of PIF3

10.1101/813840 ◽

2019 ◽

Author(s):

Jie Dong ◽

Haodong Chen ◽

Xing Wang Deng ◽

Vivian F. Irish ◽

Ning Wei

Keyword(s):

Alternative Splicing ◽

Protein Degradation ◽

Intron Retention ◽

Red Light ◽

Light Response ◽

Protein Translation ◽

Light Cycle ◽

Light Responses ◽

E3 Ligases ◽

Short Day

AbstractThe phytochrome B (phyB) photoreceptor stimulates light responses in plants in part by inactivating repressors of light responses such as phytochrome-interacting factor 3 (PIF3). It has been established that activated phyB inhibits PIF3 by rapid protein degradation and decreased transcription. PIF3 protein degradation has been shown to be mediated by EIN3-BINDING F-BOX PROTEIN (EBF) and LIGHT-RESPONSE BTB (LRB) E3 ligases, the latter simultaneously targeting phyB for degradation. In this study, we show that PIF3 level is additionally regulated by alternative splicing and protein translation. Overaccumulation of photo-activated phyB, which occur in the mutant defective for LRB genes under continuous red light (Rc), induces a specific alternative splicing of PIF3 that results in retention of an intron in the 5’UTR of PIF3 mRNA. In turn, the upstream opening reading frames (uORF) contained within this intron inhibit PIF3 protein synthesis. The phyB-dependent alternative splicing of PIF3 is diurnally regulated under the short-day light cycle. We hypothesize that this reversible regulatory mechanism may be utilized to fine-tune the level of PIF3 protein in light-grown plants, and may contribute to the oscillation of PIF3 protein abundance under the short-day environment.One Sentence SummaryLight down-regulates PIF3 by multiple mechanisms. We show that phyB induces an alternative splicing event that inhibits PIF3 protein translation, and that is regulated by short-day diurnal cycle.

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Targeting the Ubiquitin-Proteasome System for Cancer Therapeutics by Small-Molecule Inhibitors

Cancers ◽

10.3390/cancers13123079 ◽

2021 ◽

Vol 13 (12) ◽

pp. 3079

Author(s):

Gabriel LaPlante ◽

Wei Zhang

Keyword(s):

Protein Degradation ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Proteasome Inhibitors ◽

Cancer Therapeutics ◽

Ubiquitin Proteasome System ◽

Cellular Protein ◽

E3 Ligases ◽

Protein Levels ◽

Ubiquitin Proteasome

The ubiquitin-proteasome system (UPS) is a critical regulator of cellular protein levels and activity. It is, therefore, not surprising that its dysregulation is implicated in numerous human diseases, including many types of cancer. Moreover, since cancer cells exhibit increased rates of protein turnover, their heightened dependence on the UPS makes it an attractive target for inhibition via targeted therapeutics. Indeed, the clinical application of proteasome inhibitors in treatment of multiple myeloma has been very successful, stimulating the development of small-molecule inhibitors targeting other UPS components. On the other hand, while the discovery of potent and selective chemical compounds can be both challenging and time consuming, the area of targeted protein degradation through utilization of the UPS machinery has seen promising developments in recent years. The repertoire of proteolysis-targeting chimeras (PROTACs), which employ E3 ligases for the degradation of cancer-related proteins via the proteasome, continues to grow. In this review, we will provide a thorough overview of small-molecule UPS inhibitors and highlight advancements in the development of targeted protein degradation strategies for cancer therapeutics.

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A Phenotypic Approach for the Identification of New Molecules for Targeted Protein Degradation Applications

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211017517 ◽

2021 ◽

pp. 247255522110175

Author(s):

Peter Stacey ◽

Hannah Lithgow ◽

Xiao Lewell ◽

Agnieszka Konopacka ◽

Stephen Besley ◽

...

Keyword(s):

Fusion Protein ◽

Protein Degradation ◽

E3 Ligase ◽

Ubiquitin Proteasome System ◽

E3 Ligases ◽

Von Hippel Lindau ◽

Protein Levels ◽

New Strategy ◽

Cellular Context

Targeted protein degradation is an emerging new strategy for the modulation of intracellular protein levels with applications in chemical biology and drug discovery. One approach to enable this strategy is to redirect the ubiquitin–proteasome system to mark and degrade target proteins of interest (POIs) through the use of proteolysis targeting chimeras (PROTACs). Although great progress has been made in enabling PROTACs as a platform, there are still a limited number of E3 ligases that have been employed for PROTAC design. Herein we report a novel phenotypic screening approach for the identification of E3 ligase binders. The key concept underlying this approach is the high-throughput modification of screening compounds with a chloroalkane moiety to generate HaloPROTACs in situ, which were then evaluated for their ability to degrade a GFP-HaloTag fusion protein in a cellular context. As proof of concept, we demonstrated that we could generate and detect functional HaloPROTACs in situ, using a validated Von Hippel–Lindau (VHL) binder that successfully degraded the GFP-HaloTag fusion protein in living cells. We then used this method to prepare and screen a library of approximately 2000 prospective E3 ligase-recruiting molecules.

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Transcriptional activation of muscle atrophy promotes cardiac muscle remodeling during mammalian hibernation

PeerJ ◽

10.7717/peerj.2317 ◽

2016 ◽

Vol 4 ◽

pp. e2317 ◽

Cited By ~ 10

Author(s):

Yichi Zhang ◽

Oscar A. Aguilar ◽

Kenneth B. Storey

Keyword(s):

Protein Degradation ◽

Cardiac Muscle ◽

Muscle Atrophy ◽

Total Protein ◽

Ubiquitin Proteasome System ◽

Ground Squirrels ◽

E3 Ligases ◽

Protein Levels ◽

Ubiquitin Proteasome ◽

Mammalian Hibernation

Background.Mammalian hibernation in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is characterized by dramatic changes on a physiological and molecular level. During hibernation, mammalian hearts show a propensity to hypertrophy due to the need for increasing contractility to pump colder and more viscous blood. While cardiac hypertrophy is quite often a process characterized by decompensation, the ground squirrel studied is an excellent model of cardiac plasticity and cardioprotection under conditions of hypothermia and ischemia. The forkhead box O (Foxo) family of proteins and myogenin (MyoG) are transcription factors that control protein degradation and muscle atrophy by regulating the expression of the E3 ubiquitin ligases, MAFbx and MuRF1. These ligases are part of the ubiquitin proteasome system by transferring ubiquitin to proteins and targeting these proteins for degradation. Regulation of Foxo1 and 3a occurs through phosphorylation at different residues. The threonine-24 (Thr-24) and serine-319 (Ser-319) residues on Foxo1, and the Thr-32 residue on Foxo3a are phosphorylated by Akt, leading to cytoplasmic localization of Foxo. We propose that the described mechanism contributes to the changes taking place in cardiac muscle throughout hibernation.Methods.Total and phosphorylated protein levels of Foxo1 and Foxo3a, as well as total protein levels of MyoG, MAFbx, and MuRF1, were studied using immunoblotting.Results.Immunoblotting results demonstrated upregulations in Foxo1 and Foxo3a total protein levels (1.3- and 4.5-fold increases relative to euthermic control, for Foxo1 and 3a respectively) during late torpor, and protein levels remained elevated throughout the rest of torpor and at interbout arousal. We also observed decreases in inactive, phosphorylated Foxo1 and 3a proteins during throughout torpor, where levels of p-Foxo1 Ser319and Thr24, as well as p-Foxo3a Thr32decreased by at least 45% throughout torpor. MyoG was upregulated only during late torpor by 2.4-fold. Protein levels of MAFbx and MuRF1 increased in late torpor as well as during early arousal by as much as 2.8-fold, and MAFbx levels remained elevated during interbout arousal, whereas MuRF1 levels returned to control levels.Discussion.The present results indicate that upregulation and activation of Foxo1 and 3a, in addition to the increase in MyoG levels at late torpor, may be upregulating the expression of MAFbx and MuRF1. These findings suggest that there is activation of the ubiquitin proteasome system (UPS) as ground squirrels arouse from torpor. Therefore, the signalling pathway involving MyoG, and the E3 ligases MAFbx and MuRF1, plays a significant role in cardiac muscle remodelling during hibernation. These findings provide insights into the regulation of protein degradation and turnover in the cardiac muscle of a hibernator model.

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