scholarly journals Cereblon versus VHL: Hijacking E3 Ligases Against Each Other Using PROTACs

2019 ◽  
Author(s):  
Miriam Girardini ◽  
Chiara Maniaci ◽  
Scott J. Hughes ◽  
Andrea Testa ◽  
Alessio Ciulli
Keyword(s):  
Ubiquitin Ligase ◽  
Active Compound ◽  
Substrate Recognition ◽  
E3 Ubiquitin Ligase ◽  
Cellular Activity ◽  
Proof Of Concept ◽  
Design Synthesis ◽  
E3 Ligases ◽  
Von Hippel Lindau ◽  
Ring E3

<div> <div> <div> <p>The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degradation. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compound 14a induced potent, rapid and profound preferential degradation of CRBN over VHL in cancer cell lines. At lower concentrations, weaker degradation of VHL was instead observed. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown. </p> </div> </div> </div>

scholarly journals Cereblon versus VHL: Hijacking E3 Ligases Against Each Other Using PROTACs

2019 ◽  
Author(s):  
Miriam Girardini ◽  
Chiara Maniaci ◽  
Scott J. Hughes ◽  
Andrea Testa ◽  
Alessio Ciulli
Keyword(s):  
Ubiquitin Ligase ◽  
Active Compound ◽  
Substrate Recognition ◽  
E3 Ubiquitin Ligase ◽  
Cellular Activity ◽  
Proof Of Concept ◽  
Design Synthesis ◽  
E3 Ligases ◽  
Von Hippel Lindau ◽  
Ring E3

<div> <div> <div> <p>The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degradation. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compound 14a induced potent, rapid and profound preferential degradation of CRBN over VHL in cancer cell lines. At lower concentrations, weaker degradation of VHL was instead observed. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown. </p> </div> </div> </div>

scholarly journals Cereblon vs VHL: Hijacking E3 Ligases Against Each Other Using PROTACs

2019 ◽  
Author(s):  
Miriam Girardini ◽  
Chiara Maniaci ◽  
Scott J. Hughes ◽  
Andrea Testa ◽  
Alessio Ciulli
Keyword(s):  
Ubiquitin Ligase ◽  
Active Compound ◽  
Substrate Recognition ◽  
E3 Ubiquitin Ligase ◽  
Cellular Activity ◽  
Proof Of Concept ◽  
Design Synthesis ◽  
E3 Ligases ◽  
Von Hippel Lindau ◽  
Ring E3

<div> <div> <div> <p>The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degradation. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compound 14a induced potent, rapid and profound preferential degradation of CRBN over VHL in cancer cell lines. At lower concentrations, weaker degradation of VHL was instead observed. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown. </p> </div> </div> </div>

scholarly journals The von Hippel–Lindau Cullin-RING E3 ubiquitin ligase regulates APOBEC3 cytidine deaminases

2021 ◽  
Author(s):  
Gaël K. Scholtès ◽  
Aubrey M. Sawyer ◽  
Cristina C. Vaca ◽  
Isabelle Clerc ◽  
Meejeon Roh ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Cytidine Deaminases ◽  
Von Hippel Lindau ◽  
Ring E3

scholarly journals Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12

Open Biology ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 200041 ◽  
Author(s):  
Zhuoyao Chen ◽  
Gregory A. Wasney ◽  
Sarah Picaud ◽  
Panagis Filippakopoulos ◽  
Masoud Vedadi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Hydrophobic Interactions ◽  
E3 Ubiquitin Ligase ◽  
Structural Basis ◽  
E3 Ligases ◽  
Kelch Domain ◽  
Ring E3 Ligases ◽  
Ring E3 ◽  
New Protein

Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a ‘PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.

scholarly journals Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer

2019 ◽  
Author(s):  
Paul Murphy ◽  
Yingqi Xu ◽  
Sarah L. Rouse ◽  
Steve J. Matthews ◽  
J Carlos Penedo ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Single Molecule ◽  
Electrostatic Interactions ◽  
Substrate Recognition ◽  
E3 Ligase ◽  
Nucleophilic Attack ◽  
E3 Ligases ◽  
Intrinsically Disordered ◽  
Ring E3 ◽  
Basic Region

AbstractPost-translational modification of proteins with ubiquitin represents a widely used mechanism for cellular regulation. Ubiquitin is activated by an E1 enzyme, transferred to an E2 conjugating enzyme and covalently linked to substrates by one of an estimated 600 E3 ligases (1). RING E3 ligases play a pivotal role in selecting substrates and priming the ubiquitin loaded E2 (E2~Ub) for catalysis (2,3). RING E3 RNF4 is a SUMO targeted ubiquitin ligase (4) with important roles in arsenic therapy for cancer (4,5) and in DNA damage responses (6,7). RNF4 has a RING domain and a substrate recognition domain containing multiple SUMO Interaction Motifs (SIMs) embedded in a region thought to be intrinsically disordered (8). While molecular details of SUMO recognition by the SIMs (8–10) and RING engagement of ubiquitin loaded E2 (3,11–15) have been determined, the mechanism by which SUMO substrate is delivered to the RING to facilitate ubiquitin transfer is an important question to be answered. Here, we show that the intrinsically disordered substrate-recognition domain of RNF4 maintains the SIMs in a compact global architecture that facilitates SUMO binding, while a highly-basic region positions substrate for nucleophilic attack on RING-bound ubiquitin loaded E2. Contrary to our expectation that the substrate recognition domain of RNF4 was completely disordered, distance measurements using single molecule Fluorescence Resonance Energy Transfer (smFRET) and NMR paramagnetic relaxation enhancement (PRE) revealed that it adopts a defined conformation primed for SUMO interaction. Mutational and biochemical analysis indicated that electrostatic interactions involving the highly basic region linking the substrate recognition and RING domains juxtaposed those regions and mediated substrate ubiquitination. Our results offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.

scholarly journals HIV-1 Vpr Loads Uracil DNA Glycosylase-2 onto DCAF1, a Substrate Recognition Subunit of a Cullin 4A-RING E3 Ubiquitin Ligase for Proteasome-dependent Degradation

2010 ◽  
Vol 285 (48) ◽  
pp. 37333-37341 ◽  
Author(s):  
Jinwoo Ahn ◽  
Thomas Vu ◽  
Zach Novince ◽  
Jennifer Guerrero-Santoro ◽  
Vesna Rapic-Otrin ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Substrate Recognition ◽  
E3 Ubiquitin Ligase ◽  
Dna Glycosylase ◽  
Uracil Dna Glycosylase ◽  
Cullin 4A ◽  
Ring E3 ◽  
Hiv 1

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

scholarly journals Therapeutically actionable signaling node to rescue AURKA driven loss of primary cilia in VHL-deficient cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pratim Chowdhury ◽  
Dimuthu Perera ◽  
Reid T. Powell ◽  
Tia Talley ◽  
Durga Nand Tripathi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Primary Cilia ◽  
Mtor Inhibitor ◽  
E3 Ubiquitin Ligase ◽  
Xenograft Model ◽  
Aurora Kinase ◽  
Tumor Burden ◽  
Aurora Kinase A ◽  
Von Hippel Lindau ◽  
In Cells

AbstractLoss of primary cilia in cells deficient for the tumor suppressor von Hippel Lindau (VHL) arise from elevated Aurora Kinase A (AURKA) levels. VHL in its role as an E3 ubiquitin ligase targets AURKA for degradation and in the absence of VHL, high levels of AURKA result in destabilization of the primary cilium. We identified NVP-BEZ235, a dual PI3K/AKT and mTOR inhibitor, in an image-based high throughput screen, as a small molecule that restored primary cilia in VHL-deficient cells. We identified the ability of AKT to modulate AURKA expression at the transcript and protein level. Independent modulation of AKT and mTOR signaling decreased AURKA expression in cells confirming AURKA as a new signaling node downstream of the PI3K cascade. Corroborating these data, a genetic knockdown of AKT in cells deficient for VHL rescued the ability of these cells to ciliate. Finally, inhibition of AKT/mTOR using NVP-BEZ235 was efficacious in reducing tumor burden in a 786-0 xenograft model of renal cell carcinoma. These data highlight a previously unappreciated signaling node downstream of the AKT/mTOR pathway via AURKA that can be targeted in VHL-null cells to restore ciliogenesis.

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