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

ABSTRACTWnt signaling is critically dependent on dishevelled proteins (DVL1-3), which are required to assemble an intracellular Wnt signalosome at the plasma membrane. The levels of dishevelled proteins are strictly regulated by multiple E3 ubiquitin ligases that target DVL1-3 for ubiquitination and proteasomal degradation. The BTB-Kelch family protein KLHL12 is a substrate-specific adaptor for a Cullin-RING E3 ligase complex that contains Cullin3 and Rbx1. KLHL12 was the first E3 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 PLEXHA4, PEF1 and SEC31. A 20-mer peptide containing the DVL1 motif bound to the Kelch domain of KLHL12 with low micromolar affinity. In cells, the mutation or deletion of this motif caused a striking reduction in the binding, ubiquitination and stability of DVL1 confirming this sequence as a degron motif for KLHL12 recruitment. 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. The structure revealed that the DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. Overall, 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.

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

10.26434/chemrxiv.7698185 ◽

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>

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

10.26434/chemrxiv.7698185.v2 ◽

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>

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

10.26434/chemrxiv.7698185.v1 ◽

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>

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Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

10.1101/090563 ◽

2016 ◽

Cited By ~ 1

Author(s):

Hui Dong ◽

Jack Dumenil ◽

Fu-Hao Lu ◽

Li Na ◽

Hannes Vanhaeren ◽

...

Keyword(s):

Cell Proliferation ◽

Molecular Mechanisms ◽

Regulatory Proteins ◽

Peptidase Activity ◽

Organ Growth ◽

E3 Ligases ◽

Promote Cell Proliferation ◽

Ring E3 Ligase ◽

Ring E3 Ligases ◽

Ring E3

ABSTRACTThe characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana. The peptidase is activated by two RING E3 ligases, BB and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PRT1 of the N-end rule pathway. DA1 peptidase activity also cleaves the de-ubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TCP15 and TCP22, which promote cell proliferation proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins.

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Structural basis for recruitment of DAPK1 to the KLHL20 E3 ligase

10.1101/414250 ◽

2018 ◽

Cited By ~ 1

Author(s):

Zhuoyao Chen ◽

Sarah Picaud ◽

Panagis Filippakopoulos ◽

Vincenzo D’Angiolella ◽

Alex N. Bullock

Keyword(s):

Hydrophobic Interactions ◽

E3 Ligase ◽

Salt Bridge ◽

Cysteine Residue ◽

Structural Basis ◽

Death Domain ◽

Binding Modes ◽

E3 Ligases ◽

Kelch Domain ◽

Helical Turn

SUMMARYBTB-Kelch proteins form the largest subfamily of Cullin-RING E3 ligases, yet their substrate complexes are mapped and structurally characterized only for KEAP1 and KLHL3. KLHL20 is a related CUL3-dependent ubiquitin ligase linked to autophagy, cancer and Alzheimer’s disease that promotes the ubiquitination and degradation of substrates including DAPK1, PML and ULK1. We identified a ‘LPDLV’-containing recruitment site in the DAPK1 death domain and determined the 1.1 Å crystal structure of a KLHL20-DAPK1 complex. DAPK1 binds to KLHL20 as a loose helical turn that inserts deeply into the central pocket of the Kelch domain to contact all six blades of the β-propeller. Here, KLHL20 forms a salt bridge as well as hydrophobic interactions that include a tryptophan and cysteine residue ideally positioned for covalent inhibitor development. The structure highlights the diverse binding modes of circular substrate pockets versus linear grooves and suggests a novel E3 ligase for protac-based drug design.

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Faculty Opinions recommendation of The Arabidopsis C3H2C3-type RING E3 ubiquitin ligase AtAIRP1 is a positive regulator of an abscisic acid-dependent response to drought stress.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.6881957.7063055 ◽

2010 ◽

Author(s):

Hanjo Hellmann

Keyword(s):

Abscisic Acid ◽

Drought Stress ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Positive Regulator ◽

Ring E3

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Faculty Opinions recommendation of Designing active RNF4 monomers by introducing a tryptophan: avidity towards E2∼Ub conjugates dictates the activity of ubiquitin RING E3 ligases.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.735681965.793570379 ◽

2020 ◽

Author(s):

Thorsten Hoppe

Keyword(s):

E3 Ligases ◽

Ring E3 Ligases ◽

Ring E3

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E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

International Journal of Molecular Sciences ◽

10.3390/ijms22115712 ◽

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.

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