Structural basis of K63-ubiquitin chain formation by the Gordon-Holmes syndrome RBR E3 ubiquitin ligase RNF216

2022 ◽  
Author(s):  
Thomas R. Cotton ◽  
Simon A. Cobbold ◽  
Jonathan P. Bernardini ◽  
Lachlan W. Richardson ◽  
Xiangyi S. Wang ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Structural Basis ◽  
Ubiquitin Chain ◽  
Chain Formation

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 Structural Basis of the Activation and Degradation Mechanisms of the E3 Ubiquitin Ligase Nedd4L

Structure ◽  
2014 ◽  
Vol 22 (10) ◽  
pp. 1446-1457 ◽  
Author(s):  
Albert Escobedo ◽  
Tiago Gomes ◽  
Eric Aragón ◽  
Pau Martín-Malpartida ◽  
Lidia Ruiz ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Structural Basis ◽  
Degradation Mechanisms

scholarly journals Linear Ubiquitination Mediates EGFR-Induced NF-κB Pathway and Tumor Development

2021 ◽  
Vol 22 (21) ◽  
pp. 11875
Author(s):  
Fang Hua ◽  
Wenzhuo Hao ◽  
Lingyan Wang ◽  
Shitao Li
Keyword(s):  
Cell Proliferation ◽  
Ubiquitin Ligase ◽  
Tumor Development ◽  
E3 Ubiquitin Ligase ◽  
Growth Factor Receptor ◽  
Ubiquitin Chain ◽  
Plakophilin 2 ◽  
Ubiquitin Ligase Activity ◽  
Signaling Axis ◽  
Differentiation And Proliferation

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that instigates several signaling cascades, including the NF-κB signaling pathway, to induce cell differentiation and proliferation. Overexpression and mutations of EGFR are found in up to 30% of solid tumors and correlate with a poor prognosis. Although it is known that EGFR-mediated NF-κB activation is involved in tumor development, the signaling axis is not well elucidated. Here, we found that plakophilin 2 (PKP2) and the linear ubiquitin chain assembly complex (LUBAC) were required for EGFR-mediated NF-κB activation. Upon EGF stimulation, EGFR recruited PKP2 to the plasma membrane, and PKP2 bridged HOIP, the catalytic E3 ubiquitin ligase in the LUBAC, to the EGFR complex. The recruitment activated the LUBAC complex and the linear ubiquitination of NEMO, leading to IκB phosphorylation and subsequent NF-κB activation. Furthermore, EGF-induced linear ubiquitination was critical for tumor cell proliferation and tumor development. Knockout of HOIP impaired EGF-induced NF-κB activity and reduced cell proliferation. HOIP knockout also abrogated the growth of A431 epidermal xenograft tumors in nude mice by more than 70%. More importantly, the HOIP inhibitor, HOIPIN-8, inhibited EGFR-mediated NF-κB activation and cell proliferation of A431, MCF-7, and MDA-MB-231 cancer cells. Overall, our study reveals a novel linear ubiquitination signaling axis of EGFR and that perturbation of HOIP E3 ubiquitin ligase activity is potential targeted cancer therapy.

scholarly journals Structural basis for feedforward control in the PINK1/parkin pathway

2021 ◽  
Author(s):  
Véronique Sauvé ◽  
George Sung ◽  
Emma MacDougall ◽  
Guennadi Kozlov ◽  
Anshu Saran ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Feedforward Control ◽  
E3 Ubiquitin Ligase ◽  
Evolutionary Development ◽  
Structural Basis ◽  
Intermediate Step ◽  
Vinyl Sulfone ◽  
Mitochondrial Quality Control ◽  
High Affinity Site ◽  
Nmr Titrations

PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observe that phospho-ubiquitin can bind to two distinct sites on parkin, a high affinity site on RING1 that controls parkin localization, and a low affinity site on RING0 that releases parkin autoinhibition. Surprisingly, NMR titrations and ubiquitin vinyl sulfone assays show that the RING0 site has higher affinity for phospho-ubiquitin than the phosphorylated Ubl. Parkin could be activated by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic a mitochondrion bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.

Structure of the HLTF HIRAN domain and its functional implications in regression of a stalled replication fork

2020 ◽  
Vol 76 (8) ◽  
pp. 729-735
Author(s):  
Asami Hishiki ◽  
Mamoru Sato ◽  
Hiroshi Hashimoto
Keyword(s):  
Dna Binding ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
Structural Information ◽  
E3 Ubiquitin Ligase ◽  
Dna Helicase ◽  
Structural Basis ◽  
The Past ◽  
Functional Implications ◽  
Fork Regression

HLTF (helicase-like transcription factor) is a yeast RAD5 homolog that is found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and is a pivotal protein in template-switched DNA synthesis that allows DNA replication to continue even in the presence of DNA damage by utilizing a newly synthesized undamaged strand as a template. In addition, HLTF has a DNA-binding domain termed HIRAN (HIP116 and RAD5 N-terminal). HIRAN has been hypothesized to play a role in DNA binding; however, the structural basis of its role in DNA binding has remained unclear. In the past five years, several crystal structures of HIRAN have been reported. These structures revealed new insights into the molecular mechanism underlying DNA binding by HIRAN. Here, the structural information on HIRAN is summarized and the function of HIRAN in recognizing the 3′-terminus of the daughter strand at a stalled replication fork and the implications for its involvement in fork regression are discussed.

scholarly journals Structural analysis of the LDL receptor–interacting FERM domain in the E3 ubiquitin ligase IDOL reveals an obscured substrate-binding site

2020 ◽  
Vol 295 (39) ◽  
pp. 13570-13583
Author(s):  
Luca Martinelli ◽  
Athanassios Adamopoulos ◽  
Patrik Johansson ◽  
Paul T. Wan ◽  
Jenny Gunnarsson ◽  
...  
Keyword(s):  
Binding Site ◽  
Ubiquitin Ligase ◽  
Substrate Recognition ◽  
E3 Ubiquitin Ligase ◽  
Full Length ◽  
Scattering Data ◽  
Lipid Lowering ◽  
Structural Basis ◽  
Ferm Domain

Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.

scholarly journals Deficiency of the E3 Ubiquitin Ligase RBCK1 Causes Diffuse Brain Polyglucosan Accumulation and Neurodegeneration

2018 ◽  
Author(s):  
Mitchell A. Sullivan ◽  
Felix Nitschke ◽  
Erin E. Chown ◽  
Laura F. DiGiovanni ◽  
Mackenzie Chown ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
E3 Ubiquitin Ligase ◽  
Branching Enzyme ◽  
Lafora Disease ◽  
Ubiquitin Chain ◽  
Polyglucosan Bodies ◽  
Muscle Tissues ◽  
Neurobehavioral Deficits

SUMMARYGlycogen synthesis is vital, malstructure resulting in precipitation and accumulation into neurotoxic polyglucosan bodies (PBs). One well-understood mechanism of PB generation is glycogen branching enzyme deficiency (GBED). Less understood is Lafora disease (LD), resulting from absence of the glycogen phosphatase laforin or the E3 ubiquitin ligase malin, and accumulation of hyperphosphorylated PBs. LD afforded first insight that glycogen sphericity depends on more than adequate branching activity. Unexpectedly, deficiencies of the Linear Ubiquitin Chain Assembly Complex (LUBAC) components RBCK1 and HOIP result in PBs in muscle tissues. Here we analyzed nervous system phenotypes of mice lacking RBCK1 and find profuse PB accumulations in brain and spinal cord with extensive neurodegeneration and neurobehavioral deficits. Brain glycogen in these mice is characterized by long chains and hyperphosphorylation, similar to LD. Like in LD, glycogen synthase and branching enzyme are unaltered. Regional PB distribution mirrors LD and not GBED. Perisynaptic PB localization is unlike LD or GBED. The results indicate that RBCK1 is part of a system supplementing laforin-malin in regulating glycogen architecture including in unique neuronal locales.

scholarly journals Structural Basis of the Binding of Merlin FERM Domain to the E3 Ubiquitin Ligase Substrate Adaptor DCAF1

2014 ◽  
Vol 289 (21) ◽  
pp. 14674-14681 ◽  
Author(s):  
Youjun Li ◽  
Zhiyi Wei ◽  
Junyi Zhang ◽  
Zhou Yang ◽  
Mingjie Zhang
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Structural Basis ◽  
Ferm Domain

scholarly journals Ubiquitin-dependent regulation of immune and inflammatory signaling pathways

2014 ◽  
Vol 70 (a1) ◽  
pp. C241-C241
Author(s):  
Katrin Rittinger
Keyword(s):  
Signaling Pathways ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
High Specificity ◽  
Cellular Functions ◽  
Inflammatory Signaling ◽  
Ubiquitin Chain ◽  
E3 Ligases ◽  
Wide Range ◽  
Chain Synthesis

Modification of proteins with ubiquitin is a key mechanism for the regulation of a wide range of cellular functions. The outcome of the modification is determined by the way ubiquitin molecules are linked to each other. Linear (M1-linked) ubiquitin chains play an important role in the regulation of immune and inflammatory signaling pathways and contribute to the activation of NF-κB. They are synthesized by the E3 ubiquitin ligase LUBAC (linear ubiquitin chain assembly complex) that is composed of at least three subunits named HOIL-1L, HOIP and SHARPIN. LUBAC belongs to the RBR (RING-inbetween-RING) family of E3 ligases that combine the properties of RING and HECT ligases and act as RING/HECT hybrids. Indeed, we have recently shown that linear ubiquitin chain synthesis proceeds via ubiquitin thioester intermediate formed by the HOIP subunit before subsequent transfer onto the target. I will present a combination of structural and biochemical data that provide a molecular explanation how this unusual E3 ligase complex promotes the synthesis of linear ubiquitin chains with high specificity, regardless of the E2 conjugating enzyme it works with.

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