RanBP9 controls the oligomeric state of CTLH complex assemblies

Journal of Biological Chemistry, Journal Year: 2023, Volume and Issue: 299(2), P. 102869 - 102869

Published: Jan. 5, 2023

The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin Wdr26, specify two alternative complexes that differ in quaternary structure, thereby allowing the presumably target different substrates. With aid of biophysical biochemical techniques, we characterized assembly pathways, focusing not only on Wdr26 but also RanBP9, Twa1, Armc8β which are critical establish scaffold this ligase. We demonstrate ability tetramerize into dimers define mutually exclusive oligomerization modules compete nanomolar affinity for RanBP9 binding. remaining scaffolding strongly interact each other again affinity. Our data organizes prevents higher order dimeric Armc8β–Twa1 heterodimer through its tight Combined, our studies pathways elucidate role governing differential oligomeric assemblies, advancing mechanistic understanding architectures. 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Hollemann RMND5 from Xenopus laevis ubiquitin-ligase early embryonic forebrain development.PLoS 10e0120342Crossref (25) 29Skraban C.M. Wells C.F. Markose Cho M.T. Nesbitt A.I. Au P.Y.B. al.WDR26 haploinsufficiency causes recognizable syndrome intellectual disability, seizures, abnormal gait, distinctive facial features.Am. Hum. Genet. 101: 139-148Abstract (35) 30Vogel T.W. Manjila Cohen A.R. Novel neurodevelopmental disorder case giant occipitoparietal meningoencephalocele.J. Neurosurg. Pediatr. 2012; 10: 25-29Crossref hematopoiesis (31Kunert Meyer I. Fleischhauer Wannack Fiedler Shivdasani R.A. microtubule modulator RanBP10 platelet discoid shape degranulation.Blood. 114: 5532-5540Crossref (37) 32Sherpa Mueller Ö. Yao al.Modular UBE2H-CTLH E2-E3 regulate erythroid maturation.eLife. 11 (e77937)https://doi.org/10.7554/eLife.77937Crossref 33Soni Bala Hanspal Requirement erythroblast-macrophage (Emp) definitive erythropoiesis.Blood Cells Dis. 2008; 41: 141-147Crossref 34Wei Boulais P.E. Pinho Tanaka Frenette P.S. Maea expressed macrophages, erythroblasts, maintains postnatal murine bone marrow erythroblastic islands.Blood. 133: 1222-1232Crossref 35Wei Dong Pierce Nakahara al.MAEA promoting autophagy maintenance haematopoietic stem cells.Nat. 12: 2522Crossref (18) 36Zhen Moo Chen Zheng al.Wdr26 condensation developing erythroblasts.Blood. 208-219Crossref (31) While name suggests there single architecture, fact multiple may lead complexes. how variety achieved, such identification being targeted cognate SR within remains elusive. Insights principles mostly derived (glucose-induced deficient) complex, extensively yeast counterpart phylogenetically conserved (37Francis Han Adams Molecular phylogeny eukaryotic dominated homologous components, muskelin/RanBPM/CTLH complex.PLoS 8e75217Crossref complex. In system, recognized selective SRs upon stimuli. Targeting superfluous gluconeogenic enzymes (e.g., fructose-1,6-bisphosphatase, Fbp1) first identified catabolite inactivation after switching glycolytic conditions (38Braun Hofmann Scheel Gid9, second finger contributes Gid degradation.FEBS 2011; 585: 3856-3861Crossref 39Menssen Schweiggert Schreiner Kusevic Reuther Braun al.Exploring topology catabolite-induced enzymes.J. 287: 25602-25614Abstract (74) 40Regelmann Schüle Josupeit F.S. Horak Rose Entian K.-D. al.Catabolite fructose-1,6-bisphosphatase Saccharomyces cerevisiae: screen eight genes existence pathways.Mol. 2003; 14: 1652-1663Crossref (122) 41Santt Juretschke Kimmig (E3) carbohydrate metabolism.Mol. 19: 3323-3333Crossref (109) Fbp1 via Gid4, N-terminal proline residues (N-degron) β-barrel serves N-recognin (Pro)/N-degron 42Chen S.-J. Wu Wadas Oh J.-H. 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U. 116: 15914-15923Crossref (30) 49Shin J.S. S.H. Kim Heo Song H.K. Crystal structure Pro/N-degron.Biochem. Biophys. 582: 86-92Crossref distinct third SR, Gid11, predicted contain WD40 repeat containing β-propeller predominantly threonine instead 50Kong K.-Y.E. Fischer Meurer Kats Rühle al.Timer-based proteomic profiling ubiquitin-proteasome system ligase.Mol. 2460-2476.e11Abstract Substrate-targeting abilities modulated recently Gid12 restricts obstructing Gid4-binding site (51Qiao Lee C.-W. Cheng Duennebacke al.Cryo-EM structures Gid12-bound reveal steric blockade inhibiting ubiquitylation.Nat. 3041Crossref no replacing identified; Gid4-independent route reported (21Mohamed Interestingly, Ypel5, additional yeast, potential blocks Wdr26-binding needs investigated. Besides recruitment crucial specific ubiquitylation. Structural cryo-EM revealed chelated center facilitating sites For closely related described could proteins. case, recruited C-terminal part Armc8α Together they comprise (SRS) module (Protein Data Bank [PDB] entry: 7NSC). catalytic domain–containing heterodimerize, bind SRS module. This oligomerized dimerization (supramolecular module, SA) bridging modules, adopting chelating "ring" space center. Noteworthy, possible Ypel5 face summary, GID–CTLH relies both suitable specifically degron sequences paired assembly, assists positioning Compared displays greater interchangeable thus possibly modulating assembly: Rmnd5b replace paralogs longer isoform muskelin, presumably, distantly 1B). Taken together, diversity functional composition, instead, assemblies possible, it expected specificity. To obtain insights factors utilized toolbox purified individual 1C) reconstitute stages vitro. Since knowledge still lacking, focused characterization Facilitated availability pure proteins, tested putative analytical size-exclusion chromatography (aSEC) native agarose gel electrophoresis (NAGE). step, determined state assembled using multiangle light scattering coupled aSEC (SEC–MALS). Finally, quantified affinities partners isothermal titration calorimetry (ITC). sought address questions integrated whether way finally, Armc8β–RanBP9–Twa1 influenced stepwise formation. study varying compositions, generated composed 1C). variants rat recombinantly Escherichia coli. Mouse when isolation, insoluble, coexpressed together Similarly, soluble insect cells. conserved, example, pairwise sequence identity mouse 99.6%. Larger Armc8α, systems, N termini unstructured (52Kelley Mezulis Yates Wass M.N. Sternberg M.J.E. Phyre2 web portal modeling, prediction analysis.Nat. Protoc. 845-858Crossref (6540) 53Varadi Anyango Deshpande Nair Natassia Yordanova al.AlphaFold database: Massively expanding structural coverage protein-sequence high-accuracy models.Nucl. Acids 50: D439-D444Crossref (1697) deleted enhance solubility. nomenclature will used: muskelin-M, Wdr26-W, RanBP9-R, Twa1-T, Armc8α/β-α/β, Rmnd5a–Maea abbreviated cat (as subscript). integration mediates previously 625 tandem, followed CRA-like repeats folding seven-bladed d

Language: Английский

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Insights into the recognition mechanism in the UBR box of UBR4 for its specific substrates

Communications Biology, Journal Year: 2023, Volume and Issue: 6(1)

Published: Nov. 29, 2023

Abstract The N-end rule pathway is a proteolytic system involving the destabilization of N-terminal amino acids, known as N-degrons, which are recognized by N-recognins. Dysregulation results in accumulation undesired proteins, causing various diseases. E3 ligases UBR subfamily recognize and degrade N-degrons through ubiquitin-proteasome system. Herein, we investigated UBR4, has distinct mechanism for recognizing type-2 N-degrons. Structural analysis revealed that box UBR4 differs from other boxes N-degron binding sites. It recognizes acids containing an aromatic ring type-1 arginine two phenylalanines on its hydrophobic surface. We also characterized second ligand residue. This report structural basis underlying recognition with implications understanding pathway.

Language: Английский

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The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation

Journal of Experimental & Clinical Cancer Research, Journal Year: 2024, Volume and Issue: 43(1)

Published: Jan. 9, 2024

Abstract Background Metastasis has emerged as the major reason of treatment failure and mortality in patients with nasopharyngeal carcinoma (NPC). Growing evidence links abnormal DNA methylation to initiation progression NPC. However, precise regulatory mechanism behind these processes remains poorly understood. Methods Bisulfite pyrosequencing, RT-qPCR, western blot, immunohistochemistry were used test expression level NEURL3 its clinical significance. The biological function was examined both vitro vivo. Mass spectrometry, co-immunohistochemistry, immunofluorescence staining, ubiquitin assays performed explore NEURL3. Results promoter region , encoding an E3 ligase, obviously hypermethylated, leading downregulated Clinically, NPC a low indicated unfavorable prognosis prone develop distant metastasis. Overexpression could suppress epithelial mesenchymal transition metastasis cells Mechanistically, promoted Vimentin degradation by increasing K48-linked polyubiquitination at lysine 97. Specifically, restoration fully reverse tumor suppressive effect overexpression cells. Conclusions Collectively, our study uncovers novel which inhibits metastasis, thereby providing promising therapeutic target for treatment.

Language: Английский

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Skraban‐Deardorff intellectual disability syndrome‐associated mutations in WDR26 impair CTLH E3 complex assembly

FEBS Letters, Journal Year: 2024, Volume and Issue: 598(9), P. 978 - 994

Published: April 4, 2024

Patients with Skraban-Deardorff syndrome (SKDEAS), a neurodevelopmental associated spectrum of developmental and intellectual delays disabilities, harbor diverse mutations in WDR26, encoding subunit the multiprotein CTLH E3 ubiquitin ligase complex. Structural studies revealed that homodimers WDR26 bridge two core-CTLH complexes to generate giant, hollow oval-shaped supramolecular assemblies. Additionally, mediates complex binding YPEL5 functions as substrate receptor for transcriptional repressor HBP1. Here, we mapped SKDEAS-associated on structural model tested their functionality complementation using genetically engineered human cells lacking Despite diversity mutations, 15 16 mutants impaired at least one function contributing assembly interactions, thus providing first mechanistic insights into SKDEAS pathology.

Language: Английский

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Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates

Proceedings of the National Academy of Sciences, Journal Year: 2022, Volume and Issue: 119(31)

Published: July 25, 2022

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues can be recognized as destabilizing ones by the Arg/N-degron pathway. A cleavage generate Arg at N terminus resulting C-terminal (Ct) fragment either directly or after Nt-arginylation Ct-fragment Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNA cosubstrate. Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate Cys-sulfonate) short peptides. genes fungi, animals, plants have been cloned decades ago, but three-dimensional structure remained unknown. detailed mechanism arginylation unknown well. We describe here crystal R-transferase from budding yeast Kluyveromyces lactis . The 58-kDa comprises two domains recognize, together, an acidic Nt-residue acceptor substrate, residue , 3′-proximal segment tRNA moiety. enzyme’s active site located, least in part, between domains. In vitro vivo assays with site-directed mutants were suggested structural results yielded inferences about specific binding sites Ate1. also analyzed inhibition activity hemin (Fe 3+ -heme), found induced previously undescribed disulfide-mediated oligomerization Together, these advance understanding

Language: Английский

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