E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E2

Anandapadamanaban, Madhanagopal; Kyriakidis, Nikolaos C; Csizmók, Veronika; Wallenhammar, Amélie; Espinosa, Alexander; Ahlner, Alexandra; Round, Adam; Trewhella, Jill; Moche, M.; Wahren‐Herlenius, Marie; Sunnerhagen, Maria

doi:10.1074/jbc.ra119.008485

Cited by 18 publications

(15 citation statements)

References 79 publications

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“…A 2D schematic is depicted in Figure 2K and illustrates the proposed structure of the dimeric RPB1–ARMC5–CUL3–RBX1–UBE2E1–UBC complex based on our results and literature. We extracted X-ray crystallographic 3D information of the components in the complex, that is, RPB1, CUL3, RBX1, UBE2E1, and UBC from the Protein Database ( 56–59 ). The 3D structure of ARMC5 has not been resolved but is predicted by AlphaFold ( 39 ).…”

Section: Resultsmentioning

confidence: 99%

ARMC5 is part of an RPB1-specific ubiquitin ligase implicated in adrenal hyperplasia

Lao

Bourdeau

Gagliardi

et al. 2022

Nucleic Acids Research

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ARMC5 is implicated in several pathological conditions, but its function remains unknown. We have previously identified CUL3 and RPB1 (the largest subunit of RNA polymerase II (Pol II) as potential ARMC5-interacting proteins. Here, we show that ARMC5, CUL3 and RBX1 form an active E3 ligase complex specific for RPB1. ARMC5, CUL3, and RBX1 formed an active E3 specific for RPB1. Armc5 deletion caused a significant reduction in RPB1 ubiquitination and an increase in an accumulation of RPB1, and hence an enlarged Pol II pool in normal tissues and organs. The compromised RPB1 degradation did not cause generalized Pol II stalling nor depressed transcription in the adrenal glands but did result in dysregulation of a subset of genes, with most upregulated. We found RPB1 to be highly expressed in the adrenal nodules from patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) harboring germline ARMC5 mutations. Mutant ARMC5 had altered binding with RPB1. In summary, we discovered that wildtype ARMC5 was part of a novel RPB1-specific E3. ARMC5 mutations resulted in an enlarged Pol II pool, which dysregulated a subset of effector genes. Such an enlarged Pol II pool and gene dysregulation was correlated to adrenal hyperplasia in humans and KO mice.

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Section: Resultsmentioning

confidence: 99%

ARMC5 is part of an RPB1-specific ubiquitin ligase implicated in adrenal hyperplasia

Lao

Bourdeau

Gagliardi

et al. 2022

Nucleic Acids Research

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“…Phosphorylation of S80 displaces the B-box, thereby allowing E2 recruitment to restore TRIM21 ubiquitination activity. Similar to other TRIM RING/E2∼Ub complexes, the crystal structures of TRIM21 with its cognate E2s, UBE2E1 (PDB: 6FGA) [ 30 ] and a UBE2N-Ub conjugate (PDB: 6S53) [ 29 ], show symmetrical complexes surrounding a RING dimer, while in solution the RING is in a monomer-dimer equilibrium. Nevertheless, mutants that impair dimerization (M10E and M10E/M72E) remain active and able to catalyse unanchored K63-linked ubiquitin chain synthesis, UBE2W-primed anchored K63 chain extension and ubiquitin discharge, however less efficiently than the wild-type protein, suggesting that E3 activity may not strictly require a dimeric RING.…”

Section: Regulation Of Trim Ubiquitination Activitymentioning

confidence: 97%

Does it take two to tango? RING domain self-association and activity in TRIM E3 ubiquitin ligases

Fiorentini

Esposito

Rittinger

2020

Biochemical Society Transactions

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TRIM proteins form a protein family that is characterized by a conserved tripartite motif domain comprising a RING domain, one or two B-box domains and a coiled-coil region. Members of this large protein family are important regulators of numerous cellular functions including innate immune responses, transcriptional regulation and apoptosis. Key to their cellular role is their E3 ligase activity which is conferred by the RING domain. Self-association is an important characteristic of TRIM protein activity and is mediated by homodimerization via the coiled-coil region, and in some cases higher order association via additional domains of the tripartite motif. In many of the TRIM family proteins studied thus far, RING dimerization is an important prerequisite for E3 ligase enzymatic activity though the propensity of RING domains to dimerize differs significantly between different TRIMs and can be influenced by other regions of the protein.

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“…We have also shown that endoglin can interact with TRIM21, a protein involved in multiple cell functions. As other members of the TRIM family, TRIM21 can act as a ubiquitination enzyme targeting for degradation different proteins [56,57,58,90]. The TRIM family has been shown to ubiquitinate different components of the TGF-β signaling pathway [55,91], and our results suggest for the first time that TRIM21 may target for degradation the TGF-β auxiliary receptor endoglin.…”

Section: Discussionmentioning

confidence: 63%

“…Unfortunately, little is known about the connection between the TRIM family and the TGF-β auxiliary receptor endoglin of endothelial cells. Among TRIM family members, TRIM21 acts not only as an E3 ubiquitin ligase on different target proteins, but also as a substrate in auto-ubiquitination, both leading to protein degradation [56,57,58]. In addition, TRIM21 is involved in cancer [59,60], inflammation [61,62,63,64], intracellular immunity, and autoimmunity [47,65,66,67].…”

Section: Resultsmentioning

confidence: 99%

Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

Gallardo-Vara

Ruíz-Llorente

Casado‐Vela

et al. 2019

Cells

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Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-β receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-β receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-β family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation.

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E3 ubiquitin-protein ligase TRIM21-mediated lysine capture by UBE2E1 reveals substrate-targeting mode of a ubiquitin-conjugating E2

Cited by 18 publications

References 79 publications

ARMC5 is part of an RPB1-specific ubiquitin ligase implicated in adrenal hyperplasia

ARMC5 is part of an RPB1-specific ubiquitin ligase implicated in adrenal hyperplasia

Does it take two to tango? RING domain self-association and activity in TRIM E3 ubiquitin ligases

Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

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