Generation of free ubiquitin chains is up-regulated in stress and facilitated by the HECT domain ubiquitin ligases UFD4 and HUL5

2012 ◽  
Vol 444 (3) ◽  
pp. 611-617 ◽  
Author(s):  
Ori Braten ◽  
Nitzan Shabek ◽  
Yelena Kravtsova-Ivantsiv ◽  
Aaron Ciechanover
Keyword(s):  
Signal Transduction ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
26S Proteasome ◽  
Specific Substrate ◽  
Polyubiquitin Chains ◽  
Hect Domain ◽  
Protein Substrate ◽  
C Terminus ◽  
Free Ubiquitin

Polyubiquitin chains serve a variety of physiological roles. Typically the chains are bound covalently to a protein substrate and in many cases target it for degradation by the 26S proteasome. However, several studies have demonstrated the existence of free polyubiquitin chains which are not linked to a specific substrate. Several physiological functions have been attributed to these chains, among them playing a role in signal transduction and serving as storage of ubiquitin for utilization under stress. In the present study, we have established a system for the detection of free ubiquitin chains and monitoring their level under changing conditions. Using this system, we show that UFD4 (ubiquitin fusion degradation 4), a HECT (homologous with E6-AP C-terminus) domain ubiquitin ligase, is involved in free chain generation. We also show that generation of these chains is stimulated in response to a variety of stresses, particularly those caused by DNA damage. However, it appears that the stress-induced synthesis of free chains is catalysed by a different ligase, HUL5 (HECT ubiquitin ligase 5), which is also a HECT domain E3.

scholarly journals The E3 ligase HOIL-1 catalyses ester bond formation between ubiquitin and components of the Myddosome in mammalian cells

2019 ◽  
Vol 116 (27) ◽  
pp. 13293-13298 ◽  
Author(s):  
Ian R. Kelsall ◽  
Jiazhen Zhang ◽  
Axel Knebel ◽  
J. Simon C. Arthur ◽  
Philip Cohen
Keyword(s):  
Ubiquitin Ligase ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
De Novo ◽  
E3 Ligase ◽  
Toll Like Receptor ◽  
Signaling Mechanism ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
Physiological Substrates

The linear ubiquitin assembly complex (LUBAC) comprises 3 components: HOIP, HOIL-1, and Sharpin, of which HOIP and HOIL-1 are both members of the RBR subfamily of E3 ubiquitin ligases. HOIP catalyses the formation of Met1-linked ubiquitin oligomers (also called linear ubiquitin), but the function of the E3 ligase activity of HOIL-1 is unknown. Here, we report that HOIL-1 is an atypical E3 ligase that forms oxyester bonds between the C terminus of ubiquitin and serine and threonine residues in its substrates. Exploiting the sensitivity of HOIL-1–generated oxyester bonds to cleavage by hydroxylamine, and macrophages from knock-in mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant, we identify IRAK1, IRAK2, and MyD88 as physiological substrates of the HOIL-1 E3 ligase during Toll-like receptor signaling. HOIL-1 is a monoubiquitylating E3 ubiquitin ligase that initiates the de novo synthesis of polyubiquitin chains that are attached to these proteins in macrophages. HOIL-1 also catalyses its own monoubiquitylation in cells and most probably the monoubiquitylation of Sharpin, in which ubiquitin is also attached by an oxyester bond. Our study establishes that oxyester-linked ubiquitylation is used as an intracellular signaling mechanism.

scholarly journals Structural Basis of Ubiquitin Recognition by the Ubiquitin-associated (UBA) Domain of the Ubiquitin Ligase EDD

2007 ◽  
Vol 282 (49) ◽  
pp. 35787-35795 ◽  
Author(s):  
Guennadi Kozlov ◽  
Long Nguyen ◽  
Tong Lin ◽  
Gregory De Crescenzo ◽  
Morag Park ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
Damage Repair ◽  
The Family ◽  
Uba Domain ◽  
Ordered Water

EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85Å crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices α1 and α3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.

scholarly journals Cooperation of HECT-domain Ubiquitin Ligase hHYD and DNA Topoisomerase II-binding Protein for DNA Damage Response

2001 ◽  
Vol 277 (5) ◽  
pp. 3599-3605 ◽  
Author(s):  
Yoshiomi Honda ◽  
Masahide Tojo ◽  
Kazuhito Matsuzaki ◽  
Tadashi Anan ◽  
Mitsuhiro Matsumoto ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Ubiquitin Ligase ◽  
Topoisomerase Ii ◽  
Binding Protein ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Hect Domain ◽  
Damage Response

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain

2008 ◽  
Vol 415 (1) ◽  
pp. 155-163 ◽  
Author(s):  
M. Christine Bruce ◽  
Voula Kanelis ◽  
Fatemeh Fouladkou ◽  
Anne Debonneville ◽  
Olivier Staub ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Down Regulation ◽  
Hect Domain ◽  
Ww Domains ◽  
Protein Protein Interaction ◽  
C Terminus ◽  
Subsequent Degradation ◽  
Interaction Domains ◽  
The Stability ◽  
Py Motif

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein–protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse–chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain–HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.

scholarly journals Cdc6 degradation requires phosphodegron created by GSK-3 and Cdk1 for SCFCdc4 recognition in Saccharomyces cerevisiae

2015 ◽  
Vol 26 (14) ◽  
pp. 2609-2619 ◽  
Author(s):  
Amr Al-Zain ◽  
Lea Schroeder ◽  
Alina Sheglov ◽  
Amy E. Ikui
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Genome Integrity ◽  
Binding Motif ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Origin Firing ◽  
C Terminus ◽  
Prereplicative Complex

To ensure genome integrity, DNA replication takes place only once per cell cycle and is tightly controlled by cyclin-dependent kinase (Cdk1). Cdc6p is part of the prereplicative complex, which is essential for DNA replication. Cdc6 is phosphorylated by cyclin-Cdk1 to promote its degradation after origin firing to prevent DNA rereplication. We previously showed that a yeast GSK-3 homologue, Mck1 kinase, promotes Cdc6 degradation in a SCFCdc4-dependent manner, therefore preventing rereplication. Here we present evidence that Mck1 directly phosphorylates a GSK-3 consensus site in the C-terminus of Cdc6. The Mck1-dependent Cdc6 phosphorylation required priming by cyclin/Cdk1 at an adjacent CDK consensus site. The sequential phosphorylation by Mck1 and Clb2/Cdk1 generated a Cdc4 E3 ubiquitin ligase–binding motif to promote Cdc6 degradation during mitosis. We further revealed that Cdc6 degradation triggered by Mck1 kinase was enhanced upon DNA damage caused by the alkylating agent methyl methanesulfonate and that the resulting degradation was mediated through Cdc4. Thus, Mck1 kinase ensures proper DNA replication, prevents DNA damage, and maintains genome integrity by inhibiting Cdc6.

scholarly journals SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage, while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

2021 ◽  
Vol 118 (6) ◽  
pp. e2015654118
Author(s):  
Ukhyun Jo ◽  
Yasuhisa Murai ◽  
Sirisha Chakka ◽  
Lu Chen ◽  
Ken Cheng ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Synthetic Lethality ◽  
E3 Ubiquitin Ligase ◽  
Replication Forks ◽  
Atpase Domain ◽  
C Terminus ◽  
Genome Wide ◽  
Schlafen 11

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4CDT2 ubiquitin ligase.

scholarly journals The SCFHOS/β-TRCP-ROC1 E3 Ubiquitin Ligase Utilizes Two Distinct Domains within CUL1 for Substrate Targeting and Ubiquitin Ligation

2000 ◽  
Vol 20 (4) ◽  
pp. 1382-1393 ◽  
Author(s):  
Kenneth Wu ◽  
Serge Y. Fuchs ◽  
Angus Chen ◽  
Peilin Tan ◽  
Carlos Gomez ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Binding Activity ◽  
Dual Function ◽  
Necrosis Factor Alpha ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
N Terminus ◽  
Factor Alpha ◽  
F Box Protein ◽  
Necessary And Sufficient

ABSTRACT We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IκBα. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCFHOS/β-TRCP-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/β-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IκB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.

Novel players fine-tune plant trade-offs

2015 ◽  
Vol 58 ◽  
pp. 83-100 ◽  
Author(s):  
Selena Gimenez-Ibanez ◽  
Marta Boter ◽  
Roberto Solano
Keyword(s):  
Ubiquitin Ligase ◽  
Feedback Loop ◽  
Molecular Level ◽  
26S Proteasome ◽  
Signalling Molecules ◽  
Transcriptional Reprogramming ◽  
Trade Offs ◽  
Jaz Proteins ◽  
Regulatory Feedback Loop ◽  
Fine Tune

Jasmonates (JAs) are essential signalling molecules that co-ordinate the plant response to biotic and abiotic challenges, as well as co-ordinating several developmental processes. Huge progress has been made over the last decade in understanding the components and mechanisms that govern JA perception and signalling. The bioactive form of the hormone, (+)-7-iso-jasmonyl-l-isoleucine (JA-Ile), is perceived by the COI1–JAZ co-receptor complex. JASMONATE ZIM DOMAIN (JAZ) proteins also act as direct repressors of transcriptional activators such as MYC2. In the emerging picture of JA-Ile perception and signalling, COI1 operates as an E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ repressors for degradation by the 26S proteasome, thereby derepressing transcription factors such as MYC2, which in turn activate JA-Ile-dependent transcriptional reprogramming. It is noteworthy that MYCs and different spliced variants of the JAZ proteins are involved in a negative regulatory feedback loop, which suggests a model that rapidly turns the transcriptional JA-Ile responses on and off and thereby avoids a detrimental overactivation of the pathway. This chapter highlights the most recent advances in our understanding of JA-Ile signalling, focusing on the latest repertoire of new targets of JAZ proteins to control different sets of JA-Ile-mediated responses, novel mechanisms of negative regulation of JA-Ile signalling, and hormonal cross-talk at the molecular level that ultimately determines plant adaptability and survival.

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