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

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m705655200 ◽

2007 ◽

Vol 282 (49) ◽

pp. 35787-35795 ◽

Cited By ~ 39

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.

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

Biochemical Journal ◽

10.1042/bj20111840 ◽

2012 ◽

Vol 444 (3) ◽

pp. 611-617 ◽

Cited By ~ 15

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.

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The S. Typhi effector StoD is an E3/E4 ubiquitin ligase which binds K48- and K63-linked diubiquitin

Life Science Alliance ◽

10.26508/lsa.201800272 ◽

2019 ◽

Vol 2 (3) ◽

pp. e201800272

Author(s):

Melanie A McDowell ◽

Alexander MP Byrne ◽

Elli Mylona ◽

Rebecca Johnson ◽

Agnes Sagfors ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Type Iii Secretion ◽

Intracellular Signaling ◽

Binding Assays ◽

Secretion Systems ◽

E Coli ◽

T3ss Effector ◽

C Terminus ◽

Type Iii Secretion Systems ◽

Β Sheet

Salmonella enterica (e.g., serovars Typhi and Typhimurium) relies on translocation of effectors via type III secretion systems (T3SS). Specialization of typhoidal serovars is thought to be mediated via pseudogenesis. Here, we show that the Salmonella Typhi STY1076/t1865 protein, named StoD, a homologue of the enteropathogenic Escherichia coli/enterohemorrhagic E. coli/Citrobacter rodentium NleG, is a T3SS effector. The StoD C terminus (StoD-C) is a U-box E3 ubiquitin ligase, capable of autoubiquitination in the presence of multiple E2s. The crystal structure of the StoD N terminus (StoD-N) at 2.5 Å resolution revealed a ubiquitin-like fold. In HeLa cells expressing StoD, ubiquitin is redistributed into puncta that colocalize with StoD. Binding assays showed that StoD-N and StoD-C bind the same exposed surface of the β-sheet of ubiquitin, suggesting that StoD could simultaneously interact with two ubiquitin molecules. Consistently, StoD interacted with both K63- (KD = 5.6 ± 1 μM) and K48-linked diubiquitin (KD = 15 ± 4 μM). Accordingly, we report the first S. Typhi–specific T3SS effector. We suggest that StoD recognizes and ubiquitinates pre-ubiquitinated targets, thus subverting intracellular signaling by functioning as an E4 enzyme.

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

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1382-1393.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1382-1393 ◽

Cited By ~ 72

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.

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Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity

Journal of Biological Chemistry ◽

10.1074/jbc.m115.687319 ◽

2015 ◽

Vol 291 (4) ◽

pp. 1803-1816 ◽

Cited By ~ 14

Author(s):

Su Jin Ham ◽

Soo Young Lee ◽

Saera Song ◽

Ju-Ryung Chung ◽

Sekyu Choi ◽

...

Keyword(s):

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Mitochondrial Outer Membrane ◽

Missense Mutations ◽

Mitochondrial Translocation ◽

C Terminus ◽

Carbonyl Cyanide ◽

N Terminus ◽

Upstream Kinase

Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis.

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Characterization of a Novel Member of the DOK Family That Binds and Modulates Abl Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8314 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8314-8325 ◽

Cited By ~ 78

Author(s):

Feng Cong ◽

Bing Yuan ◽

Stephen P. Goff

Keyword(s):

Tyrosine Kinase ◽

Map Kinase ◽

Intracellular Signaling ◽

Mammalian Cells ◽

Inhibitory Effect ◽

Pleckstrin Homology Domain ◽

Dependent Manner ◽

Kinase Activation ◽

C Terminus

ABSTRACT A novel member of the p62 dok family of proteins, termed DOKL, is described. DOKL contains features of intracellular signaling molecules, including an N-terminal PH (pleckstrin homology) domain, a central PTB (phosphotyrosine binding) domain, and a C-terminal domain with multiple potential tyrosine phosphorylation sites and proline-rich regions, which might serve as docking sites for SH2- and SH3-containing proteins. The DOKL gene is predominantly expressed in bone marrow, spleen, and lung, although low-level expression of the RNA can also be detected in other tissues. DOKL and p62 dok bind through their PTB domains to the Abelson tyrosine kinase in a kinase-dependent manner in both yeast and mammalian cells. DOKL is phosphorylated by the Abl tyrosine kinase in vivo. In contrast to p62 dok , DOKL lacks YxxP motifs in the C terminus and does not bind to Ras GTPase-activating protein (RasGAP) upon phosphorylation. Overexpression of DOKL, but not p62 dok , suppresses v-Abl-induced mitogen-activated protein (MAP) kinase activation but has no effect on constitutively activated Ras- and epidermal growth factor-induced MAP kinase activation. The inhibitory effect requires the PTB domain of DOKL. Finally, overexpression of DOKL in NIH 3T3 cells inhibits the transforming activity of v-Abl. These results suggest that DOKL may modulate Abl function.

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Hakai is required for stabilization of core components of the m6A mRNA methylation machinery

Nature Communications ◽

10.1038/s41467-021-23892-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Praveen Bawankar ◽

Tina Lence ◽

Chiara Paolantoni ◽

Irmgard U. Haussmann ◽

Migle Kazlauskiene ◽

...

Keyword(s):

Catalytic Activity ◽

Sex Determination ◽

Ubiquitin Ligase ◽

Mammalian Cells ◽

E3 Ubiquitin Ligase ◽

Human Cells ◽

Biological Functions ◽

Mrna Metabolism ◽

Mrna Methylation ◽

Core Components

AbstractN6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.

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A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽

10.3390/cancers13133317 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3317

Author(s):

Eric Moeglin ◽

Dominique Desplancq ◽

Audrey Stoessel ◽

Christian Massute ◽

Jeremy Ranniger ◽

...

Keyword(s):

Cancer Cells ◽

Mammalian Cells ◽

Chromatin Modification ◽

Replication Stress ◽

Living Cells ◽

Single Step ◽

Dna Double Strand Breaks ◽

Drug Induced ◽

Histone H2ax ◽

C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

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UBE4B, a microRNA-9 target gene, promotes autophagy-mediated Tau degradation

Nature Communications ◽

10.1038/s41467-021-23597-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Manivannan Subramanian ◽

Seung Jae Hyeon ◽

Tanuza Das ◽

Yoon Seok Suh ◽

Yun Kyung Kim ◽

...

Keyword(s):

Mouse Model ◽

Ubiquitin Ligase ◽

Therapeutic Approach ◽

Target Gene ◽

Neuroblastoma Cells ◽

E3 Ligase ◽

Tau Hyperphosphorylation ◽

Phosphorylated Tau ◽

Major Pathway ◽

The Brain

AbstractThe formation of hyperphosphorylated intracellular Tau tangles in the brain is a hallmark of Alzheimer’s disease (AD). Tau hyperphosphorylation destabilizes microtubules, promoting neurodegeneration in AD patients. To identify suppressors of tau-mediated AD, we perform a screen using a microRNA (miR) library in Drosophila and identify the miR-9 family as suppressors of human tau overexpression phenotypes. CG11070, a miR-9a target gene, and its mammalian orthologue UBE4B, an E3/E4 ubiquitin ligase, alleviate eye neurodegeneration, synaptic bouton defects, and crawling phenotypes in Drosophila human tau overexpression models. Total and phosphorylated Tau levels also decrease upon CG11070 or UBE4B overexpression. In mammalian neuroblastoma cells, overexpression of UBE4B and STUB1, which encodes the E3 ligase CHIP, increases the ubiquitination and degradation of Tau. In the Tau-BiFC mouse model, UBE4B and STUB1 overexpression also increase oligomeric Tau degradation. Inhibitor assays of the autophagy and proteasome systems reveal that the autophagy-lysosome system is the major pathway for Tau degradation in this context. These results demonstrate that UBE4B, a miR-9 target gene, promotes autophagy-mediated Tau degradation together with STUB1, and is thus an innovative therapeutic approach for AD.

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Autophagy induction in atrophic muscle cells requires ULK1 activation by TRIM32 through unanchored K63-linked polyubiquitin chains

Science Advances ◽

10.1126/sciadv.aau8857 ◽

2019 ◽

Vol 5 (5) ◽

pp. eaau8857 ◽

Cited By ~ 30

Author(s):

M. Di Rienzo ◽

M. Antonioli ◽

C. Fusco ◽

Y. Liu ◽

M. Mari ◽

...

Keyword(s):

Mouse Models ◽

Muscle Atrophy ◽

Ubiquitin Ligase ◽

Muscle Dystrophy ◽

Polyubiquitin Chains ◽

Autophagy Induction ◽

Atrophic Muscle ◽

Autophagic Activity

Optimal autophagic activity is crucial to maintain muscle integrity, with either reduced or excessive levels leading to specific myopathies. LGMD2H is a muscle dystrophy caused by mutations in the ubiquitin ligase TRIM32, whose function in muscles remains not fully understood. Here, we show that TRIM32 is required for the induction of muscle autophagy in atrophic conditions using both in vitro and in vivo mouse models. Trim32 inhibition results in a defective autophagy response to muscle atrophy, associated with increased ROS and MuRF1 levels. The proautophagic function of TRIM32 relies on its ability to bind the autophagy proteins AMBRA1 and ULK1 and stimulate ULK1 activity via unanchored K63-linked polyubiquitin. LGMD2H-causative mutations impair TRIM32’s ability to bind ULK1 and induce autophagy. Collectively, our study revealed a role for TRIM32 in the regulation of muscle autophagy in response to atrophic stimuli, uncovering a previously unidentified mechanism by which ubiquitin ligases activate autophagy regulators.

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