The Cul3/Klhdc5 E3 Ligase Regulates p60/Katanin and Is Required for Normal Mitosis 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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Constitutive Turnover of Cyclin E by Cul3 Maintains Quiescence

Molecular and Cellular Biology ◽

10.1128/mcb.00720-06 ◽

2007 ◽

Vol 27 (10) ◽

pp. 3651-3666 ◽

Cited By ~ 36

Author(s):

Justina D. McEvoy ◽

Uta Kossatz ◽

Nisar Malek ◽

Jeffrey D. Singer

Keyword(s):

Mammalian Cells ◽

Gene Deletion ◽

Cyclin E ◽

E3 Ligase ◽

Degradation Pathway ◽

Conditional Knockout ◽

Protein Levels ◽

Primary Fibroblasts

ABSTRACT Two distinct pathways for the degradation of mammalian cyclin E have previously been described. One pathway is induced by cyclin E phosphorylation and is dependent on the Cul1/Fbw7-based E3 ligase. The other pathway is dependent on the Cul3-based E3 ligase, but the mechanistic details of this pathway have yet to be elucidated. To establish the role of Cul3 in the degradation of cyclin E in vivo, we created a conditional knockout of the Cul3 gene in mice. Interestingly, the biallelic loss of Cul3 in primary fibroblasts derived from these mice results in increased cyclin E expression and reduced cell viability, paralleling the loss of Cul3 protein expression. Cell cycle analysis of viable, Cul3 hypomorphic cells shows that decreasing the levels of Cul3 increases both cyclin E protein levels and the number of cells in S phase. In order to examine the role of Cul3 in an in vivo setting, we determined the effect of deletion of the Cul3 gene in liver. This gene deletion resulted in a dramatic increase in cyclin E levels as well as an increase in cell size and ploidy. The results we report here show that the constitutive degradation pathway for cyclin E that is regulated by the Cul3-based E3 ligase is essential to maintain quiescence in mammalian cells.

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

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736006045.793587698 ◽

2021 ◽

Author(s):

Margot Thome

Keyword(s):

Mammalian Cells ◽

Bond Formation ◽

E3 Ligase ◽

Ester Bond

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

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736006045.793561994 ◽

2019 ◽

Author(s):

John Silke

Keyword(s):

Mammalian Cells ◽

Bond Formation ◽

E3 Ligase ◽

Ester Bond

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Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen

Scientific Reports ◽

10.1038/s41598-020-76960-z ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Lina Schukur ◽

Tamara Zimmermann ◽

Ole Niewoehner ◽

Grainne Kerr ◽

Scott Gleim ◽

...

Keyword(s):

Mammalian Cells ◽

Fluorescent Protein ◽

E3 Ligase ◽

Cellular Transformation ◽

Cellular Growth ◽

Intrinsically Disordered ◽

Transcriptional Output ◽

Generation Sequencing ◽

Copy Number Amplification ◽

In Cells

AbstractMYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.

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Pc2 and SUMOylation

Biochemical Society Transactions ◽

10.1042/bst0351401 ◽

2007 ◽

Vol 35 (6) ◽

pp. 1401-1404 ◽

Cited By ~ 41

Author(s):

D. Wotton ◽

J.C. Merrill

Keyword(s):

Recent Work ◽

Histone Deacetylase ◽

Chromatin Structure ◽

Mammalian Cells ◽

Protein Complexes ◽

E3 Ligase ◽

Histone Methyltransferase ◽

Polycomb Proteins ◽

Polycomb Protein ◽

Shed Light

Polycomb proteins are key regulators of transcription in metazoan organisms. Recent work has shed light on the nature of the polycomb protein complexes in flies and mammalian cells. Multiple enzymatic activities have been shown to associate with polycomb complexes, including histone methyltransferase, histone deacetylase and ubiquitination activities, which are primarily directed towards the modification of chromatin structure. In addition to these chromatin-based functions, other potential roles for polycomb proteins exist. Here, we present a comparison of vertebrate Pc2 (polycomb 2 protein) homologues, and review the known functions of the mammalian Pc2 focusing on its role as a SUMO (small ubiquitin-related modifier) E3 ligase. Pc2 is an E3 for several SUMO substrates, but still appears to have a more limited repertoire than other SUMO E3s, perhaps due to its association with polycomb complexes. One possibility is that Pc2 represents a relatively specialized polycomb protein, which has additional functions to those associated with other mammalian Pc (polycomb protein) paralogues.

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Decoys reveal the genetic and biochemical roles of redundant plant E3 ubiquitin ligases

10.1101/115071 ◽

2017 ◽

Cited By ~ 1

Author(s):

Chin-Mei Lee ◽

Ann Feke ◽

Christopher Adamchek ◽

Kristofor Webb ◽

José Pruneda-Paz ◽

...

Keyword(s):

Circadian Clock ◽

Mammalian Cells ◽

E3 Ligase ◽

Ubiquitin Ligases ◽

E3 Ubiquitin Ligases ◽

Genetic Redundancy ◽

Clock Period ◽

E3 Ligases ◽

Wide Range

AbstractThe ubiquitin proteasome system (UPS) is the main cellular route for protein degradation in plants and is important for a wide range of biological processes including daily and seasonal timing. The UPS relies on the action of E3 ubiquitin ligases to specifically recognize substrate proteins and facilitate their ubiquitylation. In plants, there are three major challenges that inhibit studies of E3 ligase function: 1) rampant genetic redundancy, 2) labile interactions between an E3 ligase and its cognate substrates, and 3) a lack of tools for rapid validation of bona fide substrates. To overcome these 3 challenges, we have developed a decoy method that allows for rapid genetic analysis of E3 ligases, in vivo identification of substrates using immunoprecipitation followed by mass spectrometry, and reconstitution of the ubiquitylation reaction in mammalian cells to rapidly validate potential substrates. We employ the strategy to study the plant F-box proteins, ZTL, LKP2, and FKF1 revealing differential genetic impacts on circadian clock period and seasonal flowering. We identify a group of circadian clock transcriptional regulators that interact with ZTL, LKP2, and FKF1 in vivo providing a host of potential substrates that have not been seen previously. We then validate one substrate of ZTL, the plant circadian clock transcription factor CHE, and show that ZTL mediates CHE ubiquitylation and that the levels of the CHE protein cycle in daily timecourses. This work further untangles the complicated genetic roles of this family of E3 ligases and suggests that ZTL is a master regulator of a diverse set of critical clock transcription factors. Furthermore, the method that is validated here can be tool employed widely to overcome traditional challenges in studying redundant plant E3 ubiquitin ligases.

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RNA binding regulates TRIM25-mediated RIG-I ubiquitylation

10.1101/2020.05.04.070177 ◽

2020 ◽

Author(s):

Kevin Haubrich ◽

Sandra Augsten ◽

Bernd Simon ◽

Pawel Masiewicz ◽

Kathryn Perez ◽

...

Keyword(s):

Binding Site ◽

Cell Fate ◽

Mammalian Cells ◽

Rna Binding ◽

Mutational Analysis ◽

Coiled Coil ◽

E3 Ligase ◽

Cell Fate Decisions ◽

Ubiquitin E3 Ligase

ABSTRACTTRIM25 is a ubiquitin E3 ligase active in innate immunity and cell fate decisions. Mounting evidence suggests that TRIM25’s E3 ligase activity is regulated by RNAs. However, while mutations affecting RNA-binding have been described, the precise RNA binding site has not been identified nor which domains are involved. Here, we present biophysical evidence for the presence of RNA binding sites on both TRIM25 PRY/SPRY and coiled-coil domains, and map the binding site on the PRY/SPRY with residue resolution. Cooperative RNA-binding of both domains enhances their otherwise transient interaction in solution and increases the E3 ligase activity of TRIM25. Mutational analysis shows that RNA binding affects ubiquitination of RIG-I in mammalian cells. In addition, we present a simple model system for RNA-induced liquid-liquid phase separation of TRIM25 in vitro, resembling previously observed cellular RNA granules, that facilitates the recruitment of RIG-I.

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Regulation of p27 Degradation and S-Phase Progression by Ro52 RING Finger Protein

Molecular and Cellular Biology ◽

10.1128/mcb.01630-05 ◽

2006 ◽

Vol 26 (16) ◽

pp. 5994-6004 ◽

Cited By ~ 50

Author(s):

Abdelmajid Sabile ◽

Andrea Michael Meyer ◽

Christiane Wirbelauer ◽

Daniel Hess ◽

Ulrike Kogel ◽

...

Keyword(s):

Mammalian Cells ◽

Kinase Inhibitor ◽

Ring Finger ◽

E3 Ligase ◽

S Phase ◽

Dependent Manner ◽

Ring Finger Protein ◽

Catalytic Core ◽

Finger Protein ◽

Phase Progression

ABSTRACT Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27 provides a powerful route for enforcing normal progression through the mammalian cell cycle. According to a current model, the ubiquitination of p27 during S-phase progression is mediated by SCFSkp2 E3 ligase that captures Thr187-phosphorylated p27 by means of the F-box protein Skp2, which in turn couples the bound substrate via Skp1 to a catalytic core complex composed of Cul1 and the Rbx/Roc RING finger protein. Here we identify Skp2 as a component of an Skp1-cullin-F-box complex that is based on a Cul1-Ro52 RING finger B-box coiled-coil motif family protein catalytic core. Ro52-containing complexes display E3 ligase activity and promote the ubiquitination of Thr187-phosphorylated p27 in a RING-dependent manner in vitro. The knockdown of Ro52 expression in human cells with small interfering RNAs causes the accumulation of p27 and the failure of cells to enter S phase. Importantly, these effects are abrogated by the simultaneous removal of p27. Taken together, these data suggest a key role for Ro52 RING finger protein in the regulation of p27 degradation and S-phase progression in mammalian cells and provide evidence for the existence of a Cul1-based catalytic core that utilizes Ro52 RING protein to promote ubiquitination.

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