scholarly journals Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes

2021 ◽  
Author(s):  
Thanh Thi Le ◽  
Johanna Ainsworth ◽  
Cristian Polo Rivera ◽  
Thomas Macartney ◽  
Karim Labib
Keyword(s):  
Dna Replication ◽  
Ubiquitin Ligases ◽  
Human Cells ◽  
Ubiquitin Chain ◽  
Human Proteins ◽  
Mouse Cells ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes

Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination.  In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase.  Here we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase.  Using purified human proteins, including a panel of E2 ubiquitin conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1.  The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1.  CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes.  Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7.  Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.

scholarly journals OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

2018 ◽  
Vol 293 (47) ◽  
pp. 18285-18295 ◽  
Author(s):  
Nagesh Pasupala ◽  
Marie E. Morrow ◽  
Lauren T. Que ◽  
Barbara A. Malynn ◽  
Averil Ma ◽  
...  
Keyword(s):  
Polyubiquitin Chains ◽  
Protein Ubiquitination ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
In Cells ◽  
Conjugating Enzyme

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

Hug and hold tight: Dimerization controls the turnover of the ubiquitin-conjugating enzyme UBE2S

2020 ◽  
Vol 13 (654) ◽  
pp. eabd9892
Author(s):  
Anja Bremm
Keyword(s):  
Half Life ◽  
Anaphase Promoting Complex ◽  
Ubiquitin Chain ◽  
Precise Control ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
Chain Synthesis ◽  
Ubiquitin Conjugating Enzymes ◽  
Conjugating Enzyme

Precise control of the activity and abundance of ubiquitin-conjugating enzymes (E2s) ensures fidelity in ubiquitin chain synthesis. In this issue of Science Signaling, Liess et al. demonstrate that the human anaphase-promoting complex (APC/C)–associated E2 UBE2S adopts an autoinhibited dimeric state that increases the half-life of UBE2S by preventing its autoubiquitination-driven turnover.

scholarly journals E3 ubiquitin ligase components in GtoPdb v.2021.3

2021 ◽  
Vol 2021 (3) ◽  
Author(s):  
Elena Faccenda ◽  
Robert Layfield
Keyword(s):  
Cell Cycle Progression ◽  
Protein Complexes ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Post Translational Modification ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Ubiquitin Molecule ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes

Ubiquitination (a.k.a. ubiquitylation) is a protein post-translational modification that typically requires the sequential action of three enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases) [19]. Ubiquitination of proteins can target them for proteasomal degradation, or modulate cellular processes including cell cycle progression, transcriptional regulation, DNA repair and signal transduction. E3 ubiquitin ligases, of which there are >600 in humans, are a family of highly heterogeneous proteins and protein complexes that recruit ubiquitin-loaded E2 enzymes to mediate transfer of the ubiquitin molecule from the E2 to protein substrates. Target substrate specificity is determined by a substrate recognition subunit within the E3 complex.

scholarly journals Pex2 and Pex12 Function as Protein-Ubiquitin Ligases in Peroxisomal Protein Import

2009 ◽  
Vol 29 (20) ◽  
pp. 5505-5516 ◽  
Author(s):  
Harald W. Platta ◽  
Fouzi El Magraoui ◽  
Bastian E. Bäumer ◽  
Daniel Schlee ◽  
Wolfgang Girzalsky ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Ubiquitin Ligases ◽  
Receptor Protein ◽  
Receptor Complex ◽  
Peroxisomal Protein ◽  
Cargo Receptor ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
Import Receptor

ABSTRACT The PTS1-dependent peroxisomal matrix protein import is facilitated by the receptor protein Pex5 and can be divided into cargo recognition in the cytosol, membrane docking of the cargo-receptor complex, cargo release, and recycling of the receptor. The final step is controlled by the ubiquitination status of Pex5. While polyubiquitinated Pex5 is degraded by the proteasome, monoubiquitinated Pex5 is destined for a new round of the receptor cycle. Recently, the ubiquitin-conjugating enzymes involved in Pex5 ubiquitination were identified as Ubc4 and Pex4 (Ubc10), whereas the identity of the corresponding protein-ubiquitin ligases remained unknown. Here we report on the identification of the protein-ubiquitin ligases that are responsible for the ubiquitination of the peroxisomal protein import receptor Pex5. It is demonstrated that each of the three RING peroxins Pex2, Pex10, and Pex12 exhibits ubiquitin-protein isopeptide ligase activity. Our results show that Pex2 mediates the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.

scholarly journals Characterization of OTUB1 activation and inhibition by different E2 enzymes

2019 ◽  
Author(s):  
Lauren T. Que ◽  
Marie E. Morrow ◽  
Cynthia Wolberger
Keyword(s):  
Deubiquitinating Enzyme ◽  
Polyubiquitin Chains ◽  
Kinetics And Thermodynamics ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
Stimulation Of

AbstractOTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes, forming complexes in which the two enzymes can regulate one another’s activity. OTUB1 can non-catalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2~Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. In order to assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the autoubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3.

scholarly journals Ubiquitin-Conjugating Enzymes in Cancer

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1383
Author(s):  
Quyen Thu Bui ◽  
Jeong Hee Hong ◽  
Minseok Kwak ◽  
Ji Yeon Lee ◽  
Peter Chang-Whan Lee
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Therapeutic Strategies ◽  
Migration And Invasion ◽  
Cycle Arrest ◽  
Enzyme Cascade ◽  
Ubiquitination System ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes

The ubiquitin-mediated degradation system is responsible for controlling various tumor-promoting processes, including DNA repair, cell cycle arrest, cell proliferation, apoptosis, angiogenesis, migration and invasion, metastasis, and drug resistance. The conjugation of ubiquitin to a target protein is mediated sequentially by the E1 (activating)‒E2 (conjugating)‒E3 (ligating) enzyme cascade. Thus, E2 enzymes act as the central players in the ubiquitination system, modulating various pathophysiological processes in the tumor microenvironment. In this review, we summarize the types and functions of E2s in various types of cancer and discuss the possibility of E2s as targets of anticancer therapeutic strategies.

scholarly journals Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

2021 ◽  
Author(s):  
Aniruddha Das ◽  
Pankaj Thapa ◽  
Ulises Santiago ◽  
Nilesh Shanmugam ◽  
Katarzyna Banasiak ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Caenorhabditis Elegans ◽  
Molecular Mechanism ◽  
E3 Ligase ◽  
Ubiquitin Ligases ◽  
Ubiquitin Chain ◽  
Chain Formation ◽  
Assembly Mechanism ◽  
E2 Enzymes ◽  
And Function

The E3 ubiquitin ligases CHIP/CHN-1 and UFD-2 team up to accelerate ubiquitin chain formation. However, it remained largely unclear how the high processivity of this E3 set is achieved. Here we studied the molecular mechanism and function of the CHN-1/UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. The HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding and promotes the auto-inhibited CHN-1 state by acting on the conserved position of the U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitinate S-Adenosylhomocysteinase (AHCY-1), an enzyme crucial for lipid metabolism. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

scholarly journals E3 ubiquitin ligase components (version 2019.5) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (5) ◽  
Author(s):  
Elena Faccenda ◽  
Robert Layfield
Keyword(s):  
Cell Cycle Progression ◽  
Protein Complexes ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Post Translational Modification ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Ubiquitin Molecule ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes

Ubiquitination (a.k.a. ubiquitylation) is a protein post-translational modification that typically requires the sequential action of three enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases) [16]. Ubiquitination of proteins can target them for proteasomal degradation, or modulate cellular processes including cell cycle progression, transcriptional regulation, DNA repair and signal transduction. E3 ubiquitin ligases, of which there are >600 in humans, are a family of highly heterogeneous proteins and protein complexes that recruit ubiquitin-loaded E2 enzymes to mediate transfer of the ubiquitin molecule from the E2 to protein substrates. Target substrate specificity is determined by a substrate recognition subunit within the E3 complex.

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