Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology

2008 ◽  
Vol 409 (3) ◽  
pp. 723-729 ◽  
Author(s):  
Mark Windheim ◽  
Mark Peggie ◽  
Philip Cohen
Keyword(s):  
E3 Ligase ◽  
Lysine Residue ◽  
Chain Elongation ◽  
Tumour Necrosis Factor Receptor ◽  
Polyubiquitin Chain ◽  
Polyubiquitin Chains ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

RING (really interesting new gene) and U-box E3 ligases bridge E2 ubiquitin-conjugating enzymes and substrates to enable the transfer of ubiquitin to a lysine residue on the substrate or to one of the seven lysine residues of ubiquitin for polyubiquitin chain elongation. Different polyubiquitin chains have different functions. Lys48-linked chains target proteins for proteasomal degradation, and Lys63-linked chains function in signal transduction, endocytosis and DNA repair. For this reason, chain topology must be tightly controlled. Using the U-box E3 ligase CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and the RING E3 ligase TRAF6 (tumour-necrosis-factor-receptor-associated factor 6) with the E2s Ubc13 (ubiquitin-conjugating enzyme 13)–Uev1a (ubiquitin E2 variant 1a) and UbcH5a, in the present study we demonstrate that Ubc13–Uev1a supports the formation of free Lys63-linked polyubiquitin chains not attached to CHIP or TRAF6, whereas UbcH5a catalyses the formation of polyubiquitin chains linked to CHIP and TRAF6 that lack specificity for any lysine residue of ubiquitin. Therefore the abilities of these E2s to ubiquitinate a substrate and to elongate polyubiquitin chains of a specific topology appear to be mutually exclusive. Thus two different classes of E2 may be required to attach a polyubiquitin chain of a particular topology to a substrate: the properties of one E2 are designed to mono-ubiquitinate a substrate with no or little inherent specificity for an acceptor lysine residue, whereas the properties of the second E2 are tailored to the elongation of a polyubiquitin chain using a defined lysine residue of ubiquitin.

scholarly journals Mammalian DET1 Regulates Cul4A Activity and Forms Stable Complexes with E2 Ubiquitin-Conjugating Enzymes

2007 ◽  
Vol 27 (13) ◽  
pp. 4708-4719 ◽  
Author(s):  
Elah Pick ◽  
On-Sun Lau ◽  
Tomohiko Tsuge ◽  
Suchithra Menon ◽  
Yingchun Tong ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Associated Factors ◽  
Cultured Cells ◽  
Negative Regulator ◽  
Polyubiquitin Chain ◽  
Light Responses ◽  
E3 Ligases ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

ABSTRACT DET1 (de-etiolated 1) is an essential negative regulator of plant light responses, and it is a component of the Arabidopsis thaliana CDD complex containing DDB1 and COP10 ubiquitin E2 variant. Human DET1 has recently been isolated as one of the DDB1- and Cul4A-associated factors, along with an array of WD40-containing substrate receptors of the Cul4A-DDB1 ubiquitin ligase. However, DET1 differs from conventional substrate receptors of cullin E3 ligases in both biochemical behavior and activity. Here we report that mammalian DET1 forms stable DDD-E2 complexes, consisting of DDB1, DDA1 (DET1, DDB1 associated 1), and a member of the UBE2E group of canonical ubiquitin-conjugating enzymes. DDD-E2 complexes interact with multiple ubiquitin E3 ligases. We show that the E2 component cannot maintain the ubiquitin thioester linkage once bound to the DDD core, rendering mammalian DDD-E2 equivalent to the Arabidopsis CDD complex. While free UBE2E-3 is active and able to enhance UbcH5/Cul4A activity, the DDD core specifically inhibits Cul4A-dependent polyubiquitin chain assembly in vitro. Overexpression of DET1 inhibits UV-induced CDT1 degradation in cultured cells. These findings demonstrate that the conserved DET1 complex modulates Cul4A functions by a novel mechanism.

scholarly journals A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

Science ◽  
2019 ◽  
Vol 365 (6448) ◽  
pp. eaaw4912 ◽  
Author(s):  
Richard T. Timms ◽  
Zhiqian Zhang ◽  
David Y. Rhee ◽  
J. Wade Harper ◽  
Itay Koren ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Stability ◽  
Protein Degradation ◽  
E3 Ligase ◽  
Cleavage Sites ◽  
Caspase Cleavage ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
The Stability ◽  
Ring E3

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

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.

scholarly journals Crosstalk and ultrasensitivity in protein degradation pathways

2020 ◽  
Vol 16 (12) ◽  
pp. e1008492
Author(s):  
Abhishek Mallela ◽  
Maulik K. Nariya ◽  
Eric J. Deeds
Keyword(s):  
Protein Turnover ◽  
E3 Ligase ◽  
Biochemical Networks ◽  
Protein Homeostasis ◽  
Polyubiquitin Chain ◽  
Post Translational Modification ◽  
E3 Ligases ◽  
Protein Levels ◽  
Synthesis And Degradation

Protein turnover is vital to cellular homeostasis. Many proteins are degraded efficiently only after they have been post-translationally “tagged” with a polyubiquitin chain. Ubiquitylation is a form of Post-Translational Modification (PTM): addition of a ubiquitin to the chain is catalyzed by E3 ligases, and removal of ubiquitin is catalyzed by a De-UBiquitylating enzyme (DUB). Nearly four decades ago, Goldbeter and Koshland discovered that reversible PTM cycles function like on-off switches when the substrates are at saturating concentrations. Although this finding has had profound implications for the understanding of switch-like behavior in biochemical networks, the general behavior of PTM cycles subject to synthesis and degradation has not been studied. Using a mathematical modeling approach, we found that simply introducing protein turnover to a standard modification cycle has profound effects, including significantly reducing the switch-like nature of the response. Our findings suggest that many classic results on PTM cycles may not hold in vivo where protein turnover is ubiquitous. We also found that proteins sharing an E3 ligase can have closely related changes in their expression levels. These results imply that it may be difficult to interpret experimental results obtained from either overexpressing or knocking down protein levels, since changes in protein expression can be coupled via E3 ligase crosstalk. Understanding crosstalk and competition for E3 ligases will be key in ultimately developing a global picture of protein homeostasis.

scholarly journals MPSR1 is a cytoplasmic PQC E3 ligase for eliminating emergent misfolded proteins in Arabidopsis thaliana

2017 ◽  
Vol 114 (46) ◽  
pp. E10009-E10017 ◽  
Author(s):  
Jong Hum Kim ◽  
Seok Keun Cho ◽  
Tae Rin Oh ◽  
Moon Young Ryu ◽  
Seong Wook Yang ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Protein Quality ◽  
E3 Ligase ◽  
26S Proteasome ◽  
Misfolded Proteins ◽  
E3 Ligases ◽  
Proteotoxic Stress ◽  
Ring E3 Ligase ◽  
Initial Stage ◽  
Subsequent Degradation

Ubiquitin E3 ligases are crucial for eliminating misfolded proteins before they form cytotoxic aggregates that threaten cell fitness and survival. However, it remains unclear how emerging misfolded proteins in the cytoplasm can be selectively recognized and eliminated by E3 ligases in plants. We found that Misfolded Protein Sensing RING E3 ligase 1 (MPSR1) is an indispensable E3 ligase required for plant survival after protein-damaging stress. Under no stress, MPSR1 is prone to rapid degradation by the 26S proteasome, concealing its protein quality control (PQC) E3 ligase activity. Upon proteotoxic stress, MPSR1 directly senses incipient misfolded proteins and tethers ubiquitins for subsequent degradation. Furthermore, MPSR1 sustains the structural integrity of the proteasome complex at the initial stage of proteotoxic stress. Here, we suggest that the MPSR1 pathway is a constitutive mechanism for proteostasis under protein-damaging stress, as a front-line surveillance system in the cytoplasm.

scholarly journals Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~Ub conformation

2019 ◽  
Author(s):  
Emma Branigan ◽  
J. Carlos Penedo ◽  
Ronald T. Hay
Keyword(s):  
Single Molecule ◽  
Active Form ◽  
Resonance Energy Transfer ◽  
E3 Ligase ◽  
Resonance Energy ◽  
Post Translational Modification ◽  
E3 Ligases ◽  
Closed Conformation ◽  
Ring E3 Ligase ◽  
Ring E3

AbstractUbiquitination is a eukaryotic post-translational modification that modulates a host of cellular processes1. Modification is mediated by an E1 activating enzyme (E1), an E2 conjugating enzyme (E2) and an E3 ligase (E3). The E1 catalyses formation of a highly reactive thioester linked conjugate between ubiquitin and E2 (E2~Ub)2. The largest class of ubiquitin E3 ligases, which is represented by RING E3s, bind both substrate and E2~Ub and facilitate transfer of ubiquitin from the E2 to substrate. Based on extensive structural analysis3–5 it has been proposed that RING E3s prime the E2~Ub conjugate for catalysis by locking it into a “closed” conformation where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by a substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer is a model that has yet to be experimentally tested. Here we use single molecule Förster Resonance Energy Transfer (smFRET) to test this hypothesis and demonstrate that ubiquitin is transferred from the closed conformation during an E3 catalysed reaction. Using Ubc13 as an E2, we designed a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. Firstly, we defined the high FRET state as the closed conformation using a stable isopeptide linked E2~Ub conjugate, while the low FRET state represents more open conformations. Secondly, we developed a real-time smFRET assay to monitor RING E3 catalysed ubiquitination with a thioester linked E2~Ub conjugate and determined the catalytically active conformation. Our results demonstrate that the reaction proceeds from the high FRET or closed conformation and confirm the hypothesis that the closed conformation is the active form of the conjugate. These findings are not only relevant to RING E3 catalysed ubiquitination but are also broadly applicable to E3 mediated ligation of other ubiquitin-like proteins (Ubls) to substrates.

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 Crosstalk and Ultrasensitivity in Protein Degradation Pathways

2019 ◽  
Author(s):  
Abhishek Mallela ◽  
Maulik K. Nariya ◽  
Eric J. Deeds
Keyword(s):  
Protein Turnover ◽  
E3 Ligase ◽  
Biochemical Networks ◽  
Protein Homeostasis ◽  
Polyubiquitin Chain ◽  
Post Translational Modification ◽  
E3 Ligases ◽  
Protein Levels ◽  
Synthesis And Degradation

AbstractProtein turnover is vital to protein homeostasis within the cell. Many proteins are degraded efficiently only after they have been post-translationally “tagged” with a polyubiquitin chain. Ubiquitylation is a form of Post-Translational Modification (PTM): addition of a ubiquitin to the chain is catalyzed by E3 ligases, and removal of ubiquitin is catalyzed by a De-UBiquitylating enzyme (DUB). Over three decades ago, Goldbeter and Koshland discovered that reversible PTM cycles function like on-off switches when the substrates are at saturating concentrations. Although this finding has had profound implications for the understanding of switch-like behavior in biochemical networks, the general behavior of PTM cycles subject to synthesis and degradation has not been studied. Using a mathematical modeling approach, we found that simply introducing protein turnover to a standard modification cycle has profound effects, including significantly reducing the switch-like nature of the response. Our findings suggest that many classic results on PTM cycles may not hold in vivo where protein turnover is ubiquitous. We also found that proteins sharing an E3 ligase can have closely related changes in their expression levels. These results imply that it may be difficult to interpret experimental results obtained from either overexpressing or knocking down protein levels, since changes in protein expression can be coupled via E3 ligase crosstalk. Understanding crosstalk and competition for E3 ligases will be key to ultimately developing a global picture of protein homeostasis.

Orchestra for assembly and fate of polyubiquitin chains

2005 ◽  
Vol 41 ◽  
pp. 1-14 ◽  
Author(s):  
Kuhlbrodt Kirsten ◽  
Mouysset Julien ◽  
Hoppe Thorsten
Keyword(s):  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Chain Elongation ◽  
Polyubiquitin Chain ◽  
Ubiquitin Chain ◽  
Polyubiquitin Chains ◽  
Intracellular Signals ◽  
Ubiquitin Binding ◽  
Ubiquitin Ligase E3 ◽  
Ubiquitin Conjugating Enzyme

Selective protein degradation by the 26 S proteasome usually requires a polyubiquitin chain attached to the protein substrate by three classes of enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). This reaction can produce different polyubiquitin chains that, depending on size and linkage type, can provide distinct intracellular signals. Interestingly, polyubiquitination is sometimes regulated by additional conjugation factors, called E4s (polyubiquitin chain conjugation factors). Yeast UFD2 (ubiquitin fusion degradation protein-2), the first E4 to be described, binds to the ubiquitin moieties of preformed conjugates and catalyses ubiquitin-chain elongation together with E1, E2, and E3. Recent studies have illustrated that the E4 enzyme UFD2 co-operates with an orchestra of ubiquitin-binding factors in an escort pathway to transfer and deliver polyubiquitinated substrates to the 26 S proteasome. Here we propose a model in which E4-dependent polyubiquitination pathways are modulated by different ubiquitin-binding proteins, using ataxin-3 as an example.

scholarly journals RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination

2014 ◽  
Vol 1843 (1) ◽  
pp. 47-60 ◽  
Author(s):  
Meredith B. Metzger ◽  
Jonathan N. Pruneda ◽  
Rachel E. Klevit ◽  
Allan M. Weissman
Keyword(s):  
E3 Ligases ◽  
Ring Type ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes
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