A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and is essential for ER-associated degradation

Endoplasmic reticulum–associated degradation (ERAD) is a protein quality control pathway of fundamental importance to cellular homeostasis. Although multiple ERAD pathways exist for targeting topologically distinct substrates, all pathways require substrate ubiquitination. Here, we characterize a key role for the UBE2G2 Binding Region (G2BR) of the ERAD accessory protein ancient ubiquitous protein 1 (AUP1) in ERAD pathways. This 27-amino acid (aa) region of AUP1 binds with high specificity and low nanomolar affinity to the backside of the ERAD ubiquitin-conjugating enzyme (E2) UBE2G2. The structure of the AUP1 G2BR (G2BRAUP1) in complex with UBE2G2 reveals an interface that includes a network of salt bridges, hydrogen bonds, and hydrophobic interactions essential for AUP1 function in cells. The G2BRAUP1 shares significant structural conservation with the G2BR found in the E3 ubiquitin ligase gp78 and in vitro can similarly allosterically activate ubiquitination in conjunction with ERAD E3s. In cells, AUP1 is uniquely required to maintain normal levels of UBE2G2; this is due to G2BRAUP1 binding to the E2 and preventing its rapid degradation. In addition, the G2BRAUP1 is required for both ER membrane recruitment of UBE2G2 and for its activation at the ER membrane. Thus, by binding to the backside of a critical ERAD E2, G2BRAUP1 plays multiple critical roles in ERAD.

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Abstract 18: Synoviolin, an E3 Ubiquitin Ligase, Modulates Cardiac Myocyte Size and Restores Heart Function in Hypertrophic Cardiomyopathy

Circulation Research ◽

10.1161/res.111.suppl_1.a18 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Shirin Doroudgar ◽

Mirko Völkers ◽

Donna J Thuerauf ◽

Ashley Bumbar ◽

Mohsin Khan ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Protein Misfolding ◽

Cardiac Myocyte ◽

Protein Quality ◽

Heart Function ◽

E3 Ubiquitin Ligase ◽

Ubiquitin Ligases ◽

Calcium Handling ◽

Loss Of Function

The endoplasmic reticulum (ER) is essential for protein homeostasis, or proteostasis, which governs the balance of the proteome. In addition to secreted and membrane proteins, proteins bound for many other cellular locations are also made on ER-bound ribosomes, emphasizing the importance of protein quality and quantity control in the ER. Unlike cytosolic E3 ubiquitin ligases studied in the heart, synoviolin/Hrd1, which has not been studied in the heart, is an ER transmembrane E3 ubiquitin ligase, which we found to be upregulated upon protein misfolding in cardiac myocytes. Given the strategic location of synoviolin in the ER membrane, we addressed the hypothesis that synoviolin is critical for regulating the balance of the proteome, and accordingly, myocyte size. We showed that in vitro, adenovirus-mediated overexpression of synoviolin decreased cardiac myocyte size and protein synthesis, but unlike atrophy-related ubiquitin ligases, synoviolin did not increase global protein degradation. Furthermore, targeted gene therapy using adeno-associated virus 9 (AAV9) showed that overexpression of synoviolin in the left ventricle attenuated maladaptive cardiac hypertrophy and preserved cardiac function in mice subjected to trans-aortic constriction (AAV9-control TAC = 22.5 ± 6.2% decrease in EF vs. AAV9-synoviolin TAC at 6 weeks post TAC; P<0.001), and decreased mTOR activity. Since calcium is a major regulator of cardiac myocyte size, we examined the effects of synoviolin gain- or loss-of-function, using AAV9-synoviolin, or an miRNA designed to knock down synoviolin, respectively. While synoviolin gain-of-function did not affect calcium handling in isolated adult myocytes, synoviolin loss-of-function increased calcium transient amplitude (P<0.01), prolonged spark duration (P<0.001), and increased spark width (P<0.001). Spark frequency and amplitude were unaltered upon synoviolin gain- or loss-of-function. Whereas SR calcium load was unaltered by synoviolin loss-of-function, SERCA-mediated calcium removal was reduced (P<0.05). In conclusion, our studies suggest that in the heart, synoviolin is 1) a critical component of proteostasis, 2) a novel determinant of cardiac myocyte size, and 3) necessary for proper calcium handling.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004677 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18285-18295 ◽

Cited By ~ 11

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.

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In Vivo Action of the HRD Ubiquitin Ligase Complex: Mechanisms of Endoplasmic Reticulum Quality Control and Sterol Regulation

Molecular and Cellular Biology ◽

10.1128/mcb.21.13.4276-4291.2001 ◽

2001 ◽

Vol 21 (13) ◽

pp. 4276-4291 ◽

Cited By ~ 93

Author(s):

Richard G. Gardner ◽

Alexander G. Shearer ◽

Randolph Y. Hampton

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Ubiquitin Ligase ◽

Sequence Similarity ◽

High Specificity ◽

Specific Sequence ◽

Ubiquitin Ligase Complex ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzyme

ABSTRACT Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRDcomplex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent “misfolded” state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.

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The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.161283098 ◽

2001 ◽

Vol 98 (15) ◽

pp. 8815-8820 ◽

Cited By ~ 80

Author(s):

C. Van Sant ◽

R. Hagglund ◽

P. Lopez ◽

B. Roizman

Keyword(s):

Herpes Simplex ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Cell Protein ◽

Herpes Simplex Virus 1 ◽

Ubiquitin Ligase Activity ◽

Ubiquitin Conjugating Enzyme ◽

Infected Cell Protein ◽

Simplex Virus

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The APC11 RING-H2 Finger Mediates E2-Dependent Ubiquitination

Molecular Biology of the Cell ◽

10.1091/mbc.11.7.2315 ◽

2000 ◽

Vol 11 (7) ◽

pp. 2315-2325 ◽

Cited By ~ 114

Author(s):

Joel D. Leverson ◽

Claudio A.P. Joazeiro ◽

Andrew M. Page ◽

Han-kuei Huang ◽

Philip Hieter ◽

...

Keyword(s):

Ubiquitin Ligase ◽

26S Proteasome ◽

Anaphase Promoting Complex ◽

Mitotic Progression ◽

Protein Substrates ◽

Ubiquitin Ligase Activity ◽

Ubiquitin Conjugating Enzyme ◽

Ubiquitin Conjugating Enzymes

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that theSaccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

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Ubc4/5 and c-Cbl Continue to Ubiquitinate EGF Receptor after Internalization to Facilitate Polyubiquitination and Degradation

Molecular Biology of the Cell ◽

10.1091/mbc.e07-10-0988 ◽

2008 ◽

Vol 19 (8) ◽

pp. 3454-3462 ◽

Cited By ~ 69

Author(s):

Kyohei Umebayashi ◽

Harald Stenmark ◽

Tamotsu Yoshimori

Keyword(s):

Ubiquitin Ligase ◽

Time Course ◽

Kinase Activity ◽

Egf Receptor ◽

In Vitro Activity ◽

Lysosomal Sorting ◽

Time Course Experiment ◽

Ubiquitin Conjugating Enzyme ◽

Epidermal Growth

c-Cbl is the E3 ubiquitin ligase that ubiquitinates the epidermal growth factor (EGF) receptor (EGFR). On the basis of localization, knockdown, and in vitro activity analyses, we have identified the E2 ubiquitin-conjugating enzyme that cooperates with c-Cbl as Ubc4/5. Upon EGF stimulation, both Ubc4/5 and c-Cbl were relocated to the plasma membrane and then to Hrs-positive endosomes, strongly suggesting that EGFR continues to be ubiquitinated after internalization. Our time-course experiment showed that EGFR undergoes polyubiquitination, which seemed to be facilitated during the transport to Hrs-positive endosomes. Use of a conjugation-defective ubiquitin mutant suggested that receptor polyubiquitination is required for efficient interaction with Hrs and subsequent sorting to lysosomes. Abrupt inhibition of the EGFR kinase activity resulted in dissociation of c-Cbl from EGFR. Concomitantly, EGFR was rapidly deubiquitinated and its degradation was delayed. We propose that sustained tyrosine phosphorylation of EGFR facilitates its polyubiquitination in endosomes and counteracts rapid deubiquitination, thereby ensuring Hrs-dependent lysosomal sorting.

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TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum

Biochemical Journal ◽

10.1042/bj20041241 ◽

2005 ◽

Vol 388 (2) ◽

pp. 647-655 ◽

Cited By ~ 111

Author(s):

Gerco HASSINK ◽

Marjolein KIKKERT ◽

Sjaak van VOORDEN ◽

Shiow-Ju LEE ◽

Robbert SPAAPEN ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

Ring Finger ◽

Transmembrane Domains ◽

Dependent Manner ◽

N Terminus ◽

Ubiquitin Conjugating Enzyme ◽

New Gene

In the present study, the human TEB4 is identified as a novel ER (endoplasmic reticulum)-resident ubiquitin ligase. TEB4 has homologues in many species and has a number of remarkable properties. TEB4 contains a conserved RING (really interesting new gene) finger and 13 predicted transmembrane domains. The RING finger of TEB4 and its homologues is situated at the N-terminus and has the unconventional C4HC3 configuration. The N-terminus of TEB4 is located in the cytosol. We show that the isolated TEB4 RING domain catalyses ubiquitin ligation in vitro in a reaction that is ubiquitin Lys48-specific and involves UBC7 (ubiquitin-conjugating enzyme 7). These properties are reminiscent of E3 enzymes, which are involved in ER-associated protein degradation. TEB4 is an ER degradation substrate itself, promoting its own degradation in a RING finger- and proteasome-dependent manner.

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Phytophthora sojae effector Avr1d functions as E2 competitor and inhibits ubiquitination activity of GmPUB13 to facilitate infection

10.1101/2020.09.19.304535 ◽

2020 ◽

Author(s):

Yachun Lin ◽

Qinli Hu ◽

Jia Zhou ◽

Weixiao Yin ◽

Deqiang Yao ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Ubiquitin Ligase ◽

E3 Ligase ◽

Phytophthora Sojae ◽

Structural Basis ◽

Susceptibility Factor ◽

Ubiquitin Conjugating Enzyme ◽

Ubiquitination System

AbstractOomycete pathogens such as Phytophthora secrete a repertoire of effectors to host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy-roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By co-immunoprecipitation, gel infiltration and ITC assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure of Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy-roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor, negatively regulating soybean resistance against P. sojae. Altogether, this study highlights a novel virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This is the first study to unravel the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding.Significance StatementUbiquitination acts as a crucial regulator in plant immunity. Accordingly, microbial pathogens secrete effectors to hijak host ubiquitination system. However, the molecular mechanisms by which microbial effectors modulate host ubiquitination system are not yet clear. Here, we found that the Phytophthora sojae effector Avr1d physically binds to the U-box type E3 ligase GmPUB13, a susceptibility factor in soybean. The crystal structure of Avr1d in complex with GmPUB13 revealed that Avr1d occupies the binding site in GmPUB13 for the E2 ubiquitin conjugating enzyme and competes with E2 for physical binding to GmPUB13. Avr1d stabilized GmPUB13 by suppressing the self-ubiquitination activity of GmPUB13 and thereby promoting Phytophthora infection. This study provides structural basis for modulation of host targets by Phytophthora effectors.

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