Information on EC 2.3.2.26 - HECT-type E3 ubiquitin transferase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
2.3.2.26
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RECOMMENDED NAME
GeneOntology No.
HECT-type E3 ubiquitin transferase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [acceptor protein]-L-lysine = [E2 ubiquitin-conjugating enzyme]-L-cysteine + N6-ubiquitinyl-[acceptor protein]-L-lysine
show the reaction diagram
S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [HECT-type E3 ubiquitin transferase]-L-cysteine = [E2 ubiquitin-conjugating enzyme]-L-cysteine + S-ubiquitinyl-[HECT-type E3 ubiquitin transferase]-L-cysteine
show the reaction diagram
(1a)
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S-ubiquitinyl-[HECT-type E3 ubiquitin transferase]-L-cysteine + [acceptor protein]-L-lysine = [HECT-type E3 ubiquitin transferase]-L-cysteine + N6-ubiquitinyl-[acceptor protein]-L-lysine
show the reaction diagram
(1b)
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
protein ubiquitylation
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SYSTEMATIC NAME
IUBMB Comments
S-ubiquitinyl-[HECT-type E3-ubiquitin transferase]-L-cysteine:acceptor protein ubiquitin transferase (isopeptide bond-forming)
In the first step the enzyme transfers ubiquitin from the E2 ubiquitin-conjugating enzyme (EC 2.3.2.23) to a cysteine residue in its HECT domain (which is located in the C-terminal region), forming a thioester bond. In a subsequent step the enzyme transfers the ubiquitin to an acceptor protein, resulting in the formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and the epsilon-amino group of an L-lysine residue of the acceptor protein.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
Rattus novegicus
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
S-(ubiquitin)n-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [adenomatous polyposis coli]-L-lysine
[E2 ubiquitin-conjugating enzyme]-L-cysteine + N6-(ubiquitin)n-[adenomatous polyposis coli]-L-lysine
show the reaction diagram
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adenomatous polyposis coli protein functions as a negative regulator of the Wnt signaling pathway
isoform HECTD1 modifies adenomatous polyposis coli with Lys63 polyubiquitin
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?
S-(ubiquitin)n-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [Dvl2]-L-lysine
[E2 ubiquitin-conjugating enzyme]-L-cysteine + N6-(ubiquitin)n-[Dvl2]-L-lysine
show the reaction diagram
Dvl2 i.e. dishevelled, a central mediator for both Wnt/beta-catenin and Wnt/planar cell polarity pathways
isoform NEDD4L mediates polyubiquitination of Dvl2 at Lys6, Lys27, and Lys29
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?
S-(ubiquitin)n-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [Glis3]-L-lysine
[E2 ubiquitin-conjugating enzyme]-L-cysteine + N6-(ubiquitin)n-[Glis3]-L-lysine
show the reaction diagram
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Glis i.e. transcription factor Gli-similar 3
isoform Itch significantly contributes to Glis3 polyubiquitination and reduces Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 requires the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Itch dramatically inhibits Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover
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?
S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [caspase-8]-L-lysine
[E2 ubiquitin-conjugating enzyme]-L-cysteine + N6-ubiquitinyl-[caspase-8]-L-lysine
show the reaction diagram
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isoform HECTD3 ubiquitinates caspase-8 with K63-linked polyubiquitin chains that do not target caspase-8 for degradation but decrease the caspase-8 activation
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?
S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [ING2]-L-lysine
[E2 ubiquitin-conjugating enzyme]-L-cysteine + N6-ubiquitinyl-[ING2]-L-lysine
show the reaction diagram
ING2 i.e. candidate tumor suppressor Inhibitor of Growth 2
isoform Smurf1 interacts with and targets ING2 for poly-ubiquitination and proteasomal degradation. The ING2 binding domain in Smurf1 was mapped to the catalytic HECT domain. The C-terminal PHD domain of ING2 is required for Smurf1-mediated degradation
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?
S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [Sav]-L-lysine
[E2 ubiquitin-conjugating enzyme]-L-cysteine + N6-ubiquitinyl-[Sav]-L-lysine
show the reaction diagram
S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [Spry2]-L-lysine
[E2 ubiquitin-conjugating enzyme]-L-cysteine + N6-ubiquitinyl-[Spry2]-L-lysine
show the reaction diagram
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Spry2 is a regulator of receptor tyrosine kinase signaling in development and disease
isoform Nedd4 polyubiquitinates Spry2 via Lys48 on ubiquitin and decreases its stability. The Spry2/Nedd4 association involves theWW domains of Nedd4 and requires phosphorylation of the Mnk2 kinase sites, Ser112 and Ser121, on Spry2. The phospho-Ser112/121 region on Spry2 that binds WW domains of Nedd4 is a non-canonical WW domain binding region that does not contain Pro residues after phospho-Ser
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?
S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [ubiquitin]-L-lysine
[E2 ubiquitin-conjugating enzyme]-L-cysteine + N6-ubiquitinyl-[ubiquitin]-L-lysine
show the reaction diagram
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isoform NleL functionally and structurally mimics eukaryotic HECT E3 ligases and catalyzes formation of unanchored polyubiquitin chains using Lys6 and Lys48 linkage. The catalytic cysteine residue forms a thioester intermediate with ubiquitin
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?
S-ubiquitinyl-[HECT-type E3 ubiquitin transferase]-L-cysteine + [Sox6 protein]-L-lysine
[HECT-type E3 ubiquitin transferase]-L-cysteine + N6-ubiquitinyl-[Sox6 protein]-L-lysine
show the reaction diagram
S-ubiquitinyl-[Ubc-18]-L-cysteine + [IFY-1]-L-lysine
[Ubc-18]-L-cysteine + N6-ubiquitinyl-[IFY-1]-L-lysine
show the reaction diagram
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IFY-1 i.e. anaphase inhibitor securin
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?
S-ubiquitinyl-[UbcH5a]-L-cysteine + [ubiquitin mutant G76V]-L-lysine
[UbcH5a]-L-cysteine + N6-ubiquitinyl-[mutant G76V]-L-lysine
show the reaction diagram
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?
S-ubiquitinyl-[UbcH5a]-L-cysteine + [ubiquitin-DELTAGG]-L-lysine
[UbcH5a]-L-cysteine + N6-ubiquitinyl-[ubiquitin-DELTAGG]-L-lysine
show the reaction diagram
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ubiquitin-DELTAGG i.e. mutant ubiquitin lacking the two C-terminal glycine residues, cannot be conjugated to other proteins
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?
S-ubiquitinyl-[UbcH7]-L-cysteine + [endophilin A]-L-lysine
[UbcH7]-L-cysteine + N6-ubiquitinyl-[endophilin A]-L-lysine
show the reaction diagram
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isoform Itch ubiquitinates SH3 domain-containing protein endophilin A1 and the SH3/proline-rich domain interaction facilitates this activity. EGF treatment of cells stimulates endophilin A1 ubiquitination
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?
[AIP2-ubiquitin-conjugating enzyme E2]-S-ubiquitin-L-cysteine + [EGR2]-L-lysine
[AIP2-ubiquitin-conjugating enzyme E2]-L-cysteine + [EGR2]-N6-ubiquitinyl-L-lysine
show the reaction diagram
EGR2, a zinc finger transcription factor that has been found to regulate Fas ligand expression during activation-induced T-cell death
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?
[HECT-E3-ubiquitin-carrier protein NEDD4]-S-ubiquitin-L-cysteine + [gamma-epithel Na+-channel]-L-lysine
[HECT-E3-ubiquitin-carrier protein NEDD4]-L-cysteine + [gamma-epithel Na+-channel]-N6-ubiquinyl-L-lysine
show the reaction diagram
His-tagged Ube2D3, in addition the reaction mixture contains purified E1 enyzme and ubiquitin
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?
[Nedd4-1-ubiquitin-conjugating enzyme E2]-S-ubiquitin-L-cysteine + [activated Cdc42-associated tyrosine kinase]-L-lysine
[Nedd4-1ubiquitin-conjugating enzyme E2]-L-cysteine + [activated Cdc42-associated tyrosine kinase]-N6-ubiquitinyl-L-lysine
show the reaction diagram
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activated Cdc42-associated tyrosine kinase is ubiquitinated by HECT E3 ubiquitin ligase Nedd4-1 and degraded along with epidermal growth factor receptor in response to epidermal growth factor stimulation. Activated Cdc42-associated tyrosine kinase interacts with Nedd4-1 through a conserved PPXY WW-binding motif. The WW3 domain in Nedd4-1 is critical for binding to activated Cdc42-associated tyrosine kinase. Deletion of the sterile alpha motif SAM-domain at the N-terminus dramatically reduces the ubiquitination of activated Cdc42-associated tyrosine kinase by Nedd4-1, while deletion of the Uba domain dramatically enhances the ubiquitination. Activated Cdc42-associated tyrosine kinase degradation is processed by lysosomes, not proteasomes
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?
[Nedd4-1-ubiquitin-conjugating enzyme E2]-S-ubiquitin-L-cysteine + [epidermal growth factor receptor]-L-lysine
[Nedd4-1-ubiquitin-conjugating enzyme E2]-L-cysteine + [epidermal growth factor receptor]-N6-ubiquitinyl-L-lysine
show the reaction diagram
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epidermal growth factor receptor and activated Cdc42-associated tyrosine kinase are ubiquitinated by ubiquitin ligase Nedd4-1 in response to epidermal growth factor stimulation
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?
[Rsp-ubiquitin-conjugating enzyme UbcH5B]-S-ubiquitin-L-cysteine + [Sna3 cytoplasmic domain]-L-lysine
[Rsp5-ubiquitin-conjugating enzyme UbcH5B]-L-cysteine + [Sna3 cytoplasmic domain]-N6-ubiquitinyl-L-lysine
show the reaction diagram
[TRIP1-ubiquitin-conjugating enzyme E2]-S-ubiquitin-L-cysteine + [APP-BP1]-L-lysine
[TRIP12-ubiquitin-conjugating enzyme E2]-L-cysteine + [APP-BP1]-N6-ubiquitinyl-L-lysine
show the reaction diagram
ubiquitin ligase TRIP12 functions as an E3 enzyme of APP-BP1 and additionally requires an E4 activity for polyubiquitination of APP-BP1. APP-BP1 is part of the ubiquitin-like protein NEDD8 activating enzyme. TRIP12 specifically interacts with the APP-BP1 monomer but not with the APP-BP1/Uba3 heterodimer. Overexpression of TRIP12 enhances the degradation of APP-BP1, whereas knockdown of TRIP12 stabilizes it
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?
[ubiquitin ligase HECTD3]-S-ubiquitin-L-cysteine + [Tara]-L-lysine
[ubiquitin ligase HECTD3]-L-cysteine + [Tara]-N6-ubiquitinyl-L-lysine
show the reaction diagram
Tara, Trio-associated repeat on actin, is an interacting partner of guanine nucleotide exchange factors Trio and TRF1. Ubiquitin-protein ligase HECTD3 directly binds Tara in vitro and forms a complex with Tara in vivo. Overexpression of HECTD3 enhances the ubiquitination of Tara in vivo and promotes the turnover of Tara, whereas depletion of HECTD3 by small interfering RNA decreases Tara degradation
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[ubiquitin-conjugating enzyme E2D3]-S-ubiquitin-L-cysteine + [latent membrane protein 2A LMP2A]-L-lysine
[ubiquitin-conjugating enzyme E2D3]-L-cysteine + [latent membrane protein 2A LMP2A]-N6-ubiquitinyl-L-lysine
show the reaction diagram
His-tagged Ube2D3, in addition the reaction mixture contains purified E1 enyzme and ubiquitin
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?
[ubiquitin-conjugating enzyme E2]-S-ubiquitin-L-cysteine + [transcription factor WRKY53]-L-lysine
[ubiquitin-conjugating enzyme E2D3]-L-cysteine + [transcription factor WRKY53]-N6-ubiquitinyl-L-lysine
show the reaction diagram
UPL5 is able to use the WRKY53 protein as a substrate for polyubiquitination in an in vitro system, and induction of UPL5 expression by an ethanol-inducible system in upl5 plants leads to degradation of the WRKY53 protein
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additional information
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
S-ubiquitinyl-[HECT-type E3 ubiquitin transferase]-L-cysteine + [Sox6 protein]-L-lysine
[HECT-type E3 ubiquitin transferase]-L-cysteine + N6-ubiquitinyl-[Sox6 protein]-L-lysine
show the reaction diagram
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
chlorpromazine
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minimum inhibitory concentration is 0.3 mM
chlorprothixene
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minimum inhibitory concentration is 0.3 mM
clomipramine
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antidepressant drug, specifically blocks isoform ITCH auto-ubiquitylation, as well as p73 ubiquitylation. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, leads to reduced cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. Minimum inhibitory concentration is 0.3 mM
norclomipramine
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minimum inhibitory concentration is 0.3 mM
additional information
autoinhibition mechanism of domains C2-HECT is not observed in isoform Smurf1
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.6
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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assay at
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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isoform Itch co-localizes with markers of the endosomal system in a C2 domain-dependent manner and upon EGF stimulation, substrate endophilin A1 translocates to an EGF-positive endosomal compartment where it colocalizes with itch
Manually annotated by BRENDA team
the isoform Smurf1 C2 domain exerts a key role in localization to the plasma membrane. Lysine residues, Lys-28 and Lys-85, within the C2 domain are important for Smurf1 localization at the plasma membrane
Manually annotated by BRENDA team
enzyme UBR5 localizes in the nuclei of smooth muscle cells and forms a complex with myocardin in vivo and in vitro
Manually annotated by BRENDA team
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
structure of isoform NleL contains two domains, a beta-helix domain formed by pentapeptide repeats and a bilobed catalytic domain reminiscent of the N- and C-lobe architecture of HECT E3s. The C-lobe adopts a large range of different positions relative to the N-lobe, indicating that the helix linking the two lobes is extremely flexible
C2 domain, to 1.96 A resolution. The Smurf1 C2 domain possesses a typical anti-parallel beta-sandwich fold. The Smurf1 C2 domain exerts a key role in localization to the plasma membrane. Lysine residues, Lys-28 and Lys-85, within the C2 domain are important for Smurf1 localization at the plasma membrane, regulation on cell migration, and robust ligase activity toward RhoA, which further supports a Ca2+-independent localization mechanism for Smurf1
crystal structure of the HECT domain of human ubiquitin ligase WWP1/AIP5 maintains a two-lobed structure like the HECT domain of the human ubiquitin ligase E6AP. The organization of the two lobes relative one another is different from E6AP due to a rotation about a polypeptide hinge linking the N and C lobes
hanging drop vapor diffusion method, using 0.1 M HEPES pH 8.4, 0.2 M MgCl2, 15% (v/v) ethanol at 4°C
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structure of ubiquitin-loaded human neural precursor cell–expressed developmentally downregulated protein, Nedd4, to 2.51 A resolution. The Nedd4-HECT domain-ubiquitin transitory intermediate provides a structural basis for the proposed sequential addition mechanism. The donor ubiquitin, transferred from the E2, is bound to the Nedd4 C lobe with its C-terminal tail locked in an extended conformation, primed for catalysis. Nedd4-family members are Lys63-specific enzymes whose catalysis is mediated by an essential C-terminal acidic residue
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structures of Nedd4 HECT domain, alone (2.5 A) and in complex with ubiquitin (2.7 A), showing new binding modes involving two surfaces on ubiquitin and both subdomains of the HECT N-lobes, suggesting an model for the HECT-t-substrate ubiquitin transfer, in which the growing chain on the substrate is kept close to the catalytic cysteine to promote processivity HECTNedd4 displays the typical HECT fold, composed of two lobes connected by a flexible hinge. The N-lobe consists of two moieties, the large and the smal subdomains. The small domains host the E2-binding site and the large carries the catalytic cysteine
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crystal structure of a trapped complex of ubiquitin ligase Rsp5 with ubiquitin and substrate Sna3 cytoplasmic domain as a proxy for the catalytic intermediate. The covalent linkage between ubiquitin and the HECT domain is oriented by ubiquitin interactions with the HECT domain N- and C-lobes that stabilize HECT domain conformation. The HECT domain architecture of the ligase primed for ligation prioritizes potential target lysines by their placement relative to a composite catalytic center for ubiquitination
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography and Superdex 200 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
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expressed in HEK-293 cells
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expression in Escherichia coli
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expression in Escherihia coli
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
enzyme expression is increased 3 h after Dox stimulation (0.0005 mM)
UPL5 expression is reduced after hydrogen peroxide treatment
UPL5 is highly expressed in leaf tissue but expression is reduced at bolting when transcription factor WRKY53 protein levels should be high
UPL5 is induced by jasmonic acid treatment
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C2579G
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mutation in catalytic cysteine, loss of E3 ligase activity
C716A
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mutation in catalytic cysteine. Cells transfected with C716A fail to accumulate securin with significant reduction in Mad2 level. Cell show a significant increase in cells displaying misaligned chromosomes, lagging chromosomes during mitotic exit, and multinucleation
E411Q
slight decrease of affinity to human papilloma virus E6 oncogens
E411Q/E415Q
about 6fold decrease of affinity to human papilloma virus E6 oncogens
E415Q
slight decrease of affinity to human papilloma virus E6 oncogens
E415R
slight decrease of affinity to human papilloma virus E6 oncogens
F707A
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Nedd4 mutant, almost abolished HECTNedd4 binding to Lys 63 ubiquitin. Mutant F707A has defective chain elongation on substrate or shorter free chains
L409V
about 30fold decrease of affinity to human papilloma virus E6 oncogens characterised by very fast dissociation rates
L412V
moderate decrease of affinity to human papilloma virus E6 oncogens
L413S
about 40fold decrease of affinity to human papilloma virus E6 oncogens characterised by very fast dissociation rates
L413V
moderate decrease of affinity to human papilloma virus E6 oncogens
medicine
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isoform HECTD3 is frequently overexpressed in breast carcinomas suggesting that caspase-8 ubiquitination by HECTD3 confers cancer cell survival
Q410E
no decrease of affinity to human papilloma virus E6 oncogens
Y605A
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Nedd4 mutant, almost abolished HECTNedd4 binding to Lys 63 ubiquitin
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
synthesis
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construcution of a soluble HECT domain truncation of isoform WWP2 which is amendable for preparation scale expression in Escherichia coli. A relatively simple purification process achieves highly pure protein by employing immobilized metal-affinity chromatography followed by salting out, ion exchange chromatography and finally, size exclusion chromatography. Procedure allows to obtain about 60 mg/L of the soluble protein