Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

Singh, Sunil; Sivaraman, J.

doi:10.1042/bcj20200027

Cited by 19 publications

(15 citation statements)

References 42 publications

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“…A reactive site (Y1061, Figure f) was identified on the E3 ligase catalytic domain of ubiquitin-protein ligase E3C (UBE3C), an enzyme involved in protein ubiquitination. Although the site is not near the catalytic site of C1051 (>5 Å), it may affect the function of UBE3C through allosteric regulation since both Y1061 and C1051 are located on the same domain . Overall, the tyrosine residues with high reactivity were found on a wide range of protein classes, which provides valuable information for future development of covalent drugs to manipulate protein activities and expand the scope of drug targets.…”

Section: Resultsmentioning

confidence: 99%

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

Sun

Suttapitugsakul

2021

Anal. Chem.

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The tyrosine residue of proteins participates in a wide range of activities including enzymatic catalysis, protein–protein interaction, and protein–ligand binding. However, the functional annotation of the tyrosine residues on a large scale is still very challenging. Here, we report a novel method integrating azo coupling, bioorthogonal chemistry, and multiplexed proteomics to globally investigate the tyrosine reactivity in the human proteome. Based on the azo-coupling reaction between aryl diazonium salt and the tyrosine residue, two different probes were evaluated, and the probe with the best performance was employed to further study the tyrosine residues in the human proteome. Then, tagged tyrosine-containing peptides were selectively enriched using bioorthogonal chemistry, and after the cleavage, a small tag on the peptides perfectly fits for site-specific analysis by MS. Coupling with multiplexed proteomics, we quantified over 5000 tyrosine sites in MCF7 cells, and these quantified sites displayed a wide range of reactivity. The tyrosine residues with high reactivity were found on functionally and structurally diverse proteins, including those with the catalytic activity and binding property. This method can be extensively applied to advance our understanding of protein functions and facilitate the development of covalent drugs to regulate protein activity.

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Section: Resultsmentioning

confidence: 99%

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

Sun

Suttapitugsakul

2021

Anal. Chem.

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“…2013; Marin 2018). The HECT domain of UBE3C and other HECT-domain E3 ubiquitin ligases has a well-defined function; it is essential for mediating the acceptance and transfer of ubiquitin from an E2-conjugating enzyme to a degradation substrate via a cysteine residue at its catalytic site (You and Pickart 2001; Singh and Sivaraman 2020; reviewed in Lorenz 2018). The IQ domain-containing N-terminus of UBE3C mediates an interaction with the proteasome that is also important for function, but this portion of UBE3C is less well-conserved than the HECT domain and its function is less well-defined (You and Pickart 2001; You et al .…”

Section: Resultsmentioning

confidence: 99%

Ubiquitin ligases and a processive proteasome facilitate protein clearance during the oocyte-to-embryo transition in Caenorhabditis elegans

Spike

Tsukamoto

Greenstein

2021

Preprint

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The ubiquitin-mediated degradation of oocyte translational regulatory proteins is a conserved feature of the oocyte-to-embryo transition (OET). In the nematode Caenorhabditis elegans, multiple translational regulatory proteins, including the TRIM-NHL RNA-binding protein LIN-41/Trim71 and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11, are degraded during the OET. Degradation of each protein requires activation of the M-phase cyclin-dependent kinase CDK-1, is largely complete by the end of the first meiotic division and does not require the anaphase promoting complex (APC). However, only LIN-41 degradation requires the F-box protein SEL-10/FBW7/Cdc4p, the substrate recognition subunit of an SCF-type E3 ubiquitin ligase. This finding suggests that PUF-3 and PUF-11, which localize to LIN-41-containing ribonucleoprotein particles (RNPs), are independently degraded through the action of other factors and that the oocyte RNPs are disassembled in a concerted fashion during the OET. We develop and test the hypothesis that PUF-3 and PUF-11 are targeted for degradation by the proteasome-associated HECT-type ubiquitin ligase ETC-1/UBE3C/Hul5, which is broadly expressed in C. elegans. We find that several GFP-tagged fusion proteins that are degraded during the OET, including fusions with PUF-3, PUF-11, LIN-41, IFY-1/Securin and CYB-1/Cyclin B, are incompletely degraded when ETC-1 function is compromised. However, it is the fused GFP moiety that appears to be the critical determinant of this proteolysis defect. These findings are consistent with a conserved role for ETC-1 in promoting proteasome processivity and suggest that proteasomal processivity is an important element of the OET during which many key oocyte regulatory proteins are rapidly targeted for degradation.Article SummaryThe ubiquitin-mediated degradation of translational regulatory RNA-binding proteins is a conserved feature of the oocyte-to-embryo transition (OET). C. elegans LIN-41 is a master regulator of oogenesis and is found in a large translational regulatory ribonucleoprotein (RNP) complex with more than 1000 maternal transcripts and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11. We show that the concerted action of ubiquitin-mediated protein degradation and proteasome processivity rapidly disassemble LIN-41-containing RNPs during the OET thereby relieving repression of many maternal transcripts.

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“…Although the site is not near the catalytic site of C1051 (>5Å), it may affect the UBE3C's function through allosteric regulation since both Y1061 and C1051 are located on the same domain. 56 Overall, the tyrosine residues with high reactivity were found on a wide range of protein classes, which provides valuable information for future development of covalent drugs to manipulate protein activities and expands the scope of drug targets.…”

Section: Functional Analysis Of Proteins With the Quantified Tyrosine Residuesmentioning

confidence: 99%

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

Sun¹,

Suttapitugsakul²,

2021

Preprint

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<p>The tyrosine residue of proteins participates in a wide range of activities including enzymatic catalysis, protein-protein interaction, and protein-ligand binding. However, the functional annotation of the tyrosine residues on a large scale is still very challenging. Here, we report a novel method integrating azo coupling, bioorthogonal chemistry, and multiplexed proteomics to globally investigate the tyrosine reactivity in the human proteome. Based on the azo-coupling reaction between aryl diazonium salt and the tyrosine residue, the two different probes were evaluated, and the probe with the best performance was employed to specifically target the tyrosine residues. After the reaction, tagged tyrosine containing peptides were selectively enriched using bioorthogonal chemistry, and a small tag on the peptides from the cleavage perfectly fits for site-specific analysis by MS. Coupling with multiplexed proteomics, we quantified over 5,000 tyrosine sites in MCF7 cells and these quantified sites displayed a wide range of reactivity. The tyrosine residues with high reactivity were found on functionally and structurally diverse proteins, including those with the catalytic activity and binding property. This method can be extensively applied to advance our understanding of protein functions and facilitate the development of covalent drugs to regulate protein activity.</p>

show abstract

Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

Cited by 19 publications

References 42 publications

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

Ubiquitin ligases and a processive proteasome facilitate protein clearance during the oocyte-to-embryo transition in Caenorhabditis elegans

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

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