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Target specificity of the E3 ligase LUBAC for ubiquitin and NEMO relies on different minimal requirements.

Smit JJ, van Dijk WJ, El Atmioui D, Merkx R, Ovaa H, Sixma TK - J. Biol. Chem. (2013)

Bottom Line: The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway.Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin.Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Biochemistry and.

ABSTRACT
The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin.

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NEMO ubiquitination requires the top of the priming ubiquitin for NEMO.A, full-length HOIP·HOIL-1L mediated free ubiquitin chain formation and Strep-NEMO242–419 ubiquitination with different ubiquitin E16 and E18 point mutants. B, full-length HOIP·HOIL-1L does not mediate free ubiquitin chain formation and Strep-NEMO242–419 ubiquitination with N-terminally HIS-tagged ubiquitin and ubiquitin E16K. C, ubiquitin Met-(3–76) cannot be used by full-length HOIP·HOIL-1L to prime NEMO, but it can be linked to the target ubiquitinΔG76. D, HOIL-1L inhibits the transfer of wild type ubiquitin onto target ubiquitins. If the N terminus is not available (TAMRAUb, right panel), this interferes with of HOIL-1L with the transfer of the donor ubiquitin is no longer observed.
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Figure 5: NEMO ubiquitination requires the top of the priming ubiquitin for NEMO.A, full-length HOIP·HOIL-1L mediated free ubiquitin chain formation and Strep-NEMO242–419 ubiquitination with different ubiquitin E16 and E18 point mutants. B, full-length HOIP·HOIL-1L does not mediate free ubiquitin chain formation and Strep-NEMO242–419 ubiquitination with N-terminally HIS-tagged ubiquitin and ubiquitin E16K. C, ubiquitin Met-(3–76) cannot be used by full-length HOIP·HOIL-1L to prime NEMO, but it can be linked to the target ubiquitinΔG76. D, HOIL-1L inhibits the transfer of wild type ubiquitin onto target ubiquitins. If the N terminus is not available (TAMRAUb, right panel), this interferes with of HOIL-1L with the transfer of the donor ubiquitin is no longer observed.

Mentions: The NEMO ubiquitination assays showed that even at short time points, multiple ubiquitins could be transferred onto the target. Mechanistically this could indicate either a slow priming event of the first ubiquitin followed by highly processive chain formation or en-block transfer of ubiquitin chains onto NEMO. In slower reactions, however, with the relatively inefficiently used ubiquitin E16 and E18 mutants, it is clear that single ubiquitins are transferred onto NEMO, indicating that en bloc transfer is not required (Fig. 5A). These results show that the in vitro ubiquitination of NEMO is limited by the priming ubiquitination event on NEMO, explaining why mono-ubiquitinated NEMO is a minority of the NEMO population in most of the assays.


Target specificity of the E3 ligase LUBAC for ubiquitin and NEMO relies on different minimal requirements.

Smit JJ, van Dijk WJ, El Atmioui D, Merkx R, Ovaa H, Sixma TK - J. Biol. Chem. (2013)

NEMO ubiquitination requires the top of the priming ubiquitin for NEMO.A, full-length HOIP·HOIL-1L mediated free ubiquitin chain formation and Strep-NEMO242–419 ubiquitination with different ubiquitin E16 and E18 point mutants. B, full-length HOIP·HOIL-1L does not mediate free ubiquitin chain formation and Strep-NEMO242–419 ubiquitination with N-terminally HIS-tagged ubiquitin and ubiquitin E16K. C, ubiquitin Met-(3–76) cannot be used by full-length HOIP·HOIL-1L to prime NEMO, but it can be linked to the target ubiquitinΔG76. D, HOIL-1L inhibits the transfer of wild type ubiquitin onto target ubiquitins. If the N terminus is not available (TAMRAUb, right panel), this interferes with of HOIL-1L with the transfer of the donor ubiquitin is no longer observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 5: NEMO ubiquitination requires the top of the priming ubiquitin for NEMO.A, full-length HOIP·HOIL-1L mediated free ubiquitin chain formation and Strep-NEMO242–419 ubiquitination with different ubiquitin E16 and E18 point mutants. B, full-length HOIP·HOIL-1L does not mediate free ubiquitin chain formation and Strep-NEMO242–419 ubiquitination with N-terminally HIS-tagged ubiquitin and ubiquitin E16K. C, ubiquitin Met-(3–76) cannot be used by full-length HOIP·HOIL-1L to prime NEMO, but it can be linked to the target ubiquitinΔG76. D, HOIL-1L inhibits the transfer of wild type ubiquitin onto target ubiquitins. If the N terminus is not available (TAMRAUb, right panel), this interferes with of HOIL-1L with the transfer of the donor ubiquitin is no longer observed.
Mentions: The NEMO ubiquitination assays showed that even at short time points, multiple ubiquitins could be transferred onto the target. Mechanistically this could indicate either a slow priming event of the first ubiquitin followed by highly processive chain formation or en-block transfer of ubiquitin chains onto NEMO. In slower reactions, however, with the relatively inefficiently used ubiquitin E16 and E18 mutants, it is clear that single ubiquitins are transferred onto NEMO, indicating that en bloc transfer is not required (Fig. 5A). These results show that the in vitro ubiquitination of NEMO is limited by the priming ubiquitination event on NEMO, explaining why mono-ubiquitinated NEMO is a minority of the NEMO population in most of the assays.

Bottom Line: The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway.Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin.Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Biochemistry and.

ABSTRACT
The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin.

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