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In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
LUBAC synthesizes linear ubiquitin chains via a thioester intermediate
Benjamin Stieglitz1, Aylin C Morris-Davies, Marios G Koliopoulos
1Division of Molecular Structure, MRC-National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK.
EMBO Reports
|July 14, 2012
Summary
The linear ubiquitin chain assembly complex (LUBAC) generates linear ubiquitin chains via its HOIP subunit. Complex formation with HOIL-1L or SHARPIN releases inhibition, revealing LUBAC
Area of Science:
- Molecular Biology
- Biochemistry
- Immunology
Background:
- The linear ubiquitin chain assembly complex (LUBAC) is a key regulator of immune and inflammatory signaling pathways.
- LUBAC, a RING E3 ligase, uniquely dictates ubiquitin chain topology, an activity typically performed by E2 enzymes.
Purpose of the Study:
- To investigate the catalytic mechanism and regulation of LUBAC, specifically the HOIP subunit.
- To elucidate the role of the RING-in-between-RING (RBR) domain in linear ubiquitin chain formation.
- To understand how LUBAC activity is regulated by its interacting partners.
Main Methods:
- Biochemical assays to assess ubiquitin ligase activity.
- Analysis of the HOIP catalytic subunit and its functional domains.
- Investigating the effects of HOIL-1L and SHARPIN on HOIP activity.
Main Results:
- The RING-in-between-RING (RBR) region of HOIP is sufficient for generating linear ubiquitin chains.
- The N-terminal region of HOIP inhibits its catalytic activity, which is relieved by HOIL-1L or SHARPIN.
- HOIP utilizes a thioester intermediate via a conserved cysteine in the RING2 domain for ubiquitin transfer.
Conclusions:
- LUBAC's HOIP subunit possesses intrinsic linear ubiquitin chain formation capability.
- Regulation of LUBAC activity involves complex formation with HOIL-1L and SHARPIN.
- The catalytic mechanism of HOIP supports the hybrid RING/HECT model for RBR ligases.
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