Skip to Content
Merck
  • Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling.

Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling.

The EMBO journal (2020-11-21)
Lilian M Fennell, Carlos Gomez Diaz, Luiza Deszcz, Anoop Kavirayani, David Hoffmann, Kota Yanagitani, Alexander Schleiffer, Karl Mechtler, Astrid Hagelkruys, Josef Penninger, Fumiyo Ikeda
ABSTRACT

HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site-specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)-induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF-κB reporter relative to wild-type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated HoipK778R/K778R knock-in mice, which show no overt developmental phenotypes; however, in response to TNF, HoipK778R/K778R mouse embryonic fibroblasts display mildly suppressed NF-κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF-induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in HoipK778R/K778R mice, which is rescued by knockout of TNFR1. We propose that site-specific ubiquitination of HOIP regulates a LUBAC-dependent switch between survival and apoptosis in TNF signaling.

MATERIALS
Product Number
Brand
Product Description

Roche
DNase I recombinant, grade I, from bovine pancreas, expressed in Pichia pastoris
Sigma-Aldrich
Anti-RNF31 antibody produced in rabbit, affinity isolated antibody
Sigma-Aldrich
Monoclonal Anti-Vinculin antibody produced in mouse, clone hVIN-1, ascites fluid
Sigma-Aldrich
Sodium orthovanadate, ≥90% (titration)
Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Sigma-Aldrich
Cycloheximide solution, Ready-Made Solution, microbial, 100 mg/mL in DMSO, Suitable for cell culture
Millipore
ANTI-FLAG® M2 Affinity Gel, purified immunoglobulin, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-Keratin 14 antibody produced in rabbit, affinity isolated antibody
Sigma-Aldrich
Ubiquitin from bovine erythrocytes, BioUltra, ≥98% (SDS-PAGE), essentially salt-free, lyophilized powder
Sigma-Aldrich
Anti-HOIL-1 Antibody, clone 2E2, clone 2E2, from mouse
Roche
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, Protease Inhibitor Cocktail Tablets provided in a glass vial, Tablets provided in a glass vial