Merck
  • Home
  • Search Results
  • The linear ubiquitin chain assembly complex (LUBAC) generates heterotypic ubiquitin chains.

The linear ubiquitin chain assembly complex (LUBAC) generates heterotypic ubiquitin chains.

eLife (2021-06-19)
Alan Rodriguez Carvajal, Irina Grishkovskaya, Carlos Gomez Diaz, Antonia Vogel, Adar Sonn-Segev, Manish S Kushwah, Katrin Schodl, Luiza Deszcz, Zsuzsanna Orban-Nemeth, Shinji Sakamoto, Karl Mechtler, Philipp Kukura, Tim Clausen, David Haselbach, Fumiyo Ikeda
ABSTRACT

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L), was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RING-in-between-RING (RBR) domains, a coordinated ubiquitin relay mechanism between the HOIL-1-interacting protein (HOIP) and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-RNF31 antibody produced in rabbit, affinity isolated antibody
Sigma-Aldrich
GeneJuice® Transfection Reagent, Non-lipid based chemical transfection reagent optimized for maximum transfection efficiency, ease-of-use, and minimal cytotoxicity on a wide variety of mammallian cells.
Sigma-Aldrich
Monoclonal Anti-Vinculin antibody produced in mouse, clone hVIN-1, ascites fluid
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose and L-glutamine, without sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
Penicillin-Streptomycin, with 10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, 0.1 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, ≥96.0% (calc. on dry substance, AT)
Sigma-Aldrich
Anti-HOIL-1 Antibody, clone 2E2, clone 2E2, from mouse
Roche
Chymotrypsin Sequencing Grade, from bovine pancreas