The E3 ligase HOIL-1 catalyses ester bond formation between ubiquitin and components of the Myddosome in mammalian cells

Significance The formation of isopeptide bonds between the C-terminal carboxylate of ubiquitin and ε-amino groups of lysine residues on another protein is a major mechanism for regulating protein function. Ubiquitin can also form peptide bonds with the N-terminal α-amino group of another ubiquitin, a reaction catalysed by the HOIP component of the linear ubiquitin assembly complex (LUBAC). Here, we identify the HOIL-1 component of LUBAC as an unusual ligase that catalyses the formation of oxyester bonds between the C-terminal carboxylate of ubiquitin and serine and threonine residues in other proteins. We identify components of the Myddosome as physiological substrates of HOIL-1, indicating a role for HOIL-1 in regulating innate immunity.

PCR products. This generated a Rbck1 C458S founder that was backcrossed, and transmission of the Rbck1 C458S allele reconfirmed by PCR genotyping and sequencing. G1 Rbck1 C458S heterozygous mice were used for further breeding. Genotyping was routinely performed by PCR using the primers CGGCAGACGACAGAGATGC and GGCTGGACTGAGTTCATGGC followed by digestion of the PCR product with Alw N1. For a wild type allele, this results in a band of 509bp while a targeted allele gives bands of 355 and 154bp.
Mice were maintained on a C57BL/6J (Charles River UK) background and provided with free access to food (R&M3 pelleted irradiated diet) and water. Animals were kept in individually ventilated cages at 21 o C, 45-65% relative humidity and a 12h/12h light/dark cycle under specific-pathogen-free conditions in accordance with UK and European Union regulations. Experiments on mice were approved by the University of Dundee ethical review board under a UK Home Office project license.
Expression and purification of human proteins. Full length HOIL-1 and mutant proteins with single amino acid substitutions, which were preceded by the sequence MGSSHHHHHHSSGLEVLFQGPGSPEFPGVDSKAAA containing a His 6 tag for affinity purification, were expressed in BL21(DE3) E. coli cells. His 6 -UbcH7 was expressed in BL21(DE3) E. coli cells and His 6 -UBE1 was expressed in Sf21 insect cells. After purification by affinity chromatography on Ni-NTA-agarose, the purified proteins were dialysed into Phosphate Buffered Saline (PBS), 1 mM TCEP. The His 6 -tag was removed from UbcH7 by cleavage with PreScission Protease and buffer exchanged by size exclusion chromatography into PBS, 1 mM TCEP. The expression and purification of the deubiquitylases USP2-His 6 and GST-Otulin (4), and the isopeptidases vOTU (5), GST-DEN1 (also known as GST-NEDP1) (6) and SENP1 (7) as well as the preparation of Halo-NEMO affinity resin (2) have been described previously. Proteins were stored in aliquots at -80°C.
Immunoblotting and immunoprecipitation. Where indicated, cell lysates were incubated for 30 min at 37°C in the presence of 1.5 M hydroxylamine. NuPAGE LDS sample buffer was added and after incubation for 15 min at ambient temperature, the denatured samples were subjected to SDS-PAGE and immunoblotting. This was performed using NuPAGE gels and PVDF membranes as described (10). To immunoprecipitate LUBAC, 1 µg of anti-HOIP antibody was incubated for 16 h at 4°C with 1 mg of BMDM extract. 10 µl Protein G-Sepharose (packed bead volume) was added and the samples incubated for a further 60 min at 4°C with end-over-end rotation. The Protein G-Sepharose was collected by brief centrifugation, washed three times with cell lysis buffer containing 200 mM NaCl and once with 50 mM Tris-HCl pH 7.5, 50 mM NaCl and 5 mM DTT. Following the last wash, the Protein G-Sepharose was resuspended in 30 µl of 50 mM HEPES pH 7.5, 100 mM NaCl, 2 mM DTT, 1 mM MnCl 2 , 0.01% (w/v) Brij-35 with or without 100 Units of λPPase, 1 µM USP2, 1 µM Otulin or 1 M hydroxylamine. After incubation for 60 min at 37°C, reactions were terminated by denaturation in NuPAGE LDS sample buffer, the Protein G-Sepharose removed, and the supernatant analysed by SDS-PAGE followed by immunoblotting.
Mass spectrometry. Samples from in vitro ubiquitylation reactions were separated by SDS-PAGE and stained with InstantBlue Coomassie stain. Gel slices containing the proteins were excised with a scalpel, alkylated with chloroacetamide (11) and digested with trypsin for 16 h at 37°C (12). In some experiments, a further 3 h incubation with Asp-N proteinase was included to increase the sequence coverage of HOIL-1. Peptides were extracted from the gel slices using 5% formic acid and 50% acetonitrile, followed by concentration in a vacuum centrifuge. They were resuspended in 30µl of 0.1% formic acid (FA) and 10 or 15 µl was injected into the mass spectrometer.
Mass spectrometric analysis was performed by LC-MS/MS on a linear ion traporbitrap hybrid mass spectrometer (LTQ-Orbitrap Velos, Thermo Scientific) with an EASY-nESI source (Thermo Scientific) and coupled to a Dionex Ultimate 3000 nano-LC system. The samples were analysed using a 65 min method where the peptides were separated on a PepMap RSLC C18 reverse phase C18 column (2 µm, 100Å, 75 µm × 50 cm) at a flow rate of 300 nl/min and eluted with a 42 min linear gradient from 97% solvent A (0.1% FA in water) to 35% solvent B (80% acetonitrile, 0.08% FA) followed by an increase of solvent B to 99% at 47 min. To identify the most hydrophobic peptides in HOIL-1, the solvent system was changed as follows:-a 30 min linear gradient was used starting at 97% solvent A (0.1% FA in water) and finishing at 100% solvent B (0.1% FA in acetonitrile).
Data was acquired in data dependent mode, switching between MS and MS/MS acquisition automatically. Full scan MS spectra were acquired in the orbitrap over a mass range of 400-1600 m/z with resolution of 60,000 at 400 m/z (after accumulation to an FTMS Full AGC Target: 1,000,000; MSn AGC Target: 50,000). The 20 most intense ions were fragmented by collision induced dissociation with a collision energy of 35eV and analysed in the linear ion trap (Full AGC Target: 30,000. MSn AGC Target: 5,000). Raw files obtained were analysed using Proteome Discoverer 2.0, using Mascot (www.matrixscience.com) as the search engine. The database search parameters were set to detect ubiquitylation of lysine, serine, threonine and tyrosine residues.
Statistics. Pearson's chi-square ( 2 ) test (13) was used to compare observed Mendelian frequencies with those theoretical values that might be expected to arise from a random sample, with differences considered significant at p < 0.05.

Ubiquitin S20
Ubiquitin T22 Fig. S3. The hydroxylamine-induced cleavage of ubiquitylated IRAK1 is much slower at pH 6.8 than pH 9.0. WT BMDM were stimulated for 10 min with 1 µg/ml R848 and ubiquitylated proteins captured from cell extracts on Halo-NEMO beads and treated for 30 min at 37 o C with λPPase in buffer containing DTT (see Methods). The beads were incubated for 60 min without (−) or with (+) 0.5 M hydroxylamine at pH 6.8 or with 0.5 M hydroxylamine at pH 9.0, then immunoblotted with antibodies recognising IRAK1 or IKKβ. IKKβ, which binds to NEMO in a ubiquitin-independent manner, was used as a loading control. The pH 6.8 buffer was made by adding 1 ml of 15 M hydroxylamine to 29 ml of 100 mM Na 2 HPO 4 /NaH 2 PO 4 (pH 6.0) and adjusting the pH to 6.8 with 0.2 ml of 100% (v/v) acetic acid. In the absence of hydroxylamine, the buffer was 100 mM Na 2 HPO 4 /NaH 2 PO 4 pH 6.8. The buffer at pH 9.0 was 19 mM sodium carbonate, 22 mM sodium bicarbonate containing 0.5 M hydroxylamine.  S5. A more slowly migrating form of IRAK1 produced by incubation with hydroxylamine and USP2 is unaffected by isoproteases that cleave ISG15, NEDD8 and SUMO from proteins. BMDM from WT mice were stimulated for 10 min with R848 (1 µg/ml). Following cell lysis, ubiquitylated proteins were captured on Halo-NEMO beads, treated with λPPase, incubated for 60 min at pH 9.0 without (-) or with (+) 0.5 M hydroxylamine followed by incubation at pH 7.5 with 1 µM USP2. They were then incubated for a further 60 min at pH 7.5 without (-) or with (+) 0.1 µM vOTU (which hydrolyses Interferon-Stimulated Gene 15 from proteins), 1 µM DEN1 (which hydrolyses NEDD8 from proteins) or 1 µM SENP1 (which hydrolyses SUMO from proteins) at pH 7.5, and denatured in SDS. Following SDS-PAGE the captured proteins were immunoblotted with antibodies recognising IRAK1 or IKKβ as a loading control. The oxyester-linked ubiquitin attached directly to IRAK1 and IRAK2 is resistant to USP2. WT BMDM were stimulated for 10 min with 1 µg/ml R848, the ubiquitylated proteins captured on Halo-NEMO beads and treated for 30 min at 37 o C with λPPase. Following incubation for 60 min at pH 7.5 without (−) or with (+) 1 µM USP2, the beads were washed and then incubated for a further 60 min at pH 9.0 with 0.5 M hydroxylamine. Following SDS-PAGE and transfer to PVDF membranes, captured proteins were immunoblotted with antibodies recognising IRAK1, IRAK2 and IKKβ (loading control). The present study establishes that two types of Ub chain are attached to IRAK1, IRAK2 and MyD88. One is initiated by the attachment of ubiquitin to a serine or threonine residue on these proteins and is catalysed by HOIL-1, while the other is initiated by the formation of an isopeptide bond to a lysine residue. Ubiquitin is also attached covalently to HOIL-1 and Sharpin by oxyester bonds. HOIL-1 can also catalyse the formation of ubiquitin dimers linked by an oxyester bond in vitro, but whether these linkages are present in the ubiquitin chains that become attached to IRAK1, IRAK2 and MyD88 during TLR signalling is not established. In the schematic we speculatively place such linkages at the terminal K63-Ub and M1-Ub linkages as a "capping" mechanism to explain why the ubiquitin chains that become attached to IRAK1 and IRAK2 are much larger in macrophages from mice expressing an E3 ligase-inactive mutant of HOIL-1.