The ubiquitin ligase HOIL-1L regulates immune responses by interacting with linear ubiquitin chains

Abstract

Summary The Linear Ubiquitin Chain Assembly Complex (LUBAC), composed of HOIP, HOIL-1L, and SHARPIN, promotes tumor necrosis factor (TNF)-dependent NF-κB signaling in diverse cell types. HOIL-1L contains an Npl4 Zinc Finger (NZF) domain that specifically recognizes linear ubiquitin chains, but its physiological role in vivo has remained unclear. Here, we demonstrate that the HOIL-1L NZF domain has important regulatory functions in inflammation and immune responses in mice. We generated knockin mice ( Hoil-1l T201A;R208A/T201A;R208A ) expressing a HOIL-1L NZF mutant and observed attenuated responses to TNF- and LPS-induced shock, including prolonged survival, stabilized body temperature, reduced cytokine production, and liver damage markers. Cells derived from Hoil-1l T201A;R208A/T201A;R208A mice show reduced TNF-dependent NF-κB activation and incomplete recruitment of HOIL-1L into TNF Receptor (TNFR) Complex I. We further show that HOIL-1L NZF cooperates with SHARPIN to prevent TNFR-dependent skin inflammation. Collectively, our data suggest that linear ubiquitin-chain binding by HOIL-1L regulates immune responses and inflammation in vivo .

HOIL‐1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation

“…In agreement with the data published using ubiquitin dimers (Sato et al , 2011 ; Gomez‐Diaz et al , 2021 ), we found that the mutation or loss of the NZF domain was sufficient to disrupt the binding of HOIL‐1 to immobilised Halo‐tagged M1‐Ub tetramers (Fig EV4C and D ). This indicates that constructs containing the RBR domain, but lacking the NZF domain of HOIL‐1, have a much lower affinity for the M1‐Ub tetramer than constructs containing the NZF domain.…”

“… HOIL‐1[T203A/F204A/R210A] was assayed at 30°C in the absence or presence of the indicated ubiquitin dimers and tetramers. These mutations have been reported to impair the binding of ubiquitin to the NZF domain (Sato et al , 2011 ; Gomez‐Diaz et al , 2021 ) and their approximate location in HOIL‐1 is indicated (schematic not to scale). Assay of HOIL‐1[Δ194‐222] lacking the NZF core domain at 30°C in the absence or presence of the indicated ubiquitin oligomers.…”

“…To investigate whether the M1‐Ub and K63‐Ub oligomers exerted their effects on HOIL‐1 activity by binding to the NZF domain, we made mutations in this domain that have been reported to impair the binding of M1‐Ub dimers (Sato et al , 2011 ; Gomez‐Diaz et al , 2021 ). Surprisingly, these mutations did not reduce the rate of ubiquitylation of maltoheptaose by either M1‐Ub or K63‐Ub oligomers (Fig 5C ).…”

“… Maltoheptaose ubiquitylation by HOIL‐1 was assayed at 30°C in the presence of Met1‐ or Lys63‐linked ubiquitin dimers for the indicated times and visualised by Coomassie staining. Maltoheptaose ubiquitylation by HOIL‐1 was assayed at 30°C in the presence of Met1‐ or Lys63‐linked ubiquitin tetramers for the indicated times and visualised by Coomassie staining. HOIL‐1[T203A/F204A/R210A] was assayed at 30°C in the absence or presence of the indicated ubiquitin dimers and tetramers. These mutations have been reported to impair the binding of ubiquitin to the NZF domain (Sato et al , 2011; Gomez‐Diaz et al , 2021) and their approximate location in HOIL‐1 is indicated (schematic not to scale). Assay of HOIL‐1[Δ194‐222] lacking the NZF core domain at 30°C in the absence or presence of the indicated ubiquitin oligomers. Allosteric activation of HOIL‐1[233‐510] (lacking the LTM, UBL and NZF domains at the N‐terminus) by ubiquitin oligomers. …”

“…The E2-conjugating enzyme UBE2D3 (UbcH5c) has been reported to permit ester-linked ubiquitylation when acting in combination with the E3 ligases MYCBP2 and RNF213 (Pao et al, 2018;Otten et al, 2021). We found that the rate of conversion of ubiquitin to ubiquitylated maltoheptaose was accelerated similarly by M1-Ub or K63-Ub oligomers when UBE2L3 was replaced by UBE2D3 (Fig To investigate whether the M1-Ub and K63-Ub oligomers exerted their effects on HOIL-1 activity by binding to the NZF domain, we made mutations in this domain that have been reported to impair the binding of M1-Ub dimers (Sato et al, 2011;Gomez-Diaz et al, 2021). Surprisingly, these mutations did not reduce the rate of ubiquitylation of maltoheptaose by either M1-Ub or K63-Ub oligomers (Fig 5C).…”