Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Non-lysine ubiquitination: Biochemical characterisation of the RING-between-RING E3 ubiquitin ligase, HOIL-1L (#215)

Xiangyi Wang 1 2 , Bernhard Lechtenberg 2
  1. Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne, VIC, Australia
  2. Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

Ubiquitination is a post-translational modification that controls almost every process inside eukaryotic cells. It involves the attachment of the small protein ubiquitin to substrates, which can be either a different protein or another ubiquitin molecule to form polyubiquitin chains. Conventionally, linkages are formed as isopeptide bonds between the ubiquitin C-terminus and a lysine side chain of the substrate; additionally, peptide bonds can be formed with the first methionine residue of a ubiquitin to form Met1-linked or linear ubiquitin chains. Ubiquitination is brought about by the cooperation of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases. HOIL-1L (heme-oxidised IRP2 ubiquitin ligase-1L) belongs to the RING-between-RING (RBR) E3 ligase family characterised by the catalytic RING1-IBR-RING2 domains. HOIL-1L, together with another RBR E3 ligase, HOIP, forms part of the linear ubiquitin chain assembly complex (LUBAC). LUBAC is so far the only known E3 entity catalysing the formation of Met-1 linked ubiquitin chains, which have been demonstrated to regulate inflammation and immune signalling. In LUBAC, HOIP catalyses Met1-linked ubiquitin chain formation, while, intriguingly, HOIL-1L has recently been shown to catalyse oxyester linkage formation between the ubiquitin C-terminus and the Ser/Thr side chains of the substrate. Exciting as it is, the molecular mechanism and catalytic cycle of HOIL-1L’s atypical activity remain largely unknown.

Using molecular and biochemical approaches, I confirmed HOIL-1L’s esterification activity and further demonstrated that HOIL-1L has a preference towards Ser over Thr residues. I have also shown that HOIL-1L mediates discharge of ubiquitin from the E2 UbcH7 and that this activity can be allosterically activated by specific di-ubiquitin moieties, which has been proposed as a common feature of the RBR E3 ligase family. Furthermore, my preliminary data suggest that HOIL-1L may use a unique catalytic mechanism which resembles that of a structurally distinct Ser/Thr-specific E3 ligase, MYCPB2.

 

 

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