Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Biochemical study of HOIL-1L, an unconventional RING-between-RING (RBR) E3 ubiquitin ligase (#134)

Xiangyi Susie Wang 1 2 , Simon Cobbold 1 2 , Bernhard Lechtenberg 1 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 carrying broad signal transduction functions in eukaryotic cells. The process is dependent on the attachment of the small protein ubiquitin to a substrate via the catalysis of E3 ubiquitin ligases. Ubiquitin is most commonly conjugated onto substrate protein lysine residues, resulting in a multitude of consequences, for example, substrate degradation, trafficking and association with other proteins. Intriguingly, recent studies demonstrate that specialised E3 ligases can use substrate serine and threonine residues as ubiquitination sites [1, 2]. The repertoire of the ubiquitin machinery is further expanded by the discovery of host cells modifying Salmonella lipopolysaccharide (LPS) with ubiquitin earlier this year [3].

 

Heme-oxidised IRP2 ubiquitin ligase-1L (HOIL-1L) belongs to the RING-between-RING (RBR) E3 ligase family. HOIL-1L constitutes part of the linear ubiquitin chain assembly complex (LUBAC), a central regulator of innate immune signalling. Recent evidence suggests that HOIL-1L regulates LUBAC activity via ubiquitination of serine or threonine residues[4, 5]. An unpublished study further links HOIL-1L's unconventional ubiquitination activity to non-proteinaceous substrates in vitro [6]. However, deeper understanding of HOIL-1L's catalytic mechanism and function is prohibited by lack of molecular details.

 

I have established biochemistry methods to investigate different steps of HOIL-1L’s catalytic activity and regulation. Using such methods in combination with mass spectrometry, I have characterised ubiquitination targets by HOIL-1L. I have also obtained molecular information of how HOIL-1L engages ubiquitin for substrate modification using structural biology approaches.

 

 

  1. Pao, K.C., et al., Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity.Nature, 2018. 556(7701): p. 381-385.
  2. Kelsall, I.R., et al., The E3 ligase HOIL-1 catalyses ester bond formation between ubiquitin and components of the Myddosome in mammalian cells. Proc Natl Acad Sci U S A, 2019. 116(27): p. 13293-13298.
  3. Otten, E.G., et al., Ubiquitylation of lipopolysaccharide by RNF213 during bacterial infection. Nature, 2021. 594(7861): p. 111-116.
  4. Rodriguez Carvajal, A., et al., The linear ubiquitin chain assembly complex (LUBAC) generates heterotypic ubiquitin chains. Elife, 2021. 10.
  5. Fuseya, Y., et al., The HOIL-1L ligase modulates immune signalling and cell death via monoubiquitination of LUBAC. Nat Cell Biol, 2020. 22(6): p. 663-673.
  6. Ian R. Kelsall, E.H.M., Yingqi Xu, Cheryl L. Scudamore, Sambit K. Nanda, Paula Mancebo-Gamella, Nicola T. Wood, Axel Knebel, Stephen J. Matthews, Philip Cohen, HOIL-1-catalysed ubiquitylation of unbranched glucosaccharides and its activation by ubiquitin oligomers. 2021.