Ubiquitin interactions of HOIP, the catalytical subunit of the linear ubiquitin chain assembly complex : Structural and biophysical approach
Salminen, Elina (2021)
Salminen, Elina
2021
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2021-05-11
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202104273957
https://urn.fi/URN:NBN:fi:tuni-202104273957
Tiivistelmä
Ubiquitination forms a complex signalling system that regulates various cellular processes. Eight linkage types of ubiquitin chains exist, and each ubiquitin chain type is thought to encode distinctive signalling functions in cells. LUBAC is the only E3 ligase complex that generates Met1-linked, or linear, ubiquitin chains, and consists of the catalytical subunit HOIP and two accessory proteins HOIL-1 and SHARPIN. Linear ubiquitination has crucial roles in immune signalling pathways, where it is essential for pathways that activate NF-B transcription factors due to various stimuli. Upon stimulus by e.g. cytokines or bacterial peptides, receptor signalling complexes form at the receptors, consisting of different signal mediators including protein kinases and ubiquitin ligases. LUBAC generates linear ubiquitin chains on various substrates at these complexes, and the chains serve as recruitment and activation platform for subsequent signalling proteins in the pathways. LUBAC gets recruited to the receptor signalling complexes through interaction with ubiquitin chains, but the details of this process are not understood. Lys63-linked ubiquitin chains are essential for the recruitment of LUBAC, but also other chain types may be involved in the process. To understand how LUBAC gets recruited to these complexes, it is important to know the ubiquitin binding properties of LUBAC subunits. Limited information about the ubiquitin binding properties of the catalytical subunit HOIP is available, and to expand our understanding about the subject, this thesis studied how HOIP interacts with ubiquitin chains through its N-terminal zinc finger region. Fragments of the zinc finger region and different ubiquitin chains were produced, and their interactions were studied with ITC and NMR. It was found that two NZF domains of HOIP interact with ubiquitin and that HOIP interacts with Lys48, Lys63 and Met1-linked ubiquitin chains with high affinity but with different reaction stoichiometry, indicating that the binding mode is different between different chain types. Mutation that mimics phosphorylation between the NZF domains of HOIP did not affect binding of Met1-linked ubiquitin. HOIP forms a weak dimer through its B-box domain in vitro. In cells, LUBAC functions as a complex containing dimers of each three subunit, suggesting that something may stabilise HOIP dimer in cells. To study if binding to ubiquitin chains could stabilise HOIP dimer, the structure of the B-box domain was determined by X-ray crystallography. This enabled generation of HOIP mutant that was unable to dimerise. Interestingly, this mutation did not influence binding of HOIP zinc finger region to long linear ubiquitin chains. The results of this thesis provide insight into the ubiquitin interactions and dimerisation of HOIP, and can guide further studies in eventually understanding how LUBAC gets recruited to immune receptor signalling complexes.