Dissertation: ”Exploring conditional motif-based protein interactions in health and disease”

Johanna Kliche defends her doctoral thesis, entitled ”Exploring conditional motif-based protein interactions in health and disease”, within the subject of Biochemistry.

Opponent: Prof. Ulrich Stelzl, University of Graz, Austria

Supervisor: Prof. Ylva Ivarsson, Biochemistry, Department of Chemistry – BMC, Uppsala University

Protein-protein interactions (PPIs) orchestrate a variety of cellular events, ranging from signal transduction, scaffolding to subcellular localisation. A subclass of PPIs is mediated by short linear motifs, which are short amino acid stretches found in the intrinsically disordered regions of the proteome. Regulation of these interactions, which determines which proteins interact, as well as when and where interactions occur, is vital for performing cellular tasks. Phosphorylation can act as cue for this regulation, creating, breaking or fine-tuning a given interaction site. Disease-associated mutations may, in turn, deregulate motif-based PPIs. The Ivarsson lab established proteomic peptide-phage display (ProP-PD) for the discovery of binding peptides and motifs of protein domains. I extended the approach to kinase domains to assess their peptide binding properties and uncovered potential docking interactions of CASK and MAPK8.I further investigated the modulation of motif-based PPIs by phosphorylation guided either by bioinformatic predictions or by phosphomimetic ProP-PD. This led to unravelling of phospho-modulated binding motifs in the cytoplasmic tails of coronavirus host receptors. In addition, I developed an improved phosphomimetic phage library combining the intrinsically disordered regions of the human proteome with functionally prioritised phosphosites. Screening protein domains against the phosphomimetic library suggested novel interaction partners and their phospho-modulation. I demonstrated the dependency of clathrin binding on S839 HURP phosphorylation, which was further found to be required for the mitotic function of HURP. Lastly, I assessed, with the genetic variation phage library, whether mutational ProP-PD is suited to capture changes in motif-based PPIs as a consequence of disease-associated mutation. The method can with high confidence identify PPI-disruptive mutations, such as the P348L SQSTM1 mutation that diminishes binding to KEAP1 and a R157C CDC45 mutation that disrupts the nuclear localisation of CDC45 and its interaction with KPNA7. Together, I have investigated motif-based PPIs in health and disease and probed their identification by (mutational) ProP-PD. Mutational ProP-PD offers the advantage to identify conditional interaction partners, which might be overlooked in conventional ProP-PD experiments.

Link to the thesis in full text in DiVA.

Image of the thesis.