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Proteins are responsible for countless processes in living creatures, but most often they do not perform these tasks alone. Rather, they engage in interactions with other proteins, creating whole protein-protein interaction (PPI) networks. Some of these interactions are formed between a folded protein domain and a short linear motif (SLiM), which is a small, 3-10 amino acid long stretch usually in the intrinsically disordered regions of proteins. These interactions tend to be low-to-medium affinity and transient, therefore their capture requires special tools. Furthermore, viruses often hijack the human cellular machinery through PPIs as they have limited genomes and are obligate cellular parasites. Therefore, the investigation of viral-host PPIs is of great importance and can lead to the development of novel antivirals.

In my thesis, I used mostly peptide-based and mass spectrometry (MS) techniques to validate and further explore motif-based PPIs. The main objectives were to: i) evaluate and compare synthetic peptide-based pulldown approaches, ii) validate and further explore the interaction between viral peptides and human polyadenylate-binding protein (PABP) using green fluorescent protein (GFP)-tagged peptide repeats, iii) confirm interactions, define and refine human interaction motifs that engage in interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins by employing peptide SPOT (synthetic peptide arrays on membrane support technique) arrays and alanine scanning, iv) investigate the change in the interactome of the nuclear pore complex protein 153 (NUP153) between uninfected and tick-borne encephalitis virus (TBEV)-infected states using GFP-tagged full-length protein for pulldown.

First, we explored the potential of affinity purification-mass spectrometry (AP-MS) and protein interaction screen on peptide matrix (PRISMA) to capture SLiM-based PPIs. The peptide pulldown approach proved to be more applicable over a wide range of affinities and interactions, however, protein concentration and the local concentration of presented motifs were limiting factors in certain cases. We then investigated SLiM-based interactions between RNA-viruses and human proteins. Here, using green fluorescent-peptide pulldowns I confirmed the interaction between viral peptides and the human poly-A binding protein. Next, we uncovered that some human SLiMs interact with SARS-CoV-2 proteins, and I was able to highlight the interaction motif using peptide arrays when only a handful of peptides were available. Lastly, I identified different enriched proteins in NUP153-pulldowns from mock-infected and TBEV-infected cell lysate, that were complementary to the changes observed with other techniques.

In conclusion, I explored a range of techniques that are valuable in the validation of PPIs, which is crucial in combination with high-throughput approaches. As more and more SLiM-based interactions are explored and predicted, the value of these tools continues to increase.