Biology subjects

Kassa, E.

Publications and source records attributed to Kassa, E..

3 recordsLinked to original sources

bioRxiv2022

Evaluation of affinity-purification coupled to mass spectrometry approaches for capture of short linear motif-based interactions

Low affinity and transient protein-protein interactions, such as short linear motif (SLiM)-based interactions, require dedicated experimental tools for discovery and validation. Here, we evaluated and compared biotinylated peptide pulldown and protein interaction screen on peptide matrix (PRISMA) coupled to mass-spectrometry (MS) using a set of peptides containing interaction motifs. Eight different peptide sequences that engage in interactions with three distinct protein domains (KEAP1 Kelch, MDM2 SWIB, and TSG101 UEV) with a wide range of affinities were tested. We found that peptide pulldown can be an effective approach for SLiM validation, however, parameters such as protein abundance and competitive interactions can prevent the capture of known interactors. The use of tandem peptide repeats improved the capture and preservation of some interactions. When testing PRISMA, it failed to provide comparable results for a model peptide that successfully pulled down a known interactor using biotinylated peptide pulldown. Overall, in our hands, we find that albeit more laborious, biotin-peptide pulldown was more successful in terms of validation of known interactions. Our results highlight that the tested affinity-capture MS-based methods for validation of SLiM-based interactions from cell lysates are suboptimal, and we identified parameters for consideration for method development.

biochemistry↗

bioRxiv2022

Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets.

The infection and replication cycle of all viruses depend on interactions between viral and host proteins. Each of these protein-protein interactions is therefore a potential drug target. These host-virus interactions often involve a disordered protein region on one side of the interface and a folded protein domain on the other. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind peptides from human proteins. Of 281 high/medium confidence peptides, 23 interactions involving eight SARS-CoV-2 protein domains were tested by fluorescence polarization, and binding was observed with affinities spanning the whole micromolar range. The key specificity determinants were established for six of these domains, two based on ProP-PD and four by alanine scanning SPOT arrays. Finally, two cell-penetrating peptides, targeting Nsp9 and Nsp16, respectively, were shown to function as inhibitors of viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously provide information on potential host-virus interactions and identify ligands with antiviral properties.

molecular biology↗

bioRxiv2022

Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs

Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1,712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics.

biochemistry↗