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| 2. |
- Kassa, Eszter, et al.
(författare)
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Evaluation of affinity-purification coupled to mass spectrometry approaches for capture of short linear motif-based interactions
- 2024
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Kassa, Eszter, et al.
(författare)
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Evaluation of affinity-purification coupled to mass spectrometry approaches for capture of short linear motif-based interactions
- 2023
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Ingår i: Analytical Biochemistry. - : Elsevier. - 0003-2697 .- 1096-0309. ; 663
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Tidskriftsartikel (refereegranskat)abstract
- 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 massspectrometry (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 model peptides that successfully pulled down known interactors 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.
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| 4. |
- Kassa, Eszter
(författare)
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Exploration of analytical methods to study motif-mediated host-virus protein-protein interactions
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 5. |
- Mihalic, Filip, et al.
(författare)
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Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- The virus life cycle depends on host-virus protein-protein interactions, which often involve a disordered protein region binding to a folded protein domain. 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 281 peptides from human proteins, and affinities of 31 interactions involving eight SARS-CoV-2 protein domains were determined (KD ∼ 7-300 μM). Key specificity residues of the peptides were established for six of the interactions. Two of the peptides, binding Nsp9 and Nsp16, respectively, inhibited viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously identify potential host-virus interactions and peptides with antiviral properties. Furthermore, the high number of low-affinity interactions suggest that overexpression of viral proteins during infection may perturb multiple cellular pathways.
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| 6. |
- Mihalic, Filip, et al.
(författare)
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Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs
- 2022
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Ingår i: Nature Communications. - : Cold Spring Harbor Laboratory. - 2041-1723.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 7. |
- Mihalič, Filip, et al.
(författare)
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Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- 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 1712 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.
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| 8. |
- Valdés, Alberto, et al.
(författare)
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Proteomic comparison between different tissue preservation methods for identification of promising biomarkers of urothelial bladder cancer
- 2021
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1-non-muscle invasive bladder cancer (NMIBC), and T2/T3-muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers.
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