| 1. |
- Åberg, Emma
(författare)
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Evolution of Interactions Involving Intrinsically Disordered Proteins
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the evolution of intrinsically disordered proteins and their interaction partners. The work presented is a combination of phylogenetic analysis, ancestral reconstruction and biophysical characterization in order to examine the evolutionary trajectory of protein-protein interactions involving disorder. The intrinsically disordered domains, NCBD and CID are both part of transcriptional co-regulating proteins. In evolution, NCBD existed before the emergence of CID and the most ancient domains display a low affinity complex with many weak contacts and high degree of conformational heterogeneity. Later in evolution, when NCBD and CID co-exists, a few mutations have altered the interaction in a way that the affinity is increased 25-fold and the conformational heterogeneity is decreased. In the same manner, the interaction is further optimized in extant species, resulting in a high affinity complex with less contacts of higher strength and less conformational heterogeneity. The intrinsically disordered transactivation domain of the tumour suppressing protein p53 and its negative regulator MDM2 date back to the beginning of animal life. The interacting domains are either lost or conserved in distinct phyla indicating a tight co-evolution. Phylogenetic trees produced by only including phyla with a conserved interaction domain follow the species evolution. Resurrection of p53 and MDM2 in the vertebrate lineage display an evolution of a high affinity complex in the ancestor of fish and tetrapods to a slightly improved affinity in modern tetrapods but a substantially lower affinity in zebrafish. The p53 protein family, which also includes p63 and p73, diverged from a common ancestor. The individual proteins display altered affinities to MDM2 which is a result of the high sequence divergence between them. The ionic dependence for the interactions is small, and not in line with other studies of disordered proteins. In conclusion, the work in this thesis have contributed with evolutionary analysis and experimental data of interactions involving intrinsically disordered proteins.
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| 2. |
- Kruse, Thomas, et al.
(författare)
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Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities
- 2021
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, most current large-scale methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a viral peptide discovery approach covering 23 coronavirus strains that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an ΦxFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its ΦxFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction dampened SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.
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| 3. |
- Gustafsson, Robert, et al.
(författare)
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Structure and Characterization of Phosphoglucomutase 5 from Atlantic and Baltic Herring : An Inactive Enzyme with Intact Substrate Binding
- 2020
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Ingår i: Biomolecules. - : MDPI AG. - 2218-273X. ; 10:12
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Tidskriftsartikel (refereegranskat)abstract
- Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, Clupea harengus, PGM5 is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, diering by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to dierences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.
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| 4. |
- Yang, Fan, 1988-
(författare)
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Biophysical chemistry of the ALS-associated protein SOD1 : Implications for folding, aggregation and in-cell behaviour
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biophysical chemistry deals with the structural behavior, properties and molecular function of biological macromolecules. A long-standing challenge is here to establish how these macromolecular features change upon transfer from simplified conditions in vitro to the crowded and molecularly complex environment of live cells. This thesis focuses on establishing a general overview of the structural behavior and interaction properties of the ALS-associated protein superoxide dismutase 1 (SOD1) in its natural cellular environment. Importantly, SOD1 constitutes also a multifaceted model system for the yet poorly understood mechanism of protein-aggregation disease, since it is readily amenable to protein-engineering analysis. The focus is on (i) SOD1 folding, (ii) the modulation of the SOD1 properties induced by intracellular interactions and (iii) the process of SOD1 fibrillation, all of which central to the understanding of the ALS disease mechanism. First, we investigate the biophysical role of the disordered catalytic loops in the apoSOD1 monomer, what is identified as the primary aggregation precursor. The results show that these loops play a pivotal role in modulation the apoSOD1 stability due to the generic Flory-entropy penalty, shedding new light to why this species is biased to be aggregation prone. Second, we target the diffusive interactions between SOD1 and the crowded intracellular environment by in-cell NMR. Our findings are that both the rotational tumbling and in-cell stability are controlled by basic physicochemical rules relating to the SOD1 surface properties. Finally, we analyze the kinetics of the SOD1-aggregation behavior in vitro. The observations confirm that the disordered SOD1 loops indeed accelerate the aggregation process because of their penalty to the apo state stability and show, additionally, that they influence the fibril stability.The physicochemical cues exposed by this thesis work provide not only fundamental clues to our understanding of protein properties, but shed also new light on disease-promoting properties ALS-associated protein SOD1.
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| 5. |
- Hultqvist, Greta, 1980-
(författare)
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Protein Folding, Binding and Evolution : PDZ domains and paralemmins as model systems
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins present at the synapse need to be multitasking in order to perform all vital functions in this limited space. In this thesis I have analyzed the function and evolution of such proteins, focusing on the PDZ domain and the paralemmin family. The PDZ domains bind to a wide variety of interaction partners. The affinity for each partner is regulated by residues at the binding site, but also through intradomain allostery. How this intradomain allostery is transferred to the binding site is not established. I here show that side chain interactions can explain all transfer of intradomain allostery in three analyzed PDZ domains. A circularly permuted PDZ domain has an identical set of amino acids as the original protein and a very similar structure with only a few perturbed side chains. By using the circular permutant I show that a slight alteration in the position of a side chain leads to a corresponding change in allosteric signal. I further study the folding of several PDZ domains and show that they all fold via a conserved folding mechanism, supporting the notion that the final structure has a part in deciding folding mechanism. The folding mechanism of the circularly permuted PDZ domain is conserved compared to the original protein illustrating how circular permutations can be tolerated through evolution. The multifunctionality of paralemmins probably lies in their highly flexible structures. I have studied the evolution of the paralemmins and found that the four mammalian paralemmins arose in the two whole-genome duplications that occurred early in the vertebrate evolution. The fact that all four paralemmins have survived evolution since the gene duplications suggests that they have important functions, possibly in the development of the nervous system. Synaptic proteins are crucial for many biological processes, and their misfolding implicated in many diseases. The results presented here shed light on the mechanisms of action of the synaptic proteins and will help us to understand how they generate disease.
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| 6. |
- Eklund, Sandra, et al.
(författare)
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Characterization of the endopeptidase activity of tripeptidyl-peptidase II
- 2012
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 424:3, s. 503-507
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Tidskriftsartikel (refereegranskat)abstract
- Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.
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| 7. |
- Mihalič, Filip, 1992-
(författare)
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Evolution and viral mimicry of short linear motif-mediated interactions
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are one of the most fundamental building blocks of life and their interactions regulate every cellular process. Historically they have been conceptualized as predominantly folded entities with well-defined secondary and tertiary structures. However, in recent decades, up to 50% of the human proteome has been shown to contain long disordered sequences that are flexible and unstructured in their natural environment. These intrinsically disordered regions exhibit low levels of sequence conservation and are enriched in short linear motifs (SLiMs). SLiMs are typically less then 10 amino acids long and serve as docking sites recognized by various globular domains. They exhibit a high degree of sequence degeneracy and evolutionary plasticity, allowing for rapid de novo emergence. SLiMs play crucial roles in a variety of cellular processes including cellular signalling, trafficking, transcriptional modulation, and protein degradation. Because they are small and degenerate, located in disordered regions, and form relatively weak interactions, they are difficult to identify using conventional high-throughput methods such as mass spectrometry and the yeast two-hybrid system. The same attributes that make them difficult to identify also make them ideal targets for viral SLiM mimicry, of which several examples have been described to date.To address the elusive nature of SLiMs, we have developed a novel approach for the discovery of motif-mediated interactions at the proteome scale using proteomic peptide phage display. We constructed two separate phage libraries with either human or viral disordered regions displayed on their surface. These libraries were then subjected to phage display selections against over 300 globular domains, resulting in the identification of more than 1,700 potential novel interactions. We validated a subset of these interactions with affinity measurements and GST-pulldown assays, solved the crystal structure of human globular domains in complex with viral linear motifs, and demonstrated that the gained knowledge can be applied to design peptidomimetic inhibitors of viral replication. In addition, we demonstrated that direct binding of viral SLiMs to the N-terminal domain of clathrin disrupts cellular trafficking and identified the C-terminal domain of polyadenylate-binding protein 1 as a novel target for viral SLiM mimicry. Furthermore, we demonstrated that SARS-CoV-2 viral proteins possess both SLiMs that bind to human proteins, and globular domains that recognize human SLiMs, showcasing the versatility of SLiM-mediated interactions. Finally, we examined the evolutionary trajectory of the interaction between the SWIB domain of MDM2 and the SLiM of p53 TAD to describe an example of the extraordinary evolutionary plasticity of SLiM-mediated interactions.Overall, the research presented in this thesis created the basis for an atlas of human motif-mediated interactions, yielded an extensive dataset of potential and validated cases of viral SLiM mimicry, and expanded our understanding of motif-mediated interactions from an evolutionary perspective.
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| 8. |
- 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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| 9. |
- 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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| 10. |
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