| 1. |
- Benz, Caroline
(författare)
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Diving into short linear motifs : Large-scale identification of endogenous and host-pathogen protein-protein interactions and further characterized by deep mutational scanning
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Short linear motifs (SLiMs) are protein-protein interaction sites that play an essential role in distinct cellular processes. Those interactions are challenging to capture by common high-throughput methods. Therefore, we established an improved version of Proteomic Peptide Phage Display (ProP-PD) as a dedicated method to identify SLiM-based interactions. ProP-PD libraries were created for the discovery of endogenous and host-pathogen protein-protein interactions. The M13 bacteriophage libraries present 16 amino acid long peptides from the intrinsically disordered regions (IDRs) of the human (HD2) proteome or the proteomes of RNA viruses (RiboVD). Through benchmarking of the approach using 35 well-known SLiMs binding domains and the HD2 library, we defined parameters for assigning confidence levels to the results. The selections against the HD2 library revealed >2000 SLiMs-based interaction pairs. Regarding host-pathogen interactions, we focused on interactions mediated by coronavirus proteins, exploring how human proteins bind to viral peptides and how viral proteins bind to human SLiMs. By screening more than 130 human bait proteins against the RiboVD, we revealed several host proteins potentially being targeted by SARS-CoV-2 proteins. Viral hijacking of human G3BP1/2 by the N-protein from SARS-CoV-2 impacted stress granule formation, and inhibition of the interaction was found to have an antiviral effect. Using SARS-CoV-2 proteins in selections with our HD2 library, we found that viral proteins may bind host SLiMs. Selected interactions were validated via affinity measurements revealing a wide range of affinities. Finally, we uncovered that a peptide binding to the NSP9 has an antiviral effect. It is not always possible to establish binding determinants directly from ProP-PD derived peptides. Therefore, we developed a deep mutational scanning (DMS) by phage display protocol. To test the approach, we designed libraries in which all amino acid positions of binding peptides were individually mutated, and the effect on binding was investigated through peptide phage selection. The approach was validated against well-studied interactions and applied to SLiM-based interactions between human proteins and SARS-CoV-2 proteins. Based on the DMS by phage display data we could create a higher affinity binder for NSP9 with increased antiviral effects. The research presented in this thesis has established a platform for large-scale interaction screening through phage display. The results contribute to a deeper understanding of the SLiMs binding and function and also pinpoint novel potential targets for the development of antiviral agents.
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| 2. |
- Cragnell, Carolina, et al.
(författare)
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Dynamical Oligomerisation of Histidine Rich Intrinsically Disordered ProteinS Is Regulated through Zinc-Histidine Interactions
- 2019
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Ingår i: Biomolecules. - : MDPI AG. - 2218-273X. ; 9:5
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Tidskriftsartikel (refereegranskat)abstract
- Intrinsically disordered proteins (IDPs) can form functional oligomers and in some cases, insoluble disease related aggregates. It is therefore vital to understand processes and mechanisms that control pathway distribution. Divalent cations including Zn2+ can initiate IDP oligomerisation through the interaction with histidine residues but the mechanisms of doing so are far from understood. Here we apply a multi-disciplinary approach using small angle X-ray scattering, nuclear magnetic resonance spectroscopy, calorimetry and computations to show that that saliva protein Histatin 5 forms highly dynamic oligomers in the presence of Zn2+. The process is critically dependent upon interaction between Zn2+ ions and distinct histidine rich binding motifs which allows for thermodynamic switching between states. We propose a molecular mechanism of oligomerisation, which may be generally applicable to other histidine rich IDPs. Finally, as Histatin 5 is an important saliva component, we suggest that Zn2+ induced oligomerisation may be crucial for maintaining saliva homeostasis.
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| 5. |
- Edström, Anneli, et al.
(författare)
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beta-Microseminoprotein Endows Post Coital Seminal Plasma with Potent Candidacidal Activity by a Calcium- and pH-Dependent Mechanism
- 2012
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 8:4, s. e1002625-
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Tidskriftsartikel (refereegranskat)abstract
- The innate immune factors controlling Candida albicans are mostly unknown. Vulvovaginal candidiasis is common in women and affects approximately 70-75% of all women at least once. Despite the propensity of Candida to colonize the vagina, transmission of Candida albicans following sexual intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as beta-microseminoprotein. At neutral pH, the fungicidal activity of beta-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E-71 was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of beta-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. A homologous fragment from porcine beta-microseminoprotein demonstrated calcium-dependent fungicidal activity in a CFU assay, suggesting this may be a common feature for members of the beta-microseminoprotein family. By electron microscopy, beta-microseminoprotein was found to cause lysis of Candida. Liposome experiments demonstrated that beta-microseminoprotein was active towards ergosterol-containing liposomes that mimic fungal membranes, offering an explanation for the selectivity against fungi. These data identify beta-microseminoprotein as an important innate immune factor active against C. albicans and may help explain the low sexual transmission rate of Candida.
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| 6. |
- Hendus-Altenburger, Ruth, et al.
(författare)
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The intracellular lipid-binding domain of human Na+/H+ exchanger 1 forms a lipid-protein co-structure essential for activity
- 2020
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Ingår i: Communications Biology. - : Nature Publishing Group. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical αα-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pHi and the rate of pHi recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins.
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| 7. |
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| 8. |
- Kristensen, Kristian K., et al.
(författare)
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A disordered acidic domain in GPIHBP1 harboring a sulfated tyrosine regulates lipoprotein lipase
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:26, s. E6020-E6029
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Tidskriftsartikel (refereegranskat)abstract
- The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against. ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant (k(on)) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity.
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| 9. |
- Maxwell, Karen L., et al.
(författare)
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Protein folding : Defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins
- 2005
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 14, s. 602-16
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Tidskriftsartikel (refereegranskat)abstract
- Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25°C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.
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| 10. |
- Niclasen, Louise Meinert, et al.
(författare)
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Streptococcal pyogenic exotoxin B (SpeB) boosts the contact system via binding of alpha-1 antitrypsin
- 2011
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Ingår i: Biochemical Journal. - 0264-6021. ; 434, s. 123-132
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Tidskriftsartikel (refereegranskat)abstract
- The Streptococcus pyogenes cysteine protease SpeB (streptococcal pyrogenic exotoxin B) is important for the invasive potential of the bacteria, but its production is down-regulated following systemic infection. This prompted us to investigate if SpeB potentiated the host immune response after systemic spreading. Addition of Spa to human plasma increased plasma-mediated bacterial killing and prolonged coagulation time through the intrinsic pathway of coagulation. This effect was independent of the enzymatic activity of SpeB and was mediated by a non-covalent medium-affinity binding and modification of the serpin A1AT (alpha-1 antitrypsin). Consequently, addition of A1AT to plasma increased bacterial survival. Sequestration of A1AT by SpeB led to enhanced contact system activation, supported by increased bacterial growth in prekallikrein deficient plasma. In a mouse model of systemic infection, administration of SpeB reduced significantly bacterial dissemination. The findings reveal an additional layer of complexity to host-microbe interactions that may be of benefit in the treatment of severe bacterial infections.
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