| 1. |
- Hjelm, Linnea C., 1993-
(författare)
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Development of new affinity proteins for neurodegenerative disorders
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neurodegenerative disorders include a full spectrum of diagnoses, including dementias and other neuronal diseases, characterised by degradation of neurons in the brain occurring along with disease progression. Amongst the dementias, the most prevalent are Alzheimer’s (AD) and Parkinson’s disease (PD) that affect millions of people worldwide. During the last years, advancements in potential treatments have been made where the first two clinical antibodies have been approved by the US Food and Drug Administration (FDA) for a disease modifying effect on Alzheimer’s disease.As alternatives to antibodies, other types of affinity reagents that are based on non-immunoglobulin protein scaffolds are also investigated. Such alternative scaffolds often demonstrate distinct and complementary properties compared to antibodies. In this thesis, the development of a new type of affinity protein scaffold called sequestrin is described. Sequestrins are derived from the affibody molecule and comprise two heterogenic subunits with truncated N-terminals fused as a head-to-tail construct. Sequestrins undergo a structural rearrangement upon target binding and forms a stabile complex. The scaffold is designed for interactions with disease-related amyloidogenic peptides e.g. amyloid beta and alpha-synuclein involved in AD and PD, respectively. In the first paper, a sequestrin library was developed and its compatibility with phage display was investigated. Successful panning against the amyloid beta peptide resulted in binders with high affinity. Further on in paper II, the alpha-synuclein peptide was targeted and sequestrins with low nanomolar affinities were obtained. All sequestrins displayed structural rearrangement upon target engagement, which stabilized the interaction to the target peptides and further inhibited toxic aggregation, opening up for future studies of disease modifying effects in vivo.When targeting the brain, passage through the blood–brain barrier (BBB) is an obstacle that needs to be addressed to reach sufficiently high therapeutic concentrations. To overcome this barrier, brain shuttles have been developed with the capability to transport a cargo over the BBB. One such mechanism of transportation is by receptor-mediated transcytosis, which is utilized by e.g. the transferrin receptor (TfR). In paper III, a TfR-targeting shuttle was investigated for BBB passage when fused to a sequestrin targeting the amyloid beta peptide, resulting in a higher penetration through the BBB, and maintained functionality of the sequestrin.High-throughput in vitro methods would facilitate development of novel brain shuttles. Thus, in paper IV, a transwell system based on nanofibrillar silkmembranes with murine brain endothelial cells was developed. Evaluation of the method using a TfR-specific antibody demonstrated higher transfer over the barrier compared to an isotype control and the method has potential to facilitate screening of transcytosis capability of brain shuttles.In paper V, TfR-specific affibody-based brain shuttles were developed and investigated for transcytosis capability using the in vitro transcytosis assay. A panel of affibody molecules were evaluated, demonstrating both cross-species reactivity to murine and human TfR and active receptor-mediated transcytosis. These candidates could thus potentially be used in further development of CNS-targeting therapeutics.In conclusion, a new sequestrin scaffold was developed that can be utilised for targeting amyloidogenic peptides found in neurodegenerative disorders. An affibody-based brain shuttle was also developed, which showed transcytosis capability. In the future, the new brain shuttle might be combined with sequestrins to create multifunctional fusion proteins for facilitated delivery over the BBB, which hopefully can result in therapeutic concentrations in the brain even when administered with a lower dosage.
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| 2. |
- Ali, Muhammad, 1990-
(författare)
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Identification of SLiMs: Mapping and characterizing motif-based protein interactions
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During the last twenty years it has become evident that about 35-40% of amino acids in the proteome are in regions that have evolved to remain unstructured. These intrinsically disordered regions contain short linear motifs (SLiMs), which serve as docking sites for protein-protein interactions. SLiMs often mediate low-to-medium affinity interactions that are transient in their nature. The characteristics of SLiM-based interactions make them difficult to be captured using conventional approaches like affinity-purification coupled to mass spectrometry or yeast-two-hybrid. We therefore used and developed a dedicated method for large-scale screening of SLiM-based interactions termed proteomic peptide phage display (ProP-PD).Using ProP-PD, We identified large sets of ligands, for the binding pocket of shank1 PDZ domain, containing C-terminal or internal binding motifs and established the consensus motifs to be xTxL/F-COOH and xTxFx respectively. We further validated interactions using biophysical affinity determinations and pulldown experiments. Using X-ray crystallization, we uncovered that shank1 PDZ binds to internal xTxFx motifs using a binding mode similar to that for C-terminal peptides.Adding a level of complexity, we explored interactions of the multiple binding pocket containing FERM domains from four closely related proteins: ezrin, radixin, moesin and merlin. We found hundreds of FERM ligands, which contained binding motifs of at least four different classes. By combining docking simulations with experiments, we established ligands binding to different pockets, and uncovered a complex interplay between distinct pockets.We further developed an optimized version of a phage library that displays intrinsically disordered regions of the human proteome. We benchmarked the library using a set of protein domains and reported better recovery of known SLiM-based interactions. Furthermore, we highlighted the functional aspects of identified SLiMs, in the case of nuclear localization signals, found for binding to importin-subunit alpha-3. Finally, we validated predicted binding of SLiMs in the Sars-CoV-2 host receptor ACE2, which illustrates the importance of fundamental knowledge for SLiMs and their binding partners.This work, taken together, contributes with method development for expansion of motifs based interactomes and provide insights into the plastic yet selective nature of peptide binding proteins.
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| 3. |
- Kliche, Johanna, 1993-
(författare)
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Exploring conditional motif-based protein interactions in health and disease
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein-protein interactions (PPIs) orchestrate a variety of cellular events, ranging from signal transduction, scaffolding to subcellular localisation. A subclass of PPIs is mediated by short linear motifs, which are short amino acid stretches found in the intrinsically disordered regions of the proteome. Regulation of these interactions, which determines which proteins interact, as well as when and where interactions occur, is vital for performing cellular tasks. Phosphorylation can act as cue for this regulation, creating, breaking or fine-tuning a given interaction site. Disease-associated mutations may, in turn, deregulate motif-based PPIs. The Ivarsson lab established proteomic peptide-phage display (ProP-PD) for the discovery of binding peptides and motifs of protein domains. I extended the approach to kinase domains to assess their peptide binding properties and uncovered potential docking interactions of CASK and MAPK8.I further investigated the modulation of motif-based PPIs by phosphorylation guided either by bioinformatic predictions or by phosphomimetic ProP-PD. This led to unravelling of phospho-modulated binding motifs in the cytoplasmic tails of coronavirus host receptors. In addition, I developed an improved phosphomimetic phage library combining the intrinsically disordered regions of the human proteome with functionally prioritised phosphosites. Screening protein domains against the phosphomimetic library suggested novel interaction partners and their phospho-modulation. I demonstrated the dependency of clathrin binding on S839 HURP phosphorylation, which was further found to be required for the mitotic function of HURP. Lastly, I assessed, with the genetic variation phage library, whether mutational ProP-PD is suited to capture changes in motif-based PPIs as a consequence of disease-associated mutation. The method can with high confidence identify PPI-disruptive mutations, such as the P348L SQSTM1 mutation that diminishes binding to KEAP1 and a R157C CDC45 mutation that disrupts the nuclear localisation of CDC45 and its interaction with KPNA7. Together, I have investigated motif-based PPIs in health and disease and probed their identification by (mutational) ProP-PD. Mutational ProP-PD offers the advantage to identify conditional interaction partners, which might be overlooked in conventional ProP-PD experiments.
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| 4. |
- 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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| 5. |
- Ivarsson, Ylva, 1976-
(författare)
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Evolutionary Analysis and Posttranslational Chemical Modifications in Protein Redesign : A Study on Mu Class Glutathione Transferases
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates.Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity.To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.
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| 6. |
- 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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| 7. |
- Nyqvist, Ida, 1991-
(författare)
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Biophysical characterization of protein-protein interactions involving intrinsically disordered proteins
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Intrinsically disordered proteins and regions (IDPs/Rs) are proteins that do not form stable and well-defined structures in their free states but rather occupy an ensemble of conformations that change over time while still staying functional. They are prevalent in the eukaryotic proteome and are involved in various vital processes in the cell where they often interact with their binding partners through coupled binding and folding reactions. The knowledge on the molecular details of these interactions is still limited as is the role of dynamics and conformational entropy changes. In this thesis binding interactions between IDPs and a folded protein domain have been studied in more detail. The rate-limiting transition states (TS) of binding have been examined using kinetic experiments and protein engineering (F-value analysis), and the picosecond to nanosecond backbone and side-chain dynamics of these interactions have been studied with nuclear magnetic resonance (NMR) spectroscopy. To study these properties the globular TAZ1 domain of the CREB binding protein (CBP) and three of its interaction partners, the disordered transactivation domains of STAT2, HIF-1a and RelA have been selected. At the rate limiting transition states of binding for TAZ1/TAD-STAT2 and TAZ1/CTAD-HIF-1a native hydrophobic binding contacts are largely absent. These interactions are instead formed cooperatively after passing the rate-limiting barrier. The results from the backbone and side-chain dynamic studies show that the internal motions for both binding partners are significantly affected by the interactions. Changes in dynamics upon binding correspond to conformational entropy changes that contribute significantly to the binding thermodynamics, and are in the same order of magnitude as the binding enthalpy. Additionally, the conformational entropy changes for TAZ1 vary when binding to the different IDPs, demonstrating the importance of conformational entropy. In conclusion, this work contributes to the understanding of the nature of binding interactions involving intrinsically disordered proteins.
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