| 1. |
- Ali, Muhammad, 1990-, et al.
(författare)
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Elucidation of Short Linear Motif-Based Interactions of the FERM Domains of Ezrin, Radixin, Moesin, and Merlin
- 2023
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 62:11, s. 1594-1607
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Tidskriftsartikel (refereegranskat)abstract
- The ERM (ezrin, radixin,and moesin) family of proteins and therelated protein merlin participate in scaffolding and signaling eventsat the cell cortex. The proteins share an N-terminal FERM [band four-point-one(4.1) ERM] domain composed of three subdomains (F1, F2, and F3) withbinding sites for short linear peptide motifs. By screening the FERMdomains of the ERMs and merlin against a phage library that displayspeptides representing the intrinsically disordered regions of thehuman proteome, we identified a large number of novel ligands. Wedetermined the affinities for the ERM and merlin FERM domains interactingwith 18 peptides and validated interactions with full-length proteinsthrough pull-down experiments. The majority of the peptides containedan apparent Yx-[FILV] motif; others show alternative motifs. We defineddistinct binding sites for two types of similar but distinct bindingmotifs (YxV and FYDF) using a combination of Rosetta FlexPepDock computationalpeptide docking protocols and mutational analysis. We provide a detailedmolecular understanding of how the two types of peptides with distinctmotifs bind to different sites on the moesin FERM phosphotyrosinebinding-like subdomain and uncover interdependencies between the differenttypes of ligands. The study expands the motif-based interactomes ofthe ERMs and merlin and suggests that the FERM domain acts as a switchableinteraction hub.
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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. |
- Ali, Muhammad, 1990-, et al.
(författare)
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Integrated analysis of Shank1 PDZ interactions with C-terminal and internal binding motifs
- 2021
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Ingår i: Current Research in Structural Biology. - : Elsevier. - 2665-928X. ; 3, s. 41-50
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Tidskriftsartikel (refereegranskat)abstract
- PDZ domains constitute a large family of modular domains that are well-known for binding C-terminal motifs of target proteins. Some of them also bind to internal PDZ binding motifs (PDZbms), but this aspect of the PDZ interactome is poorly studied. Here we explored internal PDZbm-mediated interactions using the PDZ domain of Shank1 as a model. We identified a series of human Shank1 ligands with C-terminal or internal PDZbms using proteomic peptide-phage display, and established that while the consensus sequence of C-terminal ligands is x-T-x-(L/F)-COOH, the consensus of internal PDZbm is exclusively x-T-x-F-x, where x is any amino acid. We found that the affinities of PDZbm interactions are in the low micromolar range. The crystal structure of the complex between Shank1 PDZ and an internal PDZbm revealed that the binding mode of internal PDZbms was similar to that of C-terminal ligands. Pull-down experiments confirmed that both C-terminal and internal PDZbm interactions can occur in the context of full-length proteins. Our study expands the interactome of Shank1 and hints at a largely unexplored interaction space of PDZ domains.
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| 4. |
- Benz, Caroline, et al.
(författare)
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Proteome-scale mapping of binding sites in the unstructured regions of the human proteome
- 2022
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Ingår i: Molecular Systems Biology. - : EMBO Press. - 1744-4292 .- 1744-4292. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Specific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type-specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide-phage display library that tiles all disordered regions of the human proteome and allows the screening of similar to 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)-binding domains and confirmed the quality of the produced data by complementary biophysical or cell-based assays. Finally, we show how the amino acid resolution-binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteomewide discovery of SLiM-based interactions.
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| 5. |
- Figalova, Nikol, et al.
(författare)
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Methodological Framework for Modelling and Empirical Approaches (Deliverable D1.1 in the H2020 MSCA ITN project SHAPE-IT)
- 2021
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- The progress in technology development over the past decades, both with respect to software and hardware, offers the vision of automated vehicles as means of achieving zero fatalities in traffic. However, the promises of this new technology – an increase in road safety, traffic efficiency, and user comfort – can only be realized if this technology is smoothly introduced into the existing traffic system with all its complexities, constraints, and requirements. SHAPE- IT will contribute to this major undertaking by addressing research questions relevant for the development and introduction of automated vehicles in urban traffic scenarios. Previous research has pointed out several research areas that need more attention for a successful implementation and deployment of human-centred vehicle automation in urban environments. In SHAPE-IT, for example, a better understanding of human behaviour and the underlying psychological mechanisms will lead to improved models of human behaviour that can help to predict the effects of automated systems on human behaviour already during system development. Such models can also be integrated into the algorithms of automated vehicles, enabling them to better understand the human interaction partners’ behaviours. Further, the development of vehicle automation is much about technology (software and hardware), but the users will be humans and they will interact with humans both inside and outside of the vehicle. To be successful in the development of automated vehicles functionalities, research must be performed on a variety of aspects. Actually, a highly interdisciplinary team of researchers, bringing together expertise and background from various scientific fields related to traffic safety, human factors, human-machine interaction design and evaluation, automation, computational modelling, and artificial intelligence, is likely needed to consider the human-technology aspects of vehicle automation. Accordingly, SHAPE-IT has recruited fifteen PhD candidates (Early Stage Researchers – ESRs), that work together to facilitate this integration of automated vehicles into complex urban traffic by performing research to support the development of transparent, cooperative, accepted, trustworthy, and safe automated vehicles. With their (and their supervisors’) different scientific background, the candidates bring different theoretical concepts and methodological approaches to the project. This interdisciplinarity of the project team offers the unique possibility for each PhD candidate to address research questions from a broad perspective – including theories and methodological approaches of other interrelated disciplines. This is the main reason why SHAPE-IT has been funded by the European Commission’s Marie Skłodowska-Curie Innovative Training Network (ITN) program that is aimed to train early state researchers in multidisciplinary aspects of research including transferable skills. With the unique scope of SHAPE-IT, including the human-vehicle perspective, considering different road-users (inside and outside of the vehicle), addressing for example trust, transparency, and safety, and including a wide range of methodological approaches, the project members can substantially contribute to the development and deployment of safe and appreciated vehicle automation in the cities of the future. To achieve the goal of interdisciplinary research, it is necessary to provide the individual PhD candidate with a starting point, especially on the different and diverse methodological approaches of the different disciplines. The empirical, user-centred approach for the development and evaluation of innovative automated vehicle concepts is central to SHAPE- IT. This deliverable (D1.1 “Methodological Framework for Modelling and Empirical Approaches”) provides this starting point. That is, this document provides a broad overview of approaches and methodologies used and developed by the SHAPE-IT ESRs during their research. The SHAPE-IT PhD candidates, as well as other researchers and developers outside of SHAPE-IT, can use this document when searching for appropriate methodological approaches, or simply get a brief overview of research methodologies often employed in automated vehicle research. The first chapter of the deliverable shortly describes the major methodological approaches to collect data relevant for investigating road user behaviour. Each subchapter describes one approach, ranging from naturalistic driving studies to controlled experiments in driving simulators, with the goal to provide the unfamiliar reader with a broad overview of the approach, including its scope, the type of data collected, and its limitations. Each subchapter ends with recommendations for further reading – literature that provide much more detail and examples. The second chapter explains four different highly relevant tools for data collection, such as interviews, questionnaires, physiological measures, and as other current tools (the Wizard of Oz paradigm and Augmented and Virtual Reality). As in the first chapter this chapter provides the reader with information about advantages and disadvantages of the different tools and with proposed further readings. The third chapter deals with computational models of human/agent interaction and presents in four subchapters different modelling approaches, ranging from models based on psychological mechanisms, rule-based and artificial intelligence models to simulation models of traffic interaction. The fourth chapter is devoted to Requirements Engineering and the challenge of communicating knowledge (e.g., human factors) to developers of automated vehicles. When forming the SHAPE-IT proposal it was identified that there is a lack of communication of human factors knowledge about the highly technical development of automated vehicles. This is why it is highly important that the SHAPE-IT ESRs get training in requirement engineering. Regardless of the ESRs working in academia or industry after their studies it is important to learn how to communicate and disseminate the findings to engineers. The deliverable ends with the chapter “Method Champions”. Here the expertise and association of the different PhD candidates with the different topics are made explicit to facilitate and encourage networking between PhDs with special expertise and those seeking support, especially with regards to methodological questions.
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| 6. |
- Javed, Rida, et al.
(författare)
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Enhancement mechanism of P dopant on atomically distributed FeN 4 P-C electrocatalyst over a wide pH range
- 2022
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Ingår i: Electrochimica Acta. - : Elsevier BV. - 0013-4686. ; 436
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Tidskriftsartikel (refereegranskat)abstract
- Heteroatom doping plays an essential role in improving the catalytic performance of electrocatalysts for oxygen reduction reaction (ORR). However, how to regulate heteroatom doping remains a significant challenge. This paper develops an efficient strategy by using a novel versatile chelating ligand to enhance P loading and expose more metal single Fe atom active sites of FeN4P-C catalyst. The electron distribution of active center is considerably changed by P doping, which significantly influences the catalytic ORR performance. The dopant P in the FeN4P-C catalyst induces a small number of d-electrons from t2g-orbitals around the Fermi level, making the interaction between Fe active site and O2 slightly more robust than in the FeN4[sbnd]C catalyst, as studied by DFT calculations. The as-prepared FeN4P-C catalyst exhibits excellent catalytic ORR activity in both acidic (with a half-wave potential of 0.760 V vs. RHE) and basic (with a half-wave potential of 0.885 V vs. RHE) conditions, which are superior to those of the commercial Pt/C (20 wt%) catalyst. Furthermore, this catalyst also demonstrates outstanding stability and good hydrogen peroxide and methanol tolerance. A Zinc-air battery(ZAB) assembled using the cathode catalyst has validated the high performance of this catalyst. This study provides an efficient method for generating well-defined single-atom active sites to improve catalytic performance and paves the way to identify coordinated single metal atom sites for electrocatalysis applications.
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| 7. |
- Kliche, Johanna, et al.
(författare)
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Cytoplasmic short linear motifs in ACE2 and integrin beta(3) link SARS-CoV-2 host cell receptors to mediators of endocytosis and autophagy
- 2021
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 14:665
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Tidskriftsartikel (refereegranskat)abstract
- The spike protein of SARS-CoV-2 binds the angiotensin-converting enzyme 2 (ACE2) on the host cell surface and subsequently enters host cells through receptor-mediated endocytosis. Additional cell receptors may be directly or indirectly involved, including integrins. The cytoplasmic tails of ACE2 and integrins contain several predicted short linear motifs (SLiMs) that may facilitate internalization of the virus as well as its subsequent propagation through processes such as autophagy. Here, we measured the binding affinity of predicted interactions between SLiMs in the cytoplasmic tails of ACE2 and integrin beta(3) with proteins that mediate endocytic trafficking and autophagy. We validated that a class I PDZ-binding motif mediated binding of ACE2 to the scaffolding proteins SNX27, NHERF3, and SHANK, and that a binding site for the clathrin adaptor AP2 mu 2 in ACE2 overlaps with a phospho-dependent binding site for the SH2 domains of Src family tyrosine kinases. Furthermore, we validated that an LC3-interacting region (LIR) in integrin beta(3) bound to the ATG8 domains of the autophagy receptors MAP1LC3 and GABARAP in a manner enhanced by LIR-adjacent phosphorylation. Our results provide molecular links between cell receptors and mediators of endocytosis and autophagy that may facilitate viral entry and propagation.
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| 8. |
- 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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| 9. |
- 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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