88 results on '"Ivarsson Y"'
Search Results
2. Clathrin N-terminal domain in complex with a HURP phospho-peptide
- Author
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Kliche, J., primary, Badgujar, D., additional, Dobritzsch, D., additional, and Ivarsson, Y., additional
- Published
- 2023
- Full Text
- View/download PDF
3. PP4R3A EVH1 domain bound to FxxP motif
- Author
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Ueki, Y., primary, Kruse, T., additional, Weisser, M.B., additional, Sundell, G.N., additional, Yoo Larsen, M.S., additional, Lopez Mendez, B., additional, Jenkins, N.P., additional, Garvanska, D.H., additional, Cressey, L., additional, Zhang, G., additional, Davey, N., additional, Montoya, G., additional, Ivarsson, Y., additional, Kettenbach, A., additional, and Nilsson, J., additional
- Published
- 2019
- Full Text
- View/download PDF
4. Frizzled 7 and PIP₂ binding by syntenin PDZ₂ domain supports Frizzled 7 trafficking and signalling
- Author
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Egea-Jimenez, AL, Gallardo, R, Garcia-Pino, A, Ivarsson, Y, Wawrzyniak, AM, Kashyap, R, Loris, R, Schymkowitz, J, Rousseau, F, and Zimmermann, P
- Abstract
PDZ domain-containing proteins work as intracellular scaffolds to control spatio-temporal aspects of cell signalling. This function is supported by the ability of their PDZ domains to bind other proteins such as receptors, but also phosphoinositide lipids important for membrane trafficking. Here we report a crystal structure of the syntenin PDZ tandem in complex with the carboxy-terminal fragment of Frizzled 7 and phosphatidylinositol 4,5-bisphosphate (PIP₂). The crystal structure reveals a tripartite interaction formed via the second PDZ domain of syntenin. Biophysical and biochemical experiments establish co-operative binding of the tripartite complex and identify residues crucial for membrane PIP₂-specific recognition. Experiments with cells support the importance of the syntenin–PIP₂ interaction for plasma membrane targeting of Frizzled 7 and c-jun phosphorylation. This study contributes to our understanding of the biology of PDZ proteins as key players in membrane compartmentalization and dynamics.
- Published
- 2016
5. Crystal structure of the syntenin PDZ1 and PDZ2 tandem in complex with the Frizzled 7 C-terminal fragment and PIP2
- Author
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Egea-Jimenez, A.L., primary, Gallardo, R., additional, Garcia-Pino, A., additional, Ivarsson, Y., additional, Wawrzyniak, A.M., additional, Kashyap, R., additional, Loris, R., additional, Schymkowitz, J., additional, Rousseau, F., additional, and Zimmermann, P., additional
- Published
- 2016
- Full Text
- View/download PDF
6. Folding and stability of globular proteins and implications for function
- Author
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Travaglini-Allocatelli C, Ivarsson Y, Jemth P, and Gianni S.
- Abstract
The description of protein folding pathways and the principles that govern them has proven to be one of the most difficult problems to be solved in structural biology. But the combination of experiments and simulations has now provided a clearer picture of the chemistry involved. Once folded, however, proteins remain dynamic systems making possible both small-scale and large-scale structural and/or dynamical changes upon binding or releasing of ligands and during catalysis. In this review we focus on recent advances in the field of protein folding and discuss possible links between folding, stability, and binding dynamics.
- Published
- 2009
7. Folding and misfolding in a naturally occurring circularly permuted PDZ domain
- Author
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Ivarsson Y, Travaglini-Allocatelli C, Brunori M, and Gianni S.
- Published
- 2008
8. Mechanisms of protein folding
- Author
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Ivarsson Y, Travaglini-Allocatelli C, Brunori M, and Gianni S.
- Published
- 2008
9. The folding pathway of an engineered circularly permuted PDZ domain
- Author
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Ivarsson Y, Travaglini-Allocatelli C, Morea V, Brunori M, and Gianni S.
- Abstract
To understand the role of sequence connectivity in the folding pathway of a multi-state protein, we have analysed the folding kinetics of an engineered circularly permuted PDZ domain. This variant has been designed with the specific aim of posing two of the strands participating in the stabilisation of an early folding nucleus as contiguous elements in the primary structure. Folding of the circularly permuted PDZ2 has been explored by a variety of different experimental approaches including stopped-flow and continuous-flow kinetics, as well as ligand-induced folding experiments. Data reveal that although circular permutation introduces a significant destabilisation of the native state, a folding intermediate is stabilised and accumulated prior folding. Furthermore, quantitative analysis of the observed kinetics indicates an acceleration of the early folding events by more than two orders of magnitude. The results support the importance of sequence connectivity both in the mechanism and the speed of protein folding.
- Published
- 2008
10. Identification and characterization of protein folding intermediates
- Author
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Gianni S, Ivarsson Y, Jemth P, Brunori M, and Travaglini-Allocatelli C.
- Published
- 2007
11. An on-pathway intermediate in the folding of a PDZ domain
- Author
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Ivarsson Y, Travaglini-Allocatelli C, Jemth P, Malatesta F, Brunori M, and Gianni S.
- Abstract
The folding pathways of some proteins include the population of partially structured species en route to the native state. Identification and characterization of these folding intermediates are particularly difficult as they are often only transiently populated and play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. To define the role of folding intermediates, a quantitative analysis of the folding and unfolding rate constants over a wide range of denaturant concentration is often required. Such a task is further complicated by the reversible nature of the folding reaction, which implies the observed kinetics to be governed by a complex combination of different microscopic rate constants. Here, we tackled this problem by measuring directly the folding rate constant under highly denaturing conditions, namely by inducing the folding of a PDZ domain through a quasi-irreversible binding reaction with a specific peptide. In analogy with previous works based on hydrogen exchange experiments, we present evidence that the folding pathway of the PDZ domain involves the formation of an obligatory on-pathway intermediate. The results presented exemplify a novel type of kinetic test to detect an on-pathway folding intermediate.
- Published
- 2007
12. Syntenin, a syndecan adaptor and an Arf6 phosphatidylinositol 4,5-bisphosphate effector, is essential for epiboly and gastrulation cell movements in zebrafish
- Author
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Lambaerts, K., primary, Van Dyck, S., additional, Mortier, E., additional, Ivarsson, Y., additional, Degeest, G., additional, Luyten, A., additional, Vermeiren, E., additional, Peers, B., additional, David, G., additional, and Zimmermann, P., additional
- Published
- 2012
- Full Text
- View/download PDF
13. The folding pathway of an engineered circularly permuted PDZ domain
- Author
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Ivarsson, Y., primary, Travaglini-Allocatelli, C., additional, Morea, V., additional, Brunori, M., additional, and Gianni, S., additional
- Published
- 2008
- Full Text
- View/download PDF
14. THE DEVELOPMENT AND CHARACTERIZATION OF A PEPTIDE-BASED SYNTENIN INHIBITOR - IMPLACATIONS FOR CANCER METASTASIS
- Author
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Haugaard-Kedstrom, L. M., Albertsen, L., Abalde Gil, F., Ivarsson, Y., Kristian Strømgaard, and Linda Haugaard-Kedström
15. Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain
- Author
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Alfonso De Simone, Ylva Ivarsson, Maurizio Brunori, Michele Vendruscolo, Stefano Gianni, Carlo Travaglini-Allocatelli, Gianni, S., Ivarsson, Y., De Simone, A., Travaglini-Allocatelli, C., Brunori, M., and Vendruscolo, M.
- Subjects
Models, Molecular ,Protein Folding ,Chemistry ,PDZ domain ,PDZ Domains ,Protein engineering ,Characterization (materials science) ,Protein Structure, Tertiary ,Folding (chemistry) ,Crystallography ,Structural Biology ,Mutation ,Biophysics ,Protein folding ,Molecular Biology - Abstract
Incorrectly folded states transiently populated during the protein folding process are potentially prone to aggregation and have been implicated in a range of misfolding disorders that include Alzheimer's and Parkinson's diseases. Despite their importance, however, the structures of these states and the mechanism of their formation have largely escaped detailed characterization because of their short-lived nature. Here we present the structures of all the major states involved in the folding process of a PDZ domain, which include an off-pathway misfolded intermediate. By using a combination of kinetic, protein engineering, biophysical and computational techniques, we show that the misfolded intermediate is characterized by an alternative packing of the N-terminal Î 2-hairpin onto an otherwise native-like scaffold. Our results suggest a mechanism of formation of incorrectly folded transient compact states by which misfolded structural elements are assembled together with more extended native-like regions. © 2010 Nature America, Inc. All rights reserved.
- Published
- 2010
16. Proteome-scale characterisation of motif-based interactome rewiring by disease mutations.
- Author
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Kliche J, Simonetti L, Krystkowiak I, Kuss H, Diallo M, Rask E, Nilsson J, Davey NE, and Ivarsson Y
- Subjects
- Humans, Protein Binding, Amino Acid Motifs, Intrinsically Disordered Proteins genetics, Intrinsically Disordered Proteins metabolism, Intrinsically Disordered Proteins chemistry, Peptide Library, Protein Interaction Maps, Polymorphism, Single Nucleotide, Proteome genetics, Mutation
- Abstract
Whole genome and exome sequencing are reporting on hundreds of thousands of missense mutations. Taking a pan-disease approach, we explored how mutations in intrinsically disordered regions (IDRs) break or generate protein interactions mediated by short linear motifs. We created a peptide-phage display library tiling ~57,000 peptides from the IDRs of the human proteome overlapping 12,301 single nucleotide variants associated with diverse phenotypes including cancer, metabolic diseases and neurological diseases. By screening 80 human proteins, we identified 366 mutation-modulated interactions, with half of the mutations diminishing binding, and half enhancing binding or creating novel interaction interfaces. The effects of the mutations were confirmed by affinity measurements. In cellular assays, the effects of motif-disruptive mutations were validated, including loss of a nuclear localisation signal in the cell division control protein CDC45 by a mutation associated with Meier-Gorlin syndrome. The study provides insights into how disease-associated mutations may perturb and rewire the motif-based interactome., (© 2024. The Author(s).)
- Published
- 2024
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17. ASXLs binding to the PHD2/3 fingers of MLL4 provides a mechanism for the recruitment of BAP1 to active enhancers.
- Author
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Zhang Y, Xie G, Lee JE, Zandian M, Sudarshan D, Estavoyer B, Benz C, Viita T, Asgaritarghi G, Lachance C, Messmer C, Simonetti L, Sinha VK, Lambert JP, Chen YW, Wang SP, Ivarsson Y, Affar EB, Côté J, Ge K, and Kutateladze TG
- Subjects
- Humans, Animals, Mice, Enhancer Elements, Genetic, HEK293 Cells, PHD Zinc Fingers, Histones metabolism, Tumor Suppressor Proteins metabolism, Tumor Suppressor Proteins genetics, Histone-Lysine N-Methyltransferase metabolism, Histone-Lysine N-Methyltransferase genetics, Ubiquitin Thiolesterase metabolism, Ubiquitin Thiolesterase genetics, Repressor Proteins metabolism, Repressor Proteins genetics, Protein Binding, DNA-Binding Proteins metabolism, DNA-Binding Proteins genetics
- Abstract
The human methyltransferase and transcriptional coactivator MLL4 and its paralog MLL3 are frequently mutated in cancer. MLL4 and MLL3 monomethylate histone H3K4 and contain a set of uncharacterized PHD fingers. Here, we report a novel function of the PHD2 and PHD3 (PHD2/3) fingers of MLL4 and MLL3 that bind to ASXL2, a component of the Polycomb repressive H2AK119 deubiquitinase (PR-DUB) complex. The structure of MLL4 PHD2/3 in complex with the MLL-binding helix (MBH) of ASXL2 and mutational analyses reveal the molecular mechanism which is conserved in homologous ASXL1 and ASXL3. The native interaction of the Trithorax MLL3/4 complexes with the PR-DUB complex in vivo depends solely on MBH of ASXL1/2, coupling the two histone modifying activities. ChIP-seq analysis in embryonic stem cells demonstrates that MBH of ASXL1/2 is required for the deubiquitinase BAP1 recruitment to MLL4-bound active enhancers. Our findings suggest an ASXL1/2-dependent functional link between the MLL3/4 and PR-DUB complexes., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
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18. MLL4 binds TET3.
- Author
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Becht DC, Mohid SA, Lee JE, Zandian M, Benz C, Biswas S, Sinha VK, Ivarsson Y, Ge K, Zhang Y, and Kutateladze TG
- Subjects
- Humans, Animals, Mice, Binding Sites, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins chemistry, Proto-Oncogene Proteins genetics, Models, Molecular, Myeloid-Lymphoid Leukemia Protein metabolism, Myeloid-Lymphoid Leukemia Protein chemistry, Myeloid-Lymphoid Leukemia Protein genetics, Dioxygenases metabolism, Dioxygenases chemistry, Dioxygenases genetics, Protein Binding, DNA-Binding Proteins metabolism, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, Histone-Lysine N-Methyltransferase metabolism, Histone-Lysine N-Methyltransferase chemistry, Histone-Lysine N-Methyltransferase genetics
- Abstract
Human mixed lineage leukemia 4 (MLL4), also known as KMT2D, regulates cell type specific transcriptional programs through enhancer activation. Along with the catalytic methyltransferase domain, MLL4 contains seven less characterized plant homeodomain (PHD) fingers. Here, we report that the sixth PHD finger of MLL4 (MLL4
PHD6 ) binds to the hydrophobic motif of ten-eleven translocation 3 (TET3), a dioxygenase that converts methylated cytosine into oxidized derivatives. The solution NMR structure of the TET3-MLL4PHD6 complex and binding assays show that, like histone H4 tail, TET3 occupies the hydrophobic site of MLL4PHD6 , and that this interaction is conserved in the seventh PHD finger of homologous MLL3 (MLL3PHD7 ). Analysis of genomic localization of endogenous MLL4 and ectopically expressed TET3 in mouse embryonic stem cells reveals a high degree overlap on active enhancers and suggests a potential functional relationship of MLL4 and TET3., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)- Published
- 2024
- Full Text
- View/download PDF
19. Motif-dependent binding on the intervening domain regulates O-GlcNAc transferase.
- Author
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Blankenship CM, Xie J, Benz C, Wang A, Ivarsson Y, and Jiang J
- Subjects
- Animals, Acetylglucosamine metabolism, N-Acetylglucosaminyltransferases metabolism, Peptides, Proteomics, Proteins
- Abstract
The modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) moieties is a highly dynamic process that spatiotemporally regulates nearly every important cellular program. Despite its significance, little is known about the substrate recognition and regulation modes of O-GlcNAc transferase (OGT), the primary enzyme responsible for O-GlcNAc addition. In this study, we identified the intervening domain (Int-D), a poorly understood protein fold found only in metazoan OGTs, as a specific regulator of OGT protein-protein interactions and substrate modification. Using proteomic peptide phage display (ProP-PD) coupled with structural, biochemical and cellular characterizations, we discovered a strongly enriched peptide motif, employed by the Int-D to facilitate specific O-GlcNAcylation. We further show that disruption of Int-D binding dysregulates important cellular programs, including response to nutrient deprivation and glucose metabolism. These findings illustrate a mode of OGT substrate recognition and offer key insights into the biological roles of this unique domain., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2023
- Full Text
- View/download PDF
20. Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets.
- Author
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Mihalič F, Benz C, Kassa E, Lindqvist R, Simonetti L, Inturi R, Aronsson H, Andersson E, Chi CN, Davey NE, Överby AK, Jemth P, and Ivarsson Y
- Subjects
- Humans, Protein Domains, SARS-CoV-2, Ligands, Proteomics, Peptides pharmacology, Antiviral Agents pharmacology, COVID-19
- 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 (K
D ∼ 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., (© 2023. Springer Nature Limited.)- Published
- 2023
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21. Large-scale phosphomimetic screening identifies phospho-modulated motif-based protein interactions.
- Author
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Kliche J, Garvanska DH, Simonetti L, Badgujar D, Dobritzsch D, Nilsson J, Davey NE, and Ivarsson Y
- Subjects
- Humans, Phosphorylation, Clathrin, Proteomics, Peptide Library
- Abstract
Phosphorylation is a ubiquitous post-translation modification that regulates protein function by promoting, inhibiting or modulating protein-protein interactions. Hundreds of thousands of phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide-phage display library to screen for phosphosites that modulate short linear motif-based interactions. The peptidome covers ~13,500 phospho-serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild-type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif-mediated interactions. Affinity measurements confirmed the phospho-modulation of 14 out of 18 tested interactions. We performed a detailed follow-up on a phospho-dependent interaction between clathrin and the mitotic spindle protein hepatoma-upregulated protein (HURP), demonstrating the essentiality of the phospho-dependency to the mitotic function of HURP. Structural characterisation of the clathrin-HURP complex elucidated the molecular basis for the phospho-dependency. Our work showcases the power of phosphomimetic ProP-PD to discover novel phospho-modulated interactions required for cellular function., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2023
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- View/download PDF
22. Elucidation of Short Linear Motif-Based Interactions of the FERM Domains of Ezrin, Radixin, Moesin, and Merlin.
- Author
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Ali M, Khramushin A, Yadav VK, Schueler-Furman O, and Ivarsson Y
- Subjects
- Humans, Ligands, Protein Structure, Tertiary, Peptides, Neurofibromin 2 genetics, Neurofibromin 2 chemistry, Neurofibromin 2 metabolism, FERM Domains
- Abstract
The ERM (ezrin, radixin, and moesin) family of proteins and the related protein merlin participate in scaffolding and signaling events at 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) with binding sites for short linear peptide motifs. By screening the FERM domains of the ERMs and merlin against a phage library that displays peptides representing the intrinsically disordered regions of the human proteome, we identified a large number of novel ligands. We determined the affinities for the ERM and merlin FERM domains interacting with 18 peptides and validated interactions with full-length proteins through pull-down experiments. The majority of the peptides contained an apparent Yx[FILV] motif; others show alternative motifs. We defined distinct binding sites for two types of similar but distinct binding motifs (YxV and FYDF) using a combination of Rosetta FlexPepDock computational peptide docking protocols and mutational analysis. We provide a detailed molecular understanding of how the two types of peptides with distinct motifs bind to different sites on the moesin FERM phosphotyrosine binding-like subdomain and uncover interdependencies between the different types of ligands. The study expands the motif-based interactomes of the ERMs and merlin and suggests that the FERM domain acts as a switchable interaction hub.
- Published
- 2023
- Full Text
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23. The next wave of interactomics: Mapping the SLiM-based interactions of the intrinsically disordered proteome.
- Author
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Davey NE, Simonetti L, and Ivarsson Y
- Subjects
- Humans, Amino Acid Motifs, Proteome
- Abstract
Short linear motifs (SLiMs) are a unique and ubiquitous class of protein interaction modules that perform key regulatory functions and drive dynamic complex formation. For decades, interactions mediated by SLiMs have accumulated through detailed low-throughput experiments. Recent methodological advances have opened this previously underexplored area of the human interactome to high-throughput protein-protein interaction discovery. In this article, we discuss that SLiM-based interactions represent a significant blind spot in the current interactomics data, introduce the key methods that are illuminating the elusive SLiM-mediated interactome of the human cell on a large scale, and discuss the implications for the field., Competing Interests: Declaration of competing interest Nothing declared., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
24. SLiM-binding pockets: an attractive target for broad-spectrum antivirals.
- Author
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Simonetti L, Nilsson J, McInerney G, Ivarsson Y, and Davey NE
- Subjects
- Antiviral Agents pharmacology, Amino Acid Motifs
- Abstract
Short linear motif (SLiM)-mediated interactions offer a unique strategy for viral intervention due to their compact interfaces, ease of convergent evolution, and key functional roles. Consequently, many viruses extensively mimic host SLiMs to hijack or deregulate cellular pathways and the same motif-binding pocket is often targeted by numerous unrelated viruses. A toolkit of therapeutics targeting commonly mimicked SLiMs could provide prophylactic and therapeutic broad-spectrum antivirals and vastly improve our ability to treat ongoing and future viral outbreaks. In this opinion article, we discuss the therapeutic relevance of SLiMs, advocating their suitability as targets for broad-spectrum antiviral inhibitors., Competing Interests: Declaration of interests None are declared., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
25. Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs.
- Author
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Mihalič F, Simonetti L, Giudice G, Sander MR, Lindqvist R, Peters MBA, Benz C, Kassa E, Badgujar D, Inturi R, Ali M, Krystkowiak I, Sayadi A, Andersson E, Aronsson H, Söderberg O, Dobritzsch D, Petsalaki E, Överby AK, Jemth P, Davey NE, and Ivarsson Y
- Subjects
- Amino Acid Motifs, Host-Pathogen Interactions genetics, Bacteriophages genetics, Viruses genetics, Intrinsically Disordered Proteins metabolism
- 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., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
26. Evaluation of affinity-purification coupled to mass spectrometry approaches for capture of short linear motif-based interactions.
- Author
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Kassa E, Jamshidi S, Mihalič F, Simonetti L, Kliche J, Jemth P, Sara Bergström Lind, and Ivarsson Y
- Subjects
- Kelch-Like ECH-Associated Protein 1 metabolism, Mass Spectrometry methods, Chromatography, Affinity, NF-E2-Related Factor 2 metabolism, Peptides chemistry
- 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 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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
27. A novel binding site on the cryptic intervening domain is a motif-dependent regulator of O-GlcNAc transferase.
- Author
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Blankenship C, Xie J, Benz C, Wang A, Ivarsson Y, and Jiang J
- Abstract
The modification of intracellular proteins with O-linked β- N -acetylglucosamine (O-GlcNAc) moieties is a highly dynamic process that spatiotemporally regulates nearly every important cellular program. Despite its significance, little is known about the substrate recognition and regulation modes of O-GlcNAc transferase (OGT), the primary enzyme responsible for O-GlcNAc addition. In this study, we have identified the intervening domain (Int-D), a poorly understood protein fold found only in metazoan OGTs, as a specific regulator of OGT protein-protein interactions and substrate modification. Utilizing an innovative proteomic peptide phage display (ProP-PD) coupled with structural, biochemical, and cellular characterizations, we discovered a novel peptide motif, employed by the Int-D to facilitate specific O-GlcNAcylation. We further show that disruption of Int-D binding dysregulates important cellular programs including nutrient stress response and glucose metabolism. These findings illustrate a novel mode of OGT substrate recognition and offer the first insights into the biological roles of this unique domain.
- Published
- 2023
- Full Text
- View/download PDF
28. How viral proteins bind short linear motifs and intrinsically disordered domains.
- Author
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Madhu P, Davey NE, and Ivarsson Y
- Abstract
Viruses are the obligate intracellular parasites that exploit the host cellular machinery to replicate their genome. During the viral life cycle viruses manipulate the host cell through interactions with host proteins. Many of these protein-protein interactions are mediated through the recognition of host globular domains by short linear motifs (SLiMs), or longer intrinsically disordered domains (IDD), in the disordered regions of viral proteins. However, viruses also employ their own globular domains for binding to SLiMs and IDDs present in host proteins or virus proteins. In this review, we focus on the different strategies adopted by viruses to utilize proteins or protein domains for binding to the disordered regions of human or/and viral ligands. With a set of examples, we describe viral domains that bind human SLiMs. We also provide examples of viral proteins that bind to SLiMs, or IDDs, of viral proteins as a part of complex assembly and regulation of protein functions. The protein-protein interactions are often crucial for viral replication, and may thus offer possibilities for innovative inhibitor design., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
- Published
- 2022
- Full Text
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29. A Syntenin Inhibitor Blocks Endosomal Entry of SARS-CoV-2 and a Panel of RNA Viruses.
- Author
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Lindqvist R, Benz C, Sereikaite V, Maassen L, Laursen L, Jemth P, Strømgaard K, Ivarsson Y, and Överby AK
- Subjects
- Humans, SARS-CoV-2, Syntenins, Antiviral Agents pharmacology, Antiviral Agents chemistry, Virus Internalization, COVID-19, RNA Viruses
- Abstract
Viruses are dependent on host factors in order to efficiently establish an infection and replicate. Targeting the interactions of such host factors provides an attractive strategy to develop novel antivirals. Syntenin is a protein known to regulate the architecture of cellular membranes by its involvement in protein trafficking and has previously been shown to be important for human papilloma virus (HPV) infection. Here, we show that a highly potent and metabolically stable peptide inhibitor that binds to the PDZ1 domain of syntenin inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by blocking the endosomal entry of the virus. Furthermore, we found that the inhibitor also hampered chikungunya infection and strongly reduced flavivirus infection, which is completely dependent on receptor-mediated endocytosis for their entry. In conclusion, we have identified a novel broad spectrum antiviral inhibitor that efficiently targets a broad range of RNA viruses.
- Published
- 2022
- Full Text
- View/download PDF
30. Coupling to short linear motifs creates versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition.
- Author
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Li Y, Balakrishnan VK, Rowse M, Wu CG, Bravos AP, Yadav VK, Ivarsson Y, Strack S, Novikova IV, and Xing Y
- Subjects
- Cryoelectron Microscopy, Demethylation, Holoenzymes metabolism, Methylation, Protein Phosphatase 2 metabolism
- Abstract
Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here, we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryoelectron microscopy structure of a PP2A-B56 holoenzyme-PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multipartite contacts at structured cores to activate the methylesterase. B56 interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56 interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores., Competing Interests: YL, VB, MR, CW, AB, VY, YI, SS, IN, YX No competing interests declared
- Published
- 2022
- Full Text
- View/download PDF
31. ProP-PD for proteome-wide motif-mediated interaction discovery.
- Author
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Davey NE, Simonetti L, and Ivarsson Y
- Subjects
- Amino Acid Motifs, Proteome
- Abstract
Competing Interests: Declaration of interests No interests are declared.
- Published
- 2022
- Full Text
- View/download PDF
32. Orchestrating serine/threonine phosphorylation and elucidating downstream effects by short linear motifs.
- Author
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Kliche J and Ivarsson Y
- Subjects
- Binding Sites, Humans, Phosphoric Monoester Hydrolases metabolism, Phosphorylation, Protein Processing, Post-Translational, Protein Serine-Threonine Kinases metabolism, Substrate Specificity, Protein Interaction Domains and Motifs, Serine chemistry, Serine metabolism, Threonine chemistry, Threonine metabolism
- Abstract
Cellular function is based on protein-protein interactions. A large proportion of these interactions involves the binding of short linear motifs (SLiMs) by folded globular domains. These interactions are regulated by post-translational modifications, such as phosphorylation, that create and break motif binding sites or tune the affinity of the interactions. In addition, motif-based interactions are involved in targeting serine/threonine kinases and phosphatases to their substrate and contribute to the specificity of the enzymatic actions regulating which sites are phosphorylated. Here, we review how SLiM-based interactions assist in determining the specificity of serine/threonine kinases and phosphatases, and how phosphorylation, in turn, affects motif-based interactions. We provide examples of SLiM-based interactions that are turned on/off, or are tuned by serine/threonine phosphorylation and exemplify how this affects SLiM-based protein complex formation., (© 2022 The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
33. Proteome-scale mapping of binding sites in the unstructured regions of the human proteome.
- Author
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Benz C, Ali M, Krystkowiak I, Simonetti L, Sayadi A, Mihalic F, Kliche J, Andersson E, Jemth P, Davey NE, and Ivarsson Y
- Subjects
- Binding Sites, Humans, Peptide Library, Peptides genetics, Peptides metabolism, Protein Binding, Proteome genetics, Proteome metabolism, Proteomics
- 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 ~ 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 proteome-wide discovery of SLiM-based interactions., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2022
- Full Text
- View/download PDF
34. Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities.
- Author
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Kruse T, Benz C, Garvanska DH, Lindqvist R, Mihalic F, Coscia F, Inturi R, Sayadi A, Simonetti L, Nilsson E, Ali M, Kliche J, Moliner Morro A, Mund A, Andersson E, McInerney G, Mann M, Jemth P, Davey NE, Överby AK, Nilsson J, and Ivarsson Y
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, DNA Helicases metabolism, Humans, Poly-ADP-Ribose Binding Proteins metabolism, RNA Helicases metabolism, RNA Recognition Motif Proteins metabolism, RNA-Binding Proteins metabolism, Virus Replication physiology, Integration Host Factors metabolism, SARS-CoV-2 metabolism
- 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., (© 2021. The Author(s).)
- Published
- 2021
- Full Text
- View/download PDF
35. A High-Affinity Peptide Ligand Targeting Syntenin Inhibits Glioblastoma.
- Author
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Haugaard-Kedström LM, Clemmensen LS, Sereikaite V, Jin Z, Fernandes EFA, Wind B, Abalde-Gil F, Daberger J, Vistrup-Parry M, Aguilar-Morante D, Leblanc R, Egea-Jimenez AL, Albrigtsen M, Jensen KE, Jensen TMT, Ivarsson Y, Vincentelli R, Hamerlik P, Andersen JH, Zimmermann P, Lee W, and Strømgaard K
- Subjects
- Animals, Cell Line, Tumor, Drug Delivery Systems, High-Throughput Screening Assays, Humans, Ligands, Mice, Microsomes metabolism, Models, Molecular, Mutation, Protein Binding, X-Ray Diffraction, Xenograft Model Antitumor Assays, Antineoplastic Agents chemistry, Antineoplastic Agents pharmacology, Brain Neoplasms drug therapy, Glioblastoma drug therapy, Peptides chemistry, Peptides pharmacology, Syntenins drug effects
- Abstract
Despite the recent advances in cancer therapeutics, highly aggressive cancer forms, such as glioblastoma (GBM), still have very low survival rates. The intracellular scaffold protein syntenin, comprising two postsynaptic density protein-95/discs-large/zona occludens-1 (PDZ) domains, has emerged as a novel therapeutic target in highly malignant phenotypes including GBM. Here, we report the development of a novel, highly potent, and metabolically stable peptide inhibitor of syntenin, KSL-128114, which binds the PDZ1 domain of syntenin with nanomolar affinity. KSL-128114 is resistant toward degradation in human plasma and mouse hepatic microsomes and displays a global PDZ domain selectivity for syntenin. An X-ray crystal structure reveals that KSL-128114 interacts with syntenin PDZ1 in an extended noncanonical binding mode. Treatment with KSL-128114 shows an inhibitory effect on primary GBM cell viability and significantly extends survival time in a patient-derived xenograft mouse model. Thus, KSL-128114 is a novel promising candidate with therapeutic potential for highly aggressive tumors, such as GBM.
- Published
- 2021
- Full Text
- View/download PDF
36. Cytoplasmic short linear motifs in ACE2 and integrin β 3 link SARS-CoV-2 host cell receptors to mediators of endocytosis and autophagy.
- Author
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Kliche J, Kuss H, Ali M, and Ivarsson Y
- Subjects
- Amino Acid Sequence, Angiotensin-Converting Enzyme 2 chemistry, Angiotensin-Converting Enzyme 2 genetics, Autophagy physiology, Endocytosis physiology, Host Microbial Interactions genetics, Host Microbial Interactions physiology, Humans, Integrin beta3 chemistry, Integrin beta3 genetics, Models, Molecular, Pandemics, Peptide Fragments chemistry, Peptide Fragments genetics, Peptide Fragments physiology, Phosphorylation, Protein Binding, Protein Interaction Domains and Motifs, Protein Sorting Signals genetics, Protein Sorting Signals physiology, Receptors, Virus chemistry, Receptors, Virus genetics, SARS-CoV-2 genetics, Angiotensin-Converting Enzyme 2 physiology, COVID-19 virology, Integrin beta3 physiology, Receptors, Virus physiology, SARS-CoV-2 pathogenicity, SARS-CoV-2 physiology, Virus Internalization
- 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 β
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 μ2 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 β3 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., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)- Published
- 2021
- Full Text
- View/download PDF
37. Integrated analysis of Shank1 PDZ interactions with C-terminal and internal binding motifs.
- Author
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Ali M, McAuley MM, Lüchow S, Knapp S, Joerger AC, and Ivarsson Y
- 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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)
- Published
- 2021
- Full Text
- View/download PDF
38. Identification of PDZ Interactions by Proteomic Peptide Phage Display.
- Author
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Lüchow S, Sundell GN, and Ivarsson Y
- Subjects
- Binding Sites, Humans, Protein Binding, Protein Interaction Mapping, Proteome analysis, Cell Surface Display Techniques methods, PDZ Domains, Peptide Fragments metabolism, Peptide Library, Proteome metabolism
- Abstract
PSD95-Disc large-Zonula occludens (PDZ) domains are among the most abundant modular domains in the human proteome. They typically bind short carboxy-terminal sequence motifs of their ligand proteins, which may be transmembrane proteins such as ion channels and GPCRs, as well as soluble proteins. The identity of the endogenous ligands of many PDZ domains remains unclear despite more than two decades of PDZ research. Combinatorial peptide phage display and bioinformatics predictions have contributed to shed light on PDZ-mediated interactions. However, the efficiency of these methods for the identification of interactions of potential biological relevance is hampered by different biases. Proteomic peptide-phage display (ProP-PD) was developed to overcome these limitations. Here we describe a ProP-PD protocol for the identification of C-terminal PDZ domain ligands. The method efficiently identifies peptide ligands within a proteome of interest, and pinpoint targets of potential biological relevance.
- Published
- 2021
- Full Text
- View/download PDF
39. Deciphering the Unexpected Binding Capacity of the Third PDZ Domain of Whirlin to Various Cochlear Hair Cell Partners.
- Author
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Zhu Y, Delhommel F, Cordier F, Lüchow S, Mechaly A, Colcombet-Cazenave B, Girault V, Pepermans E, Bahloul A, Gautier C, Brûlé S, Raynal B, Hoos S, Haouz A, Caillet-Saguy C, Ivarsson Y, and Wolff N
- Subjects
- Cell Cycle Proteins metabolism, Cytoskeletal Proteins metabolism, Guanylate Kinases metabolism, Humans, Myosins metabolism, Proteins, Stereocilia metabolism, Hair Cells, Auditory cytology, Hair Cells, Auditory metabolism, Membrane Proteins chemistry, Membrane Proteins metabolism, PDZ Domains physiology, Protein Binding
- Abstract
Hearing is a mechanical and neurochemical process, which occurs in the hair cells of inner ear that converts the sound vibrations into electrical signals transmitted to the brain. The multi-PDZ scaffolding protein whirlin plays a critical role in the formation and function of stereocilia exposed at the surface of hair cells. In this article, we reported seven stereociliary proteins that encode PDZ binding motifs (PBM) and interact with whirlin PDZ3, where four of them are first reported. We solved the atomic resolution structures of complexes between whirlin PDZ3 and the PBMs of myosin 15a, CASK, harmonin a1 and taperin. Interestingly, the PBM of CASK and taperin are rare non-canonical PBM, which are not localized at the extreme C terminus. This large capacity to accommodate various partners could be related to the distinct functions of whirlin at different stages of the hair cell development., (Copyright © 2020. Published by Elsevier Ltd.)
- Published
- 2020
- Full Text
- View/download PDF
40. Profiling calcium-dependent interactions between Sorcin and intrinsically disordered regions of human proteome.
- Author
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Genovese I, Carotti A, Ilari A, Fiorillo A, Battista T, Colotti G, and Ivarsson Y
- Subjects
- HeLa Cells, Humans, Calcium metabolism, Calcium-Binding Proteins metabolism, Intrinsically Disordered Proteins metabolism, Proteome metabolism
- Abstract
Background: Sorcin is a calcium sensor that exerts many calcium-related functions in the cells, e.g. it regulates calcium concentration in the cytoplasm, endoplasmic reticulum (ER) and mitochondria, by interacting with calcium pumps, exchangers and channels. Albeit Sorcin is an interesting potential cancer target, little is known about its interactors upon calcium-mediated activation. Our previous study suggested that Sorcin may recognize short linear binding motifs as the crystal structure revealed a self-interaction with a GYYPGG stretch in its N-terminus, and combinatorial peptide-phage display provided support for peptide-mediated interactions., Methods: In this study we screened for motif-based interactions between Sorcin and intrinsically disordered regions of the human proteome using proteomic peptide phage display (ProP-PD). We identified a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a potential novel interactor and confirm the interaction through biophysical and cell-based approaches, and provide structural information through molecular dynamics simulations., Results: Altogether, we identify a preferred motif in the enriched pool of binders and a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a preferred ligand., Conclusion: Through this study we gain information on a new Sorcin binding partner and profile Sorcin's motif-based interaction., General Significance: The interaction between Sorcin and PPP1R3G may suggest a close dependence between glucose homeostasis and calcium concentration in the different cell compartments, opening a completely new and interesting scenery yet to be fully disclosed., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
41. Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.
- Author
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Wigington CP, Roy J, Damle NP, Yadav VK, Blikstad C, Resch E, Wong CJ, Mackay DR, Wang JT, Krystkowiak I, Bradburn DA, Tsekitsidou E, Hong SH, Kaderali MA, Xu SL, Stearns T, Gingras AC, Ullman KS, Ivarsson Y, Davey NE, and Cyert MS
- Subjects
- Active Transport, Cell Nucleus, Amino Acid Motifs, Biotinylation, Centrosome metabolism, Computer Simulation, HEK293 Cells, HeLa Cells, Humans, Mass Spectrometry, Phosphoric Monoester Hydrolases chemistry, Phosphorylation, Protein Interaction Maps, Proteome metabolism, Receptor, Notch1 metabolism, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins metabolism, Signal Transduction, Calcineurin metabolism, Nuclear Pore Complex Proteins metabolism, Phosphoric Monoester Hydrolases metabolism
- Abstract
Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca
2+ -activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
42. Genetically Encoded Cyclic Peptide Phage Display Libraries.
- Author
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Simonetti L and Ivarsson Y
- Published
- 2020
- Full Text
- View/download PDF
43. Screening Intrinsically Disordered Regions for Short Linear Binding Motifs.
- Author
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Ali M, Simonetti L, and Ivarsson Y
- Subjects
- Bacteriophage M13 genetics, Binding Sites, Computational Biology methods, DNA Primers, Data Management methods, Electroporation methods, Escherichia coli, Guidelines as Topic, High-Throughput Nucleotide Sequencing, Information Storage and Retrieval methods, Protein Binding, Amino Acid Motifs, Cell Surface Display Techniques methods, Gene Library, High-Throughput Screening Assays methods, Intrinsically Disordered Proteins chemistry, Peptide Library
- Abstract
The intrinsically disordered regions of the proteome are enriched in short linear motifs (SLiMs) that serve as binding sites for peptide binding proteins. These interactions are often of low-to-mid micromolar affinities and are challenging to screen for experimentally. However, a range of dedicated methods have been developed recently, which open for screening of SLiM-based interactions on large scale. A variant of phage display, termed proteomic peptide phage display (ProP-PD), has proven particularly useful for the purpose. Here, we describe a complete high-throughput ProP-PD protocol for screening intrinsically disordered regions for SLiMs. The protocol requires some basic bioinformatics skills for the design of the library and for data analysis but can be performed in a standard biochemistry lab. The protocol starts from the construction of a library, followed by the high-throughput expression and purification of bait proteins, the phage selection, and the analysis of the binding-enriched phage pools using next-generation sequencing. As the protocol generates rather large data sets, we also emphasize the importance of data management and storage.
- Published
- 2020
- Full Text
- View/download PDF
44. A Consensus Binding Motif for the PP4 Protein Phosphatase.
- Author
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Ueki Y, Kruse T, Weisser MB, Sundell GN, Larsen MSY, Mendez BL, Jenkins NP, Garvanska DH, Cressey L, Zhang G, Davey N, Montoya G, Ivarsson Y, Kettenbach AN, and Nilsson J
- Subjects
- Amino Acid Sequence genetics, Binding Sites, Conserved Sequence, Crystallography, X-Ray methods, HEK293 Cells, HeLa Cells, Humans, Phosphorylation, Protein Binding genetics, Substrate Specificity, Phosphoprotein Phosphatases genetics, Phosphoprotein Phosphatases metabolism, Phosphoprotein Phosphatases ultrastructure
- Abstract
Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
45. Systematic identification of recognition motifs for the hub protein LC8.
- Author
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Jespersen N, Estelle A, Waugh N, Davey NE, Blikstad C, Ammon YC, Akhmanova A, Ivarsson Y, Hendrix DA, and Barbar E
- Subjects
- Algorithms, Calorimetry, Cell Cycle Proteins metabolism, Cytoplasmic Dyneins chemistry, Cytoplasmic Dyneins genetics, HeLa Cells, Humans, Microtubule-Associated Proteins metabolism, Models, Molecular, Protein Binding, Proteomics, Thermodynamics, Cytoplasmic Dyneins metabolism, Peptides chemistry, Protein Interaction Domains and Motifs
- Abstract
Hub proteins participate in cellular regulation by dynamic binding of multiple proteins within interaction networks. The hub protein LC8 reversibly interacts with more than 100 partners through a flexible pocket at its dimer interface. To explore the diversity of the LC8 partner pool, we screened for LC8 binding partners using a proteomic phage display library composed of peptides from the human proteome, which had no bias toward a known LC8 motif. Of the identified hits, we validated binding of 29 peptides using isothermal titration calorimetry. Of the 29 peptides, 19 were entirely novel, and all had the canonical TQT motif anchor. A striking observation is that numerous peptides containing the TQT anchor do not bind LC8, indicating that residues outside of the anchor facilitate LC8 interactions. Using both LC8-binding and nonbinding peptides containing the motif anchor, we developed the "LC8Pred" algorithm that identifies critical residues flanking the anchor and parses random sequences to predict LC8-binding motifs with ∼78% accuracy. Our findings significantly expand the scope of the LC8 hub interactome., (© 2019 Jespersen et al.)
- Published
- 2019
- Full Text
- View/download PDF
46. Affinity and specificity of motif-based protein-protein interactions.
- Author
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Ivarsson Y and Jemth P
- Subjects
- Animals, Evolution, Molecular, Humans, Protein Binding, Substrate Specificity, Protein Interaction Domains and Motifs, Proteins chemistry, Proteins metabolism
- Abstract
It is becoming increasingly clear that eukaryotic cell physiology is largely controlled by protein-protein interactions involving disordered protein regions, which usually interact with globular domains in a coupled binding and folding reaction. Several protein recognition domains are part of large families where members can interact with similar peptide ligands. Because of this, much research has been devoted to understanding how specificity can be achieved. A combination of interface complementarity, interactions outside of the core binding site, avidity from multidomain architecture and spatial and temporal regulation of expression resolves the conundrum. Here, we review recent advances in molecular aspects of affinity and specificity in such protein-protein interactions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
47. Editorial overview: Folding and binding.
- Author
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Ivarsson Y and Jemth P
- Subjects
- Protein Binding, Protein Folding, Proteins chemistry, Proteins metabolism
- Published
- 2019
- Full Text
- View/download PDF
48. Proteome-wide analysis of phospho-regulated PDZ domain interactions.
- Author
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Sundell GN, Arnold R, Ali M, Naksukpaiboon P, Orts J, Güntert P, Chi CN, and Ivarsson Y
- Subjects
- Amino Acid Sequence genetics, Binding Sites, Disks Large Homolog 4 Protein genetics, Humans, Ligands, Oligonucleotides genetics, Peptide Library, Phosphorylation, Protein Binding genetics, Protein Interaction Mapping, Zonula Occludens-1 Protein genetics, PDZ Domains genetics, Peptides genetics, Protein Interaction Maps genetics, Proteome genetics
- Abstract
A key function of reversible protein phosphorylation is to regulate protein-protein interactions, many of which involve short linear motifs (3-12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2018
- Full Text
- View/download PDF
49. High-throughput discovery of functional disordered regions.
- Author
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Ali M and Ivarsson Y
- Subjects
- Amino Acid Sequence, Protein Conformation, Databases, Protein, Proteins
- Published
- 2018
- Full Text
- View/download PDF
50. The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ1.
- Author
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Sundell GN, Vögeli B, Ivarsson Y, and Chi CN
- Subjects
- Algorithms, Binding Sites, Cyclophilin A chemistry, Cyclophilin A genetics, Cyclophilin A metabolism, Humans, Kinetics, Ligands, Magnetic Resonance Spectroscopy, Membrane Proteins chemistry, Membrane Proteins genetics, Oligopeptides chemistry, Oligopeptides genetics, PDZ Domains, Peptide Fragments chemistry, Peptide Fragments genetics, Phosphorylation, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Replication Origin, Ribosomal Protein S6 Kinases, 90-kDa chemistry, Ribosomal Protein S6 Kinases, 90-kDa genetics, Serine metabolism, Tumor Suppressor Proteins chemistry, Tumor Suppressor Proteins genetics, Membrane Proteins metabolism, Models, Molecular, Oligopeptides metabolism, Peptide Fragments metabolism, Protein Processing, Post-Translational, Ribosomal Protein S6 Kinases, 90-kDa metabolism, Tumor Suppressor Proteins metabolism
- Abstract
The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the
15 N/1 H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.- Published
- 2018
- Full Text
- View/download PDF
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