| 1. |
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| 2. |
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| 3. |
- 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
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 62:11, s. 1594-1607
-
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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| 4. |
- Ali, Muhammad, et al.
(författare)
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High-throughput discovery of functional disordered regions
- 2018
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Ingår i: Molecular Systems Biology. - : EMBO. - 1744-4292 .- 1744-4292. ; 14:5
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Partially or fully intrinsically disordered proteins are widespread in eukaryotic proteomes and play important biological functions. With the recognition that well defined protein structure is not a fundamental requirement for function come novel challenges, such as assigning function to disordered regions. In their recent work, Babu and colleagues (Ravarani etal,) took on this challenge by developing IDR-Screen, a robust high-throughput approach for identifying functions of disordered regions.
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| 5. |
- Ali, Muhammad, 1990-
(författare)
-
Identification of SLiMs: Mapping and characterizing motif-based protein interactions
- 2020
-
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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| 6. |
- Ali, Muhammad, 1990-, et al.
(författare)
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Integrated analysis of Shank1 PDZ interactions with C-terminal and internal binding motifs
- 2021
-
Ingår i: Current Research in Structural Biology. - : Elsevier. - 2665-928X. ; 3, s. 41-50
-
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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| 7. |
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| 8. |
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| 9. |
- Baietti, Maria Francesca, et al.
(författare)
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Syndecan-syntenin-ALIX regulates the biogenesis of exosomes
- 2012
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Ingår i: Nature cell biology. - : Springer Science and Business Media LLC. - 1476-4679 .- 1465-7392. ; 14:7, s. 677-685
-
Tidskriftsartikel (refereegranskat)abstract
- The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan-syntenin-ALIX in membrane transport and signalling processes.
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| 10. |
- Becht, Dustin C., et al.
(författare)
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MLL4 binds TET3
- 2024
-
Ingår i: Structure. - : Cell Press. - 0969-2126 .- 1878-4186. ; 32:6
-
Tidskriftsartikel (refereegranskat)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 MLL4 (MLL4 PHD6 ) binds to the hydrophobic motif of ten -eleven translocation 3 (TET3), a dioxygenase converts methylated cytosine into oxidized derivatives. The solution NMR structure of the TET3-MLL4 complex and binding assays show that, like histone H4 tail, TET3 occupies the hydrophobic site MLL 4 PHD6 , and that this interaction is conserved in the seventh PHD finger of homologous MLL3 (MLL3 PHD7 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.
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| 11. |
- Benz, Caroline
(författare)
-
Diving into short linear motifs : Large-scale identification of endogenous and host-pathogen protein-protein interactions and further characterized by deep mutational scanning
- 2023
-
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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| 12. |
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| 13. |
- Benz, Caroline, et al.
(författare)
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Proteome-scale mapping of binding sites in the unstructured regions of the human proteome
- 2022
-
Ingår i: Molecular Systems Biology. - : EMBO Press. - 1744-4292 .- 1744-4292. ; 18:1
-
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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| 14. |
- Blankenship, Connor M., et al.
(författare)
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Motif-dependent binding on the intervening domain regulates O-GlcNAc transferase
- 2023
-
Ingår i: Nature Chemical Biology. - : Springer Nature. - 1552-4450 .- 1552-4469. ; 19:11, s. 1423-1431
-
Tidskriftsartikel (refereegranskat)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.
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| 15. |
- Blikstad, Cecilia, et al.
(författare)
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High-throughput methods for identification of protein-protein interactions involving short linear motifs
- 2015
-
Ingår i: Cell Communication and Signaling. - : Springer Science and Business Media LLC. - 1478-811X. ; 13
-
Forskningsöversikt (refereegranskat)abstract
- Interactions between modular domains and short linear motifs (3-10 amino acids peptide stretches) are crucial for cell signaling. The motifs typically reside in the disordered regions of the proteome and the interactions are often transient, allowing for rapid changes in response to changing stimuli. The properties that make domain-motif interactions suitable for cell signaling also make them difficult to capture experimentally and they are therefore largely underrepresented in the known protein-protein interaction networks. Most of the knowledge on domain-motif interactions is derived from low-throughput studies, although there exist dedicated high-throughput methods for the identification of domain-motif interactions. The methods include arrays of peptides or proteins, display of peptides on phage or yeast, and yeast-two-hybrid experiments. We here provide a survey of scalable methods for domain-motif interaction profiling. These methods have frequently been applied to a limited number of ubiquitous domain families. It is now time to apply them to a broader set of peptide binding proteins, to provide a comprehensive picture of the linear motifs in the human proteome and to link them to their potential binding partners. Despite the plethora of methods, it is still a challenge for most approaches to identify interactions that rely on post-translational modification or context dependent or conditional interactions, suggesting directions for further method development.
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| 16. |
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| 17. |
- Davey, Norman E., et al.
(författare)
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Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome
- 2017
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 284:3, s. 485-498
-
Tidskriftsartikel (refereegranskat)abstract
- The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 mu M through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD-derived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.
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| 18. |
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| 19. |
- Davey, Norman E., et al.
(författare)
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The next wave of interactomics : Mapping the SLiM-based interactions of the intrinsically disordered proteome
- 2023
-
Ingår i: Current opinion in structural biology. - : Elsevier BV. - 0959-440X .- 1879-033X. ; 80
-
Tidskriftsartikel (refereegranskat)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.
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| 20. |
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| 21. |
- Egea-Jimenez, Antonio Luis, et al.
(författare)
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Frizzled 7 and PIP2 binding by syntenin PDZ2 domain supports Frizzled 7 trafficking and signalling
- 2016
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
-
Tidskriftsartikel (refereegranskat)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.
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| 22. |
- Ernst, Andreas, et al.
(författare)
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A Structural Portrait of the PDZ Domain Family
- 2014
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 426:21, s. 3509-3519
-
Tidskriftsartikel (refereegranskat)abstract
- PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.
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| 23. |
- Fernaeus, Ylva, 1976-, et al.
(författare)
-
Plei-Plei!
- 2012. - 1
-
Bok (övrigt vetenskapligt/konstnärligt)abstract
- Let us introduce an amazing crowd of researchers at Mobile Life Centre in Stockholm, Sweden, and some of their friends at Nokia Research Center, Microsoft Research Cambridge and Ericsson Research. These people are at the international forefront of research in the domain of mobile interactive technology – a situation that this book aims to celebrate!This is also a printed book, which means it may work a bit like a time capsule, showcasing a set of explorations that may appear peculiar and out-dated, depending on when you happen to read it. It may therefore be highlighted that all the work presented in here was conducted during the first five years of the Mobile Life Centre (2007-2012) — a time when the mobile mass market, as well as research in this field, was still new, fresh and explorative in nature.The title, Plei-Plei, refers to a playful approach towards research as characteristic in the work presented in this book. The term is also used by natives in the pacific islands of Vanuatu, to describe “mere play” in their everyday lives, as well as in their use of mobile phones. This means that the book is not just about fun and games, but rather an attempt to capture how research can be driven by a genuine curiosity of, and inspiration from, what people enjoy doing.Since many of our friends have told us that research papers are usually too long and also somewhat boring to read, we have chosen to present this work by highlighting some of our favourite results with illustrations and shorter texts that hopefully will be more inspirational and enjoyable to read. Thanks to massive help from Boris Design Studio, we are immensely impressed with the result that is now in your hand.Please Enjoy!
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| 24. |
- Fugier, Charlotte, et al.
(författare)
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Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy
- 2011
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Ingår i: Nature medicine. - : Springer Science and Business Media LLC. - 1546-170X .- 1078-8956. ; 17:6, s. 720-725
-
Tidskriftsartikel (refereegranskat)abstract
- Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.
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| 25. |
- Gallardo, Rodrigo, et al.
(författare)
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Structural diversity of PDZ-lipid interactions
- 2010
-
Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 11:4, s. 456-67
-
Tidskriftsartikel (refereegranskat)abstract
- PDZ domains are globular protein modules that are over-and-above appreciated for their interaction with short peptide motifs found in the cytosolic tail of membrane receptors, channels, and adhesion molecules. These domains predominate in scaffold molecules that control the assembly and the location of large signaling complexes. Studies have now emerged showing that PDZ domains can also interact with membrane lipids, and in particular with phosphoinositides. Phosphoinositides control various aspects of cell signaling, vesicular trafficking, and cytoskeleton remodeling. When investigated, lipid binding appears to be extremely relevant for PDZ protein functionality. Studies point to more than one mechanism for PDZ domains to associate with lipids. Few studies have been focused on the structural basis of PDZ-phosphoinositide interactions, and the biological consequences of such interactions. Using the current knowledge on syntenin-1, syntenin-2, PTP-Bas, PAR-3 and PICK1, we recapitulate our understanding of the structural and biochemical aspects of PDZ-lipid interactions and the consequences for peptide interactions.
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| 26. |
- Ganesan, Ashok, et al.
(författare)
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Selectivity of Aggregation-Determining Interactions
- 2015
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:2, s. 236-247
-
Tidskriftsartikel (refereegranskat)abstract
- Protein aggregation is sequence specific, favoring self-assembly over cross-seeding with non-homologous sequences. Still, as the majority of proteins in a proteome are aggregation prone, the high level of homogeneity of protein inclusions in vivo both during recombinant overexpression and in disease remains surprising. To investigate the selectivity of protein aggregation in a proteomic context, we here compared the selectivity of aggregation-determined interactions with antibody binding. To that purpose, we synthesized biotin-labeled peptides, corresponding to aggregation-determining sequences of the bacterial protein β-galactosidase and two human disease biomarkers: C-reactive protein and prostate-specific antigen. We analyzed the selectivity of their interactions in Escherichiacoli lysate, human serum and human seminal plasma, respectively, using a Western blot-like approach in which the aggregating peptides replace the conventional antibody. We observed specific peptide accumulation in the same bands detected by antibody staining. Combined spectroscopic and mutagenic studies confirmed accumulation resulted from binding of the peptide on the identical sequence of the immobilized target protein. Further, we analyzed the sequence redundancy of aggregating sequences and found that about 90% of them are unique within their proteome. As a result, the combined specificity and low sequence redundancy of aggregating sequences therefore contribute to the observed homogeneity of protein aggregation in vivo. This suggests that these intrinsic proteomic properties naturally compartmentalize aggregation events in sequence space. In the event of physiological stress, this might benefit the ability of cells to respond to proteostatic stress by allowing chaperones to focus on specific aggregation events rather than having to face systemic proteostatic failure.
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| 27. |
- Garrido-Urbani, Sarah, et al.
(författare)
-
Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin
- 2016
-
Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 590:1, s. 3-12
-
Tidskriftsartikel (refereegranskat)abstract
- Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance.
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| 28. |
- Genovese, Ilaria, et al.
(författare)
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Profiling calcium-dependent interactions between Sorcin and intrinsically disordered regions of human proteome
- 2020
-
Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier. - 0304-4165 .- 1872-8006. ; 1864:8
-
Tidskriftsartikel (refereegranskat)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.
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| 29. |
- Gianni, Stefano, et al.
(författare)
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Identification and characterization of protein folding intermediates
- 2007
-
Ingår i: Biophysical Chemistry. - : Elsevier BV. - 0301-4622 .- 1873-4200. ; 128:2-3, s. 105-113
-
Forskningsöversikt (refereegranskat)abstract
- In order to understand the mechanism by which a polypeptide chain folds into its functionally active native state it is necessary to characterize in detail all the species accumulated along the pathway. The elusive nature of protein folding intermediates poses their identification and characterization as an extremely difficult task in the protein folding field. In the case of small single domain proteins, the direct measurement of the thermodynamics and structural parameters of protein folding intermediates has provided new insights on the nature of the forces involved in the stabilization of nascent protein structures. Here we summarize some of the experimental approaches aimed at the detection and characterization of folding intermediates along with a discussion of some general structural features emerging from these studies.
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| 30. |
- Gianni, Stefano, et al.
(författare)
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Mechanism of Na(+) binding to thrombin resolved by ultra-rapid kinetics
- 2007
-
Ingår i: Biophysical Chemistry. - : Elsevier BV. - 0301-4622 .- 1873-4200. ; 131:1-3, s. 111-114
-
Tidskriftsartikel (refereegranskat)abstract
- The interaction of Na(+) and K(+) with proteins is at the basis of numerous processes of biological importance. However, measurement of the kinetic components of the interaction has eluded experimentalists for decades because the rate constants are too fast to resolve with conventional stopped-flow methods. Using a continuous-flow apparatus with a dead time of 50 micro s we have been able to resolve the kinetic rate constants and entire mechanism of Na(+) binding to thrombin, an interaction that is at the basis of the procoagulant and prothrombotic roles of the enzyme in the blood.
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| 31. |
- Gianni, Stefano, et al.
(författare)
-
Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain
- 2010
-
Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 17:12, s. 1431-1437
-
Tidskriftsartikel (refereegranskat)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 β-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.
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| 32. |
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| 33. |
- Haugaard-Kedström, Linda M., et al.
(författare)
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A High-Affinity Peptide Ligand Targeting Syntenin Inhibits Glioblastoma
- 2021
-
Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 64:3, s. 1423-1434
-
Tidskriftsartikel (refereegranskat)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/discslarge/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 KSL128114 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.
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| 34. |
- Hjelm, Linnea C., 1993-
(författare)
-
Development of new affinity proteins for neurodegenerative disorders
- 2023
-
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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| 35. |
- Hämälistö, Saara, et al.
(författare)
-
A ZO-1/α5β1-integrin complex regulates cytokinesis downstream of PKCε in NCI-H460 cells plated on fibronectin
- 2013
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:8, s. e70696-
-
Tidskriftsartikel (refereegranskat)abstract
- Recently, we demonstrated that integrin adhesion to the extracellular matrix at the cleavage furrow is essential for cytokinesis of adherent cells. Here, we report that tight junction protein ZO-1 (Zonula Occludens-1) is required for successful cytokinesis in NCI-H460 cells plated on fibronectin. This function of ZO-1 involves interaction with the cytoplasmic domain of α5-integrin to facilitate recruitment of active fibronectin-binding integrins to the base of the cleavage furrow. In the absence of ZO-1, or a functional ZO-1/α5β1-integrin complex, proper actin-dependent constriction between daughter cells is impaired and cells fail cytokinesis. Super-resolution microscopy reveals that in ZO-1 depleted cells the furrow becomes delocalized from the matrix. We also show that PKCε-dependent phosphorylation at Serine168 is required for ZO-1 localization to the furrow and successful cell division. Altogether, our results identify a novel regulatory pathway involving the interplay between ZO-1, α5-integrin and PKCε in the late stages of mammalian cell division.
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| 36. |
- Ilari, Andrea, et al.
(författare)
-
Structural basis of Sorcin-mediated calcium-dependent signal transduction
- 2015
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Sorcin is an essential penta-EF hand calcium binding protein, able to confer the multi-drug resistance phenotype to drug-sensitive cancer cells and to reduce Endoplasmic Reticulum stress and cell death. Sorcin silencing blocks cell cycle progression in mitosis and induces cell death by triggering apoptosis. Sorcin participates in the modulation of calcium homeostasis and in calcium-dependent cell signalling in normal and cancer cells. The molecular basis of Sorcin action is yet unknown. The X-ray structures of Sorcin in the apo (apoSor) and in calcium bound form (CaSor) reveal the structural basis of Sorcin action: calcium binding to the EF1-3 hands promotes a large conformational change, involving a movement of the long D-helix joining the EF1-EF2 sub-domain to EF3 and the opening of EF1. This movement promotes the exposure of a hydrophobic pocket, which can accommodate in CaSor the portion of its N-terminal domain displaying the consensus binding motif identified by phage display experiments. This domain inhibits the interaction of sorcin with PDCD6, a protein that carries the Sorcin consensus motif, co-localizes with Sorcin in the perinuclear region of the cell and in the midbody and is involved in the onset of apoptosis.
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| 37. |
- Ivarsson, Mats, et al.
(författare)
-
God Havsmiljö 2020 : Marin strategi för Nordsjön och Östersjön Del 1: Inledande bedömning av miljötillstånd och socioekonomisk analys
- 2012
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Havsmiljöförordningens övergripande mål är att upprätthålla eller uppnå en god miljöstatus i de svenska förvaltningsområdena Nordsjön och Östersjön till år 2020. En av uppgifterna i den första förvaltningsperioden är att göra en inledande bedömning av miljötillståndet och en ekonomisk och social analys av nyttjandet av havet. Den inledande bedömningen redovisar grundläggande egenskaper och det aktuella miljötillståndet i Nordsjön och Östersjön. Rapporten följer havsmiljödirektivets instruktioner, det vill säga det EU-direktiv som i Sverige genomförs genom havsmiljöförordningen. I den inledande bedömningen ingår en beskrivning av fysiska och kemiska förhållanden, livsmiljöer, samt biologiska förhållanden. Belastning på miljön i form av fysisk störning (t.ex. skador på bottnarna) tillförsel av näringsämnen, tillförsel och förorenande ämnen samt biologisk störning (t.ex. uttag av arter genom fiske) ingår också i analysen. Den ekonomiska och sociala analysen består tre delar. Den första delen beskriver hur förvaltningsområdena nyttjas och var i områdena som aktiviteterna sker. Den andra delen ger en bild av trender i de mänskliga aktiviteter som påverkar miljötillståndet samt en beskrivning av samhällets kostnad för en eventuell försämring av miljötillståndet. Den svenska analysen bygger på ekosystemtjänstansatsen vilket innebär att kostnaden beskrivs i termer av välfärdsförluster som kan kopplas till försämrade eller försvagade ekosystemtjänster. Den tredje delen är en social analys som behandlar direkta och indirekta drivkrafter för miljöbelastningarna. Havsmiljödirektivets krav innebär i vissa fall att nya underlag måste tas fram vilket inte alltid varit möjligt att nå i tid för den inledande bedömningen. Rapporten innehåller därför också bristanalyser som pekar ut behov av framtida informations- och kunskapsinhämtning. Bristanalysen redovisas i anslutning till respektive delkapitel. Den inledande bedömningen baseras på underlag från svenska universitet, forskningsinstitut, myndigheter, konsulter och publicerade resultat från projekt som drivits av HELCOM, OSPAR samt projektet HARMONY, där Danmark, Sverige, Norge och Tyskland deltagit för att ta fram underlag om östra Nordsjön. I rapporten återfinns Havs- och vattenmyndighetens samlade slutsatser om det aktuella miljötillståndet och den socioekonomiska analysen. Slutsatsen av den inledande bedömningen överensstämmer med vad som framkommit i senare års nationella liksom internationella tillståndsbedömningar; tillståndet i Nordsjön och Östersjön varierar visserligen mellan olika havsbassänger, liksom mellan kust- och utsjövatten, men sammanfattningsvis är nuvarande tillstånd i många fall inte förenligt med vad som kännetecknar god miljöstatus enligt havsmiljöförordningen. Det ogynnsamma tillståndet är förknippat med negativa konsekvenser för såväl marina växter, djur och livsmiljöer som de ekosystemtjänster som människan nyttjar. En sammanfattning av miljötillstånd och belastning finns i avsnitt 3.8 och slutsatser finns i kapitel 8.
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| 38. |
- Ivarsson Westerberg, Anders, et al.
(författare)
-
Det långa 1990-talet
- 2014. - 1
-
Ingår i: Det långa 1990-talet. - Umeå : Boréa Bokförlag. - 9789189140882 ; , s. 11-33
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 39. |
- Ivarsson Westerberg, Anders, et al.
(författare)
-
Vad var det som hände?
- 2014. - 1
-
Ingår i: Det långa 1990-talet. - Umeå : Boréa Bokförlag. - 9789189140882 ; , s. 441-457
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 40. |
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| 41. |
- Ivarsson, Ylva, et al.
(författare)
-
Affinity and specificity of motif-based protein-protein interactions
- 2019
-
Ingår i: Current opinion in structural biology. - : Current Biology. - 0959-440X .- 1879-033X. ; 54, s. 26-33
-
Forskningsöversikt (refereegranskat)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.
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| 42. |
- Ivarsson, Ylva, et al.
(författare)
-
An on-pathway intermediate in the folding of a PDZ domain
- 2007
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:12, s. 8568-8572
-
Tidskriftsartikel (refereegranskat)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.
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| 43. |
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| 44. |
- Ivarsson, Ylva, et al.
(författare)
-
Cooperative phosphoinositide and peptide binding by PSD-95/discs large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin
- 2011
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:52, s. 44669-78
-
Tidskriftsartikel (refereegranskat)abstract
- PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. We screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns(4,5)P(2)-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. We identified the transmembrane protein Crumbs as a Pyd PDZ2 ligand and probed the relation between peptide and PtdIns(4,5)P(2) binding. Contrary to the prevalent view on PDZ/peptide/lipid binding, we did not find competition between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns(4,5)P(2) 6-fold. Our results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns(4,5)P(2) binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals.
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| 45. |
- Ivarsson, Ylva, et al.
(författare)
-
Editorial overview : Folding and binding
- 2019
-
Ingår i: Current opinion in structural biology. - : CURRENT BIOLOGY LTD. - 0959-440X .- 1879-033X. ; 54, s. 139-140
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 46. |
- Ivarsson, Ylva, et al.
(författare)
-
Engineered symmetric connectivity of secondary structure elements highlights malleability of protein folding pathways
- 2009
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 131:33, s. 11727-33
-
Tidskriftsartikel (refereegranskat)abstract
- To understand the role of sequence connectivity in protein folding pathways, we explored by Phi-value analysis the folding pathway of an engineered circularly permuted PDZ domain. This variant has the same sequence connectivity as naturally occurring circularly permuted PDZ domains and displays a symmetrical distribution of secondary structure elements (i.e., beta beta alpha beta beta alpha beta beta) while maintaining the same tertiary interactions of the well-characterized second PDZ domain from PTP-BL (PDZ2). Reliable Phi values were obtained for both a low-energy intermediate and the late rate-limiting transition state, allowing a description of both early and late events in folding. A comparison with Phi values obtained for wild-type PDZ2 reveals that while the structure of the late transition state is robust and unaffected by circular permutation, the folding intermediate is stabilized by a different nucleus involving residues located at the new N- and C-termini. The results suggest that folding is driven by competing nuclei whose stabilities may be selectively tuned by circular permutation.
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| 47. |
- Ivarsson, Ylva, et al.
(författare)
-
Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications
- 2007
-
Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 0304-4165 .- 1872-8006. ; 1770:9, s. 1374-1381
-
Tidskriftsartikel (refereegranskat)abstract
- Based on the crystal structure of human glutathione transferase M1-1, cysteine residues were introduced in the substrate-binding site of a Cys-free mutant of the enzyme, which were subsequently alkylated with 1-iodoalkanes. By different combinations of site-specific mutations and chemical modifications of the enzyme the enantioselectivity in the conjugation of glutathione with the epoxide-containing substrates 1-phenylpropylene oxide and styrene-7,8-oxide were enhanced up to 9- and 10-fold. The results also demonstrate that the enantioselectivity can be diminished, or even reversed, by suitable modifications, which can be valuable under some conditions. The redesign of the active-site structure for enhanced or diminished enantioselectivities have divergent requirements for different epoxides, calling for a combinatorial approach involving alternative mutations and chemical modifications to optimize the enantioselectivity for a targeted substrate. This approach outlines a general method of great potential for fine-tuning substrate specificity and tailoring stereoselectivity of recombinant enzymes.
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| 48. |
- Ivarsson, Ylva, 1976-
(författare)
-
Evolutionary Analysis and Posttranslational Chemical Modifications in Protein Redesign : A Study on Mu Class Glutathione Transferases
- 2006
-
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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| 49. |
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| 50. |
- Ivarsson, Ylva, et al.
(författare)
-
Folding and misfolding in a naturally occurring circularly permuted PDZ domain
- 2008
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:14, s. 8954-60
-
Tidskriftsartikel (refereegranskat)abstract
- One of the most extreme and fascinating examples of naturally occurring mutagenesis is represented by circular permutation. Circular permutations involve the linking of two chain ends and cleavage at another site. Here we report the first description of the folding mechanism of a naturally occurring circularly permuted protein, a PDZ domain from the green alga Scenedesmus obliquus. Data reveal that the folding of the permuted protein is characterized by the presence of a low energy off-pathway kinetic trap. This finding contrasts with what was previously observed for canonical PDZ domains that, although displaying a similar primary structure when structurally re-aligned, fold via an on-pathway productive intermediate. Although circular permutation of PDZ domains may be necessary for a correct orientation of their functional sites in multi-domain protein scaffolds, such structural rearrangement may compromise their folding pathway. This study provides a straightforward example of the divergent demands of folding and function.
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