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  • 1. Baietti, Maria Francesca
    et al.
    Zhang, Zhe
    Mortier, Eva
    Melchior, Aurélie
    Degeest, Gisèle
    Geeraerts, Annelies
    Ivarsson, Ylva
    KU Leuven.
    Depoortere, Fabienne
    Coomans, Christien
    Vermeiren, Elke
    Zimmermann, Pascale
    David, Guido
    Syndecan-syntenin-ALIX regulates the biogenesis of exosomes2012In: Nature cell biology, ISSN 1476-4679, Vol. 14, no 7, p. 677-685Article in journal (Refereed)
    Abstract [en]

    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.

  • 2.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    High-throughput methods for identification of protein-protein interactions involving short linear motifs2015In: Cell Communication and Signaling, ISSN 1478-811X, E-ISSN 1478-811X, Vol. 13, article id 38Article, review/survey (Refereed)
    Abstract [en]

    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.

  • 3.
    Cho, S. Ei
    et al.
    Stanford Univ, Biol, Stanford, CA USA.
    Roy, J.
    Stanford Univ, Biol, Stanford, CA USA.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Cyert, M. S.
    Stanford Univ, Biol, Stanford, CA USA.
    Investigating the phospho-regulation of ER shaping protein RTN1A (Reticulon-1A) by the Calcineurin phosphatase.2017In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal (Other academic)
  • 4.
    Davey, Norman E.
    et al.
    Univ Coll Dublin, Conway Inst Biomol & Biomed Sci, Dublin 4, Ireland..
    Seo, Moon-Hyeong
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Yadav, Vikash Kumar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Jeon, Jouhyun
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Nim, Satra
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Krystkowiak, Izabella
    Univ Coll Dublin, Conway Inst Biomol & Biomed Sci, Dublin 4, Ireland..
    Blikstad, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Dong, Debbie
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Markova, Natalia
    Malvern Instruments Nord AB, Solna, Sweden..
    Kim, Philip M.
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada.;Univ Toronto, Dept Mol Genet, Toronto, ON M5S 1A1, Canada.;Univ Toronto, Dept Comp Sci, Toronto, ON M5S 1A1, Canada..
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 3, p. 485-498Article in journal (Refereed)
    Abstract [en]

    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.

  • 5.
    Ernst, Andreas
    et al.
    University of Toronto.
    Appleton, Brent A
    University of Toronto.
    Ivarsson, Ylva
    Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, The Donnelly Centre, 160 College Street, Toronto, ON M5S 3E1, Canada.
    Zhang, Yingnan
    University of Toronto.
    Gfeller, David
    University of Toronto.
    Wiesmann, Christian
    University of Toronto.
    Sidhu, Sachdev S
    University of Toronto.
    A Structural Portrait of the PDZ Domain Family2014In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, no 21, p. 3509-3519Article in journal (Refereed)
    Abstract [en]

    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.

  • 6. Fugier, Charlotte
    et al.
    Klein, Arnaud F
    Hammer, Caroline
    Vassilopoulos, Stéphane
    Ivarsson, Ylva
    KU Leuven.
    Toussaint, Anne
    Tosch, Valérie
    Vignaud, Alban
    Ferry, Arnaud
    Messaddeq, Nadia
    Kokunai, Yosuke
    Tsuburaya, Rie
    de la Grange, Pierre
    Dembele, Doulaye
    Francois, Virginie
    Precigout, Guillaume
    Boulade-Ladame, Charlotte
    Hummel, Marie-Christine
    Lopez de Munain, Adolfo
    Sergeant, Nicolas
    Laquerrière, Annie
    Thibault, Christelle
    Deryckere, François
    Auboeuf, Didier
    Garcia, Luis
    Zimmermann, Pascale
    Udd, Bjarne
    Schoser, Benedikt
    Takahashi, Masanori P
    Nishino, Ichizo
    Bassez, Guillaume
    Laporte, Jocelyn
    Furling, Denis
    Charlet-Berguerand, Nicolas
    Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy2011In: Nature medicine, ISSN 1546-170X, Vol. 17, no 6, p. 720-725Article in journal (Refereed)
    Abstract [en]

    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.

  • 7. Gallardo, Rodrigo
    et al.
    Ivarsson, Ylva
    KU Leuven.
    Schymkowitz, Joost
    Rousseau, Frédéric
    Zimmermann, Pascale
    Structural diversity of PDZ-lipid interactions2010In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 4, p. 456-67Article in journal (Refereed)
    Abstract [en]

    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.

  • 8. Ganesan, Ashok
    et al.
    Debulpaep, Maja
    Wilkinson, Hannah
    Van Durme, Joost
    De Baets, Greet
    Jonckheere, Wim
    Ramakers, Meine
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Zimmermann, Pascale
    Van Eldere, Johan
    Schymkowitz, Joost
    Rousseau, Frederic
    Selectivity of Aggregation-Determining Interactions2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 2, p. 236-247Article in journal (Refereed)
    Abstract [en]

    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.

  • 9. Garrido-Urbani, Sarah
    et al.
    Garg, Pankaj
    Ghossoub, Rania
    Arnold, Roland
    Lembo, Frédérique
    Sundell, Gustav N
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kim, Philip M
    Lopez, Marc
    Zimmermann, Pascale
    Sidhu, Sachdev S
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin2016In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 590, no 1, p. 3-12Article in journal (Refereed)
    Abstract [en]

    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.

  • 10.
    Gianni, Stefano
    et al.
    University of Rome, La Sapienza.
    Ivarsson, Ylva
    Bah, Alaji
    Bush-Pelc, Leslie A
    Di Cera, Enrico
    Mechanism of Na(+) binding to thrombin resolved by ultra-rapid kinetics2007In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 131, no 1-3, p. 111-114Article in journal (Refereed)
    Abstract [en]

    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.

  • 11. Gianni, Stefano
    et al.
    Ivarsson, Ylva
    University of Rome, La Sapienza.
    De Simone, Alfonso
    Travaglini-Allocatelli, Carlo
    Brunori, Maurizio
    Vendruscolo, Michele
    Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain2010In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 17, no 12, p. 1431-1437Article in journal (Refereed)
    Abstract [en]

    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.

  • 12. Gianni, Stefano
    et al.
    Ivarsson, Ylva
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brunori, Maurizio
    Travaglini-Allocatelli, Carlo
    Identification and characterization of protein folding intermediates2007In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 128, no 2-3, p. 105-113Article, review/survey (Refereed)
    Abstract [en]

    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.

  • 13. Haugaard-Kedstrom, L. M.
    et al.
    Albertsen, L.
    Gil, F. Abalde
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Stromgaard, K.
    The Development and Characterization of a Peptide-Based Syntenin Inhibitor: Implacations for Cancer Metastasis2014In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 20, p. S39-S39Article in journal (Other academic)
  • 14. Hämälistö, Saara
    et al.
    Pouwels, Jeroen
    de Franceschi, Nicola
    Saari, Markku
    Ivarsson, Ylva
    Zimmermann, Pascale
    Brech, Andreas
    Stenmark, Harald
    Ivaska, Johanna
    A ZO-1/α5β1-integrin complex regulates cytokinesis downstream of PKCε in NCI-H460 cells plated on fibronectin2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 8, p. e70696-Article in journal (Refereed)
    Abstract [en]

    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.

  • 15.
    Ilari, Andrea
    et al.
    Univ Roma La Sapienza, CNR, Inst Mol Biol & Pathol, I-00185 Rome, Italy.;Univ Roma La Sapienza, Dept Biochem Sci, I-00185 Rome, Italy..
    Fiorillo, Annarita
    Univ Roma La Sapienza, CNR, Inst Mol Biol & Pathol, I-00185 Rome, Italy.;Univ Roma La Sapienza, Dept Biochem Sci, I-00185 Rome, Italy..
    Poser, Elena
    Univ Roma La Sapienza, CNR, Inst Mol Biol & Pathol, I-00185 Rome, Italy.;Univ Roma La Sapienza, Dept Biochem Sci, I-00185 Rome, Italy..
    Lalioti, Vasiliki S.
    Univ Autonoma Madrid, CSIC, Ctr Biol Mol Severo Ochoa, Dept Biol Celular & Inmunol, Canto Blanco, Spain.;Ctr Invest Biomed Red Enfermedades Hepat & Digest, Madrid, Spain..
    Sundell, Gustav N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala Univ, Dept Chem BMC, S-75123 Uppsala, Sweden..
    Genovese, Ilaria
    Univ Roma La Sapienza, CNR, Inst Mol Biol & Pathol, I-00185 Rome, Italy.;Univ Roma La Sapienza, Dept Biochem Sci, I-00185 Rome, Italy..
    Colotti, Gianni
    Univ Roma La Sapienza, CNR, Inst Mol Biol & Pathol, I-00185 Rome, Italy.;Univ Roma La Sapienza, Dept Biochem Sci, I-00185 Rome, Italy..
    Structural basis of Sorcin-mediated calcium-dependent signal transduction2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 16828Article in journal (Refereed)
    Abstract [en]

    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.

  • 16. Ivarsson, Ylva
    Plasticity of PDZ domains in ligand recognition and signaling2012In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 586, no 17, p. 2638-47Article in journal (Refereed)
    Abstract [en]

    The PDZ domain is a protein-protein interacting module that plays an important role in the organization of signaling complexes. The recognition of short intrinsically disordered C-terminal peptide motifs is the archetypical PDZ function, but the functional repertoire of this versatile module also includes recognition of internal peptide sequences, dimerization and phospholipid binding. The PDZ function can be tuned by various means such as allosteric effects, changes of physiological buffer conditions and phosphorylation of PDZ domains and/or ligands, which poses PDZ domains as dynamic regulators of cell signaling. This review is focused on the plasticity of the PDZ interactions.

  • 17.
    Ivarsson, Ylva
    et al.
    Donnelly Centre, University of Toronto, Toronto, ON, Canada M5S 3E1; .
    Arnold, Roland
    University of Toronto.
    McLaughlin, Megan
    University of Toronto.
    Nim, Satra
    University of Toronto.
    Joshi, Rakesh
    Western University, London, ON .
    Ray, Debashish
    University of Toronto.
    Liu, Bernard
    University of Toronto.
    Teyra, Joan
    University of Toronto.
    Pawson, Tony
    University of Toronto.
    Moffat, Jason
    University of Toronto.
    Li, Shawn Shun-Cheng
    Western University, London, ON .
    Sidhu, Sachdev S
    University of Toronto.
    Kim, Philip M
    University of Toronto.
    Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 7, p. 2542-2547Article in journal (Refereed)
    Abstract [en]

    The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.

  • 18.
    Ivarsson, Ylva
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Mackey, Aaron J.
    Edalat, Maryam
    Pearson, William R.
    Mannervik, Bengt
    Identification of residues in glutathione transferase capable of driving functional diversification in evolution: A novel approach to protein redesign2003In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 278, no 10, p. 8733-8738Article in journal (Refereed)
  • 19.
    Ivarsson, Ylva
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Combinatorial protein chemistry in three dimensions: a paradigm for the evolution of glutathione transferases with novel activities2006In: Toxicology of Glutathione Transferases / [ed] Awasthi, Yogesh C., CRC Taylor & Francis, Boca Raton , 2006, p. 47-69Chapter in book (Other academic)
  • 20.
    Ivarsson, Ylva
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Exploring the importance of an active site residue on the stereo and regio selectivity of Mu class glutathione transferases2005In: FEBS Journal, Vol. 272, p. 88 Suppl-Article in journal (Refereed)
  • 21.
    Ivarsson, Ylva
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Regio- and enantioselectivities in epoxide conjugations are modulated by residue 210 in Mu class glutathione transferases.2005In: Protein Eng Des Sel, ISSN 1741-0126, Vol. 18, no 12, p. 607-16Article in journal (Refereed)
    Abstract [en]

    The homologous human glutathione transferases (GSTs) M1-1 and M2-2 have similar catalytic activities with many electrophilic substrates, but differ strikingly in their conjugation of epoxides with glutathione. Residue 210, Thr in GST M2-2 and Ser in GST M1-1, is a key active-site component in determining the activity profile with epoxide substrates. This residue is hypervariable in Mu class GSTs, suggesting that it has special significance in the evolution of new functions. The present study shows that minor modifications of this residue can have major consequences for the enzyme-catalyzed epoxide conjugations. In general, a Ser at position 210 gives the highest catalytic efficiency, but the relatively high activity with an Ala placed on this position demonstrates that a hydroxyl group is not required. In contrast, a Thr residue suppresses the activity with epoxides by several orders of magnitude without major effects on the activity with alternative GST substrates. Residue 210 influences both the regio- and enantioselectivity with chiral and prochiral epoxides of stilbene and styrene and influences the distribution of isomeric glutathione conjugates. Thus, residue 210 contributes to both stereoselective recognition of the substrates and to partitioning of the isomeric reactants to the alternative transition states leading to separate chiral products.

  • 22.
    Ivarsson, Ylva
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Mannervik, Bengt
    Regio- and enantioselectivity in epoxide conjugations are modulated by residue 210 in Mu class glutathione transferases2006In: Protein Engineering Design and Selection, ISSN 1741-0126, Vol. 8, no 12, p. 607-616Article in journal (Refereed)
  • 23.
    Ivarsson, Ylva
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Norrgård, Malena A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications2007In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1770, no 9, p. 1374-1381Article in journal (Refereed)
    Abstract [en]

    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.

  • 24.
    Ivarsson, Ylva
    et al.
    University of Rome, La Sapienza.
    Travaglini-Allocatelli, Carlo
    Brunori, Maurizio
    Gianni, Stefano
    Engineered symmetric connectivity of secondary structure elements highlights malleability of protein folding pathways2009In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 33, p. 11727-33Article in journal (Refereed)
    Abstract [en]

    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.

  • 25.
    Ivarsson, Ylva
    et al.
    University of Rome, La Sapienza.
    Travaglini-Allocatelli, Carlo
    Brunori, Maurizio
    Gianni, Stefano
    Folding and misfolding in a naturally occurring circularly permuted PDZ domain2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 14, p. 8954-60Article in journal (Refereed)
    Abstract [en]

    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.

  • 26.
    Ivarsson, Ylva
    et al.
    University of Rome, La Sapienza.
    Travaglini-Allocatelli, Carlo
    Brunori, Maurizio
    Gianni, Stefano
    Mechanisms of protein folding2008In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 37, no 6, p. 721-728Article in journal (Refereed)
    Abstract [en]

    Understanding the mechanism by which a polypeptide chain folds into its native structure is a central problem of modern biophysics. The collaborative efforts of experimental and theoretical studies recently raised the tantalizing possibility to define a unifying mechanism for protein folding. In this review we summarize some of these intriguing advances and analyze them together with a discussion on the new findings concerning the so-called downhill folding.

  • 27. Ivarsson, Ylva
    et al.
    Travaglini-Allocatelli, Carlo
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Malatesta, Francesco
    Brunori, Maurizio
    Gianni, Stefano
    An on-pathway intermediate in the folding of a PDZ domain2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 12, p. 8568-8572Article in journal (Refereed)
    Abstract [en]

    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.

  • 28. Ivarsson, Ylva
    et al.
    Travaglini-Allocatelli, Carlo
    Morea, Veronica
    Brunori, Maurizio
    Gianni, Stefano
    The folding pathway of an engineered circularly permuted PDZ domain2008In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, no 3, p. 155-160Article in journal (Refereed)
    Abstract [en]

    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.

  • 29.
    Ivarsson, Ylva
    et al.
    KU Leuven.
    Wawrzyniak, Anna Maria
    Kashyap, Rudra
    Polanowska, Jolanta
    Betzi, Stéphane
    Lembo, Frédérique
    Vermeiren, Elke
    Chiheb, Driss
    Lenfant, Nicolas
    Morelli, Xavier
    Borg, Jean-Paul
    Reboul, Jérôme
    Zimmermann, Pascale
    Prevalence, specificity and determinants of lipid-interacting PDZ domains from an in-cell screen and in vitro binding experiments2013In: PloS one, ISSN 1932-6203, Vol. 8, no 2, p. e54581-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: PDZ domains are highly abundant protein-protein interaction modules involved in the wiring of protein networks. Emerging evidence indicates that some PDZ domains also interact with phosphoinositides (PtdInsPs), important regulators of cell polarization and signaling. Yet our knowledge on the prevalence, specificity, affinity, and molecular determinants of PDZ-PtdInsPs interactions and on their impact on PDZ-protein interactions is very limited.

    METHODOLOGY/PRINCIPAL FINDINGS: We screened the human proteome for PtdInsPs interacting PDZ domains by a combination of in vivo cell-localization studies and in vitro dot blot and Surface Plasmon Resonance (SPR) experiments using synthetic lipids and recombinant proteins. We found that PtdInsPs interactions contribute to the cellular distribution of some PDZ domains, intriguingly also in nuclear organelles, and that a significant subgroup of PDZ domains interacts with PtdInsPs with affinities in the low-to-mid micromolar range. In vitro specificity for the head group is low, but with a trend of higher affinities for more phosphorylated PtdInsPs species. Other membrane lipids can assist PtdInsPs-interactions. PtdInsPs-interacting PDZ domains have generally high pI values and contain characteristic clusters of basic residues, hallmarks that may be used to predict additional PtdInsPs interacting PDZ domains. In tripartite binding experiments we established that peptide binding can either compete or cooperate with PtdInsPs binding depending on the combination of ligands.

    CONCLUSIONS/SIGNIFICANCE: Our screen substantially expands the set of PtdInsPs interacting PDZ domains, and shows that a full understanding of the biology of PDZ proteins will require a comprehensive insight into the intricate relationships between PDZ domains and their peptide and lipid ligands.

  • 30.
    Ivarsson, Ylva
    et al.
    KU Leuven.
    Wawrzyniak, Anna Maria
    Wuytens, Gunther
    Kosloff, Mickey
    Vermeiren, Elke
    Raport, Marie
    Zimmermann, Pascale
    Cooperative phosphoinositide and peptide binding by PSD-95/discs large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 52, p. 44669-78Article in journal (Refereed)
    Abstract [en]

    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.

  • 31. Lambaerts, Kathleen
    et al.
    Van Dyck, Stijn
    Mortier, Eva
    Ivarsson, Ylva
    KU Leuven.
    Degeest, Gisèle
    Luyten, Annouck
    Vermeiren, Elke
    Peers, Bernard
    David, Guido
    Zimmermann, Pascale
    Syntenin, a syndecan adaptor and an Arf6 phosphatidylinositol 4,5-bisphosphate effector, is essential for epiboly and gastrulation cell movements in zebrafish2012In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 125, no Pt 5, p. 1129-1140Article in journal (Refereed)
    Abstract [en]

    Epiboly, the spreading and the thinning of the blastoderm to cover the yolk cell and close the blastopore in fish embryos, is central to the process of gastrulation. Despite its fundamental importance, little is known about the molecular mechanisms that control this coordinated cell movement. By a combination of knockdown studies and rescue experiments in zebrafish (Danio rerio), we show that epiboly relies on the molecular networking of syntenin with syndecan heparan sulphate proteoglycans, which act as co-receptors for adhesion molecules and growth factors. Furthermore, we show that the interaction of syntenin with phosphatidylinositol 4,5-bisphosphate (PIP2) and with the small GTPase ADP-ribosylation factor 6 (Arf6), which regulate the endocytic recycling of syndecan, is necessary for epiboly progression. Analysis of the earliest cellular defects suggests a role for syntenin in the autonomous vegetal expansion of the yolk syncytial layer and the rearrangement of the actin cytoskeleton in extra-embryonic tissues, but not in embryonic cell fate determination. This study identifies the importance of the syntenin-syndecan-PIP2-Arf6 complex for the progression of fish epiboly and establishes its key role in directional cell movements during early development.

  • 32. Nim, Satra
    et al.
    Jeon, Jouhyun
    Corbi-Verge, Carles
    Seo, Moon-Hyeong
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Moffat, Jason
    Tarasova, Nadya
    Kim, Philip M
    Pooled screening for antiproliferative inhibitors of protein-protein interactions.2016In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 12, no 4, p. 275-281Article in journal (Refereed)
    Abstract [en]

    Protein-protein interactions (PPIs) are emerging as a promising new class of drug targets. Here, we present a novel high-throughput approach to screen inhibitors of PPIs in cells. We designed a library of 50,000 human peptide-binding motifs and used a pooled lentiviral system to express them intracellularly and screen for their effects on cell proliferation. We thereby identified inhibitors that drastically reduced the viability of a pancreatic cancer line (RWP1) while leaving a control line virtually unaffected. We identified their target interactions computationally, and validated a subset in experiments. We also discovered their potential mechanisms of action, including apoptosis and cell cycle arrest. Finally, we confirmed that synthetic lipopeptide versions of our inhibitors have similarly specific and dosage-dependent effects on cancer cell growth. Our screen reveals new drug targets and peptide drug leads, and it provides a rich data set covering phenotypes for the inhibition of thousands of interactions.

  • 33. Norrgård, Malena A.
    et al.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Tars, Kaspars
    Mannervik, Bengt
    Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution2006In: Proceedings of the National Academy of Sciences of the USA, ISSN 0027-8424, Vol. 103, no 13, p. 4876-4881Article in journal (Refereed)
  • 34.
    Norrgård, Malena A
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Tars, Kaspars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution2006In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 13, p. 4876-4881Article in journal (Refereed)
    Abstract [en]

    All molecular species in an organism are connected physically and functionally to other molecules. In evolving systems, it is not obvious to what extent functional properties of a protein can change to selective advantage and leave intact favorable traits previously acquired. This uncertainty has particular significance in the evolution of novel pathways for detoxication, because an organism challenged with new xenobiotics in the environment may still require biotransformation of previously encountered toxins. Positive selection has been proposed as an evolutionary mechanism for facile adaptive responses of proteins to changing conditions. Here, we show, by saturation mutagenesis, that mutations of a hypervariable residue in human glutathione transferase M2-2 can differentially change the enzyme's substrate-activity profile with alternative substrates and, furthermore, enable or disable dissimilar chemical reactions. Crystal structures demonstrate that activity with epoxides is enabled through removal of steric hindrance from a methyl group, whereas activities with an orthoquinone and a nitroso donor are maintained in the variant enzymes. Given the diversity of cellular activities in which a single protein can be engaged, the selective transmutation of functional properties has general significance in molecular evolution.

  • 35.
    Raykova, Doroteya
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Koos, Bjoern
    Max Planck Inst Mol Physiol, Dept Syst Cell Biol, Otto Hahn Str 11, D-44227 Dortmund, Germany..
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Gelleri, Marton
    Max Planck Inst Mol Physiol, Dept Syst Cell Biol, Otto Hahn Str 11, D-44227 Dortmund, Germany.;Tech Univ Dortmund, Fac Chem & Chem Biol, Otto Hahn Str 6, D-44221 Dortmund, Germany..
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Let There Be Light!2016In: Proteomes, ISSN 2227-7382, Vol. 4, no 4, article id 36Article, review/survey (Refereed)
    Abstract [en]

    The invention of the microscope has been fundamental for the understanding of tissue architecture and subcellular structures. With the advancement of higher magnification microscopes came the development of various molecular biology tools such as Forster resonance energy transfer (FRET) and in situ proximity ligation assay (in situ PLA) to monitor protein interactions. Microscopy has become a commonly used method for the investigation of molecular events within the cell, for the identification of key players in signaling networks, and the activation of these pathways. Multiple approaches are available for functional analyses in single cells. They provide information not only on the localization of proteins at a given time point, but also on their expression levels and activity states, allowing us to pinpoint hallmarks of different cellular identities within tissues in health and disease. Clever solutions to increase the sensitivity of molecular tools, the possibilities for multiplexing, as well as image resolution have recently been introduced; however, these methods have their pros and cons. Therefore, one needs to carefully consider the biological question of interest along with the nature of the sample before choosing the most suitable method or combination of methods. Herein, we review a few of the most exciting microscopy-based molecular techniques for proteomic analysis and cover the benefits as well as the disadvantages of their use.

  • 36.
    Roy, J.
    et al.
    Stanford Univ, Biol, Stanford, CA USA.
    Wigington, C. P.
    Stanford Univ, Biol, Stanford, CA USA.
    Damle, N. P.
    Stanford Univ, Biol, Stanford, CA USA.
    Ulengin-Talkish, I.
    Stanford Univ, Biol, Stanford, CA USA.
    Cho, S. Ei
    Stanford Univ, Biol, Stanford, CA USA.
    Davey, N. E.
    Univ Coll Dublin, Conway Inst Biomol & Biomed Res, Dublin, North Ireland.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Wong, C. J.
    Univ Toronto, Mt Sinai Hosp, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada.
    Gingras, A.
    Univ Toronto, Mt Sinai Hosp, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada.
    Cyert, M. S.
    Stanford Univ, Biol, Stanford, CA USA.
    Mapping the human calcineurin phosphatase signaling network through global identification of short linear motifs that mediate substrate recognition2017In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal (Other academic)
  • 37.
    Sundell, Gustav
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Interaction Analysis through Proteomic Phage Display2014In: BioMed Research International, ISSN 2314-6133, E-ISSN 2314-6141, p. 176172-Article, review/survey (Refereed)
    Abstract [en]

    Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance.

  • 38.
    Sundell, Gustav
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Vögeli, Beat
    Department of Biochemistry and Molecular Genetics, University of Colorado at Denver, 12801 East 17th Avenue, Aurora, Colorado 80045, United States.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    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 PDZ12018In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, no 1, p. 66-71Article in journal (Refereed)
    Abstract [en]

    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 15N/1H 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.

  • 39. Travaglini-Allocatelli, Carlo
    et al.
    Ivarsson, Ylva
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gianni, Stefano
    Folding and stability of globular proteins and implications for function2009In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 19, no 1, p. 3-7Article in journal (Refereed)
    Abstract [en]

    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.

  • 40. Wawrzyniak, Anna Maria
    et al.
    Vermeiren, Elke
    Zimmermann, Pascale
    Ivarsson, Ylva
    KU Leuven.
    Extensions of PSD-95/discs large/ZO-1 (PDZ) domains influence lipid binding and membrane targeting of syntenin-12012In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 586, no 10, p. 1445-1451Article in journal (Refereed)
    Abstract [en]

    Syntenin-1 is a PDZ protein involved in receptor recycling and clustering. Its two PDZ domains interact with various receptors and phosphoinositides, and are flanked by N- and C-terminal regions. Here, we report the identification of an autoinhibitory peptide stretch in the N-terminus that might be regulated by phosphorylation. We further establish that basic residues in the C-terminal region mediate electrostatic interactions with reconstituted liposomes and contribute to the plasma membrane targeting. Our study adds new components to the multi-dentate membrane targeting mechanism and highlights the role of N- and C-terminal PDZ extensions in the regulation of syntenin-1 plasma membrane localization.

  • 41.
    Wigington, C. P.
    et al.
    Stanford Univ, Biol, Stanford, CA 94305 USA..
    Damle, N. P.
    Stanford Univ, Biol, Stanford, CA 94305 USA..
    Roy, J.
    Stanford Univ, Biol, Stanford, CA 94305 USA..
    Cho, S. E.
    Stanford Univ, Biol, Stanford, CA 94305 USA..
    Davey, N. E.
    Univ Coll, Conway Inst Biomol & Biomed Sci, Dublin, Ireland..
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Cyert, M. S.
    Stanford Univ, Biol, Stanford, CA 94305 USA..
    Uncovering novel substrates and functions for the calcineurin phosphatase in human cells.2016In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 27, no 25, article id 3947Article in journal (Refereed)
  • 42.
    Wigington, C. P.
    et al.
    Stanford Univ, Biol, Stanford, CA USA.
    Roy, J.
    Stanford Univ, Biol, Stanford, CA USA.
    Damle, N. P.
    Stanford Univ, Biol, Stanford, CA USA.
    Ulengin-Talkish, I.
    Stanford Univ, Biol, Stanford, CA USA.
    Cho, S. Ei
    Stanford Univ, Biol, Stanford, CA USA.
    Davey, N. E.
    Univ Coll Dublin, Conway Inst Bimol & Biomed Res, Dublin, Ireland.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Wong, C. J.
    Univ Toronto, Lunenfeld Tanenbaum Res Inst, Mt Sinai Hosp, Toronto, ON, Canada.
    Gingras, A.
    Univ Toronto, Lunenfeld Tanenbaum Res Inst, Mt Sinai Hosp, Toronto, ON, Canada.
    Cyert, M. S.
    Stanford Univ, Biol, Stanford, CA USA.
    A novel role for the Calcineurin phosphatase at the nuclear pore2017In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal (Other academic)
  • 43.
    Wigington, Callie P.
    et al.
    Stanford Univ, Biol, Stanford, CA 94305 USA..
    Roy, Jagoree
    Stanford Univ, Biol, Stanford, CA 94305 USA..
    Damle, Nikhil P.
    Stanford Univ, Biol, Stanford, CA 94305 USA..
    El Cho, Shein
    Stanford Univ, Biol, Stanford, CA 94305 USA..
    Davey, Norman
    Univ Coll Dublin, Conway Inst Biomol & Biomed Res, Dublin, Ireland..
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Wong, Cassandra
    Univ Toronto, Lunenfeld Tanenbaum Res Inst, Mt Sinai Hosp, Toronto, ON, Canada..
    Gingras, Anne-Claude
    Univ Toronto, Lunenfeld Tanenbaum Res Inst, Mt Sinai Hosp, Toronto, ON, Canada..
    Cyert, Martha S.
    Stanford Univ, Biol, Stanford, CA 94305 USA..
    Uncovering Novel Substrates and Functions for the Calcineurin Phosphatase in Human Cells2017In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 31, no 1, article id 771.5Article in journal (Other academic)
  • 44.
    Wu, Cheng-Guo
    et al.
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.; Univ Wisconsin Madison, Biophys Program, Madison, WI 53706 USA.; DuPont Ind Biosci, Genencor Div, 925 Page Mill Rd, Palo Alto, CA 94304 USA.
    Chen, Hui
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.; DuPont Ind Biosci, Genencor Div, 925 Page Mill Rd, Palo Alto, CA 94304 USA.
    Guo, Feng
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.; DuPont Ind Biosci, Genencor Div, 925 Page Mill Rd, Palo Alto, CA 94304 USA.; Univ Pittsburgh, Dept Microbiol & Mol Genet, Pittsburgh, PA USA.
    Yadav, Vikash K
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. DuPont Ind Biosci, Genencor Div, 925 Page Mill Rd, Palo Alto, CA 94304 USA.
    Mcilwain, Sean J
    Univ Wisconsin Madison, Sch Med & Publ Hlth, Biostat & Med Informat, Wisconsin Inst Med Res, Madison, WI USA.
    Rowse, Michael
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.
    Choudhary, Alka
    Univ Wisconsin Madison, Sch Med & Publ Hlth, Dept Med, UW Carbone Canc Ctr,Hematol Oncol, Madison, WI USA.
    Lin, Ziqing
    Sch Med & Publ Hlth, Dept Cell & Regenerat Biol, Human Prote Program, Madison, WI USA.
    Li, Yitong
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.
    Gu, Tingjia
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.
    Zheng, Aiping
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.; Univ Pittsburgh, Dept Microbiol & Mol Genet, Pittsburgh, PA USA.
    Xu, Qingge
    Sch Med & Publ Hlth, Dept Cell & Regenerat Biol, Human Prote Program, Madison, WI USA.
    Lee, Woojong
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.
    Resch, Eduard
    Fraunhofer Inst Mol Biol & Appl Ecol IME, Project Grp Translat Med & Pharmacol TMP, Frankfurt, Germany.
    Johnson, Benjamin
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.
    Day, Jenny
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.
    Ge, Ying
    Sch Med & Publ Hlth, Dept Cell & Regenerat Biol, Human Prote Program, Madison, WI USA.
    Ong, Irene M
    Univ Wisconsin Madison, Sch Med & Publ Hlth, Biostat & Med Informat, Wisconsin Inst Med Res, Madison, WI USA.
    Burkard, Mark E
    Univ Wisconsin Madison, Sch Med & Publ Hlth, Dept Med, UW Carbone Canc Ctr,Hematol Oncol, Madison, WI USA.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Xing, Yongna
    Univ Wisconsin Madison, McArdle Lab Canc Res, Dept Oncol, Sch Med & Publ Hlth, Madison, WI 53706 USA.; Univ Wisconsin Madison, Biophys Program, Madison, WI 53706 USA.
    PP2A-B' holoenzyme substrate recognition, regulation and role in cytokinesis2017In: Cell Discovery, ISSN 2056-5968, Vol. 3, article id 17027Article in journal (Refereed)
    Abstract [en]

    Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase; it forms diverse heterotrimeric holoenzymes that counteract kinase actions. Using a peptidome that tiles the disordered regions of the human proteome, we identified proteins containing [LMFI]xx[ILV]xEx motifs that serve as interaction sites for B'-family PP2A regulatory subunits and holoenzymes. The B'-binding motifs have important roles in substrate recognition and in competitive inhibition of substrate binding. With more than 100 novel ligands identified, we confirmed that the recently identified LxxIxEx B'α-binding motifs serve as common binding sites for B' subunits with minor variations, and that S/T phosphorylation or D/E residues at positions 2, 7, 8 and 9 of the motifs reinforce interactions. Hundreds of proteins in the human proteome harbor intrinsic or phosphorylation-responsive B'-interaction motifs, and localize at distinct cellular organelles, such as midbody, predicting kinase-facilitated recruitment of PP2A-B' holoenzymes for tight spatiotemporal control of phosphorylation at mitosis and cytokinesis. Moroever, Polo-like kinase 1-mediated phosphorylation of Cyk4/RACGAP1, a centralspindlin component at the midbody, facilitates binding of both RhoA guanine nucleotide exchange factor (epithelial cell transforming sequence 2 (Ect2)) and PP2A-B' that in turn dephosphorylates Cyk4 and disrupts Ect2 binding. This feedback signaling loop precisely controls RhoA activation and specifies a restricted region for cleavage furrow ingression. Our results provide a framework for further investigation of diverse signaling circuits formed by PP2A-B' holoenzymes in various cellular processes.

1 - 44 of 44
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