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  • 1.
    Eildal, Jonas N. N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Balle, Thomas
    Stuhr-Hansen, Nicolai
    Padrah, Shahrokh
    Gianni, Stefano
    Stromgaard, Kristian
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Probing the Role of Backbone Hydrogen Bonds in Protein-Peptide Interactions by Amide-to-Ester Mutations2013In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 135, no 35, p. 12998-13007Article in journal (Refereed)
    Abstract [en]

    One of the most frequent protein-protein interaction modules in mammalian cells is the postsynaptic density 95/discs large/zonula occludens 1 (PDZ) domain, involved in scaffolding and signaling and emerging as an important drug target for several diseases. Like many other protein-protein interactions, those of the PDZ domain family involve formation of intermolecular hydrogen bonds: C-termini or internal linear motifs of proteins bind as beta-strands to form an extended antiparallel beta-sheet with the PDZ domain. Whereas extensive work has focused on the importance of the, amino acid side chains of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding to natural protein ligands. Amide-to-ester mutations of the three C-terminal amides of the peptide ligand severely affected the affinity with the PDZ domain, demonstrating that hydrogen bonds contribute significantly to ligand binding (apparent changes in binding energy, Delta Delta G = 1.3 to >3.8 kcal mol(-1)). This decrease in affinity was mainly due to an increase in the dissociation rate constant, but a significant decrease in the association rate constant was found for some amide-to-ester mutations Suggesting that native hydrogen bonds have begun to form in the transition state of the binding reaction. This study provides a general framework for studying the role of backbone hydrogen bonds in protein-peptide interactions and for the first time specifically addresses these for PDZ domain-peptide interactions.

  • 2.
    Eriksson, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Fang, Xiaotian T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Olberg, D. E.
    Univ Oslo, Dept Pharm, Oslo, Norway.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    [F-18]Tetrazine-trans-cyclooctene mediated labelling of antibodies for PET imaging of amyloid-beta2018In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, p. S643-S643Article in journal (Other academic)
  • 3.
    Fang, Xiaotian
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Meier, Silvio
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    A small bispecific antibody-based construct based on bapineuzumab as a PET tracer for amyloid beta pathology in brain2017In: Meeting abstractArticle in journal (Other academic)
  • 4.
    Fang, Xiaotian T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Meier, Silvio R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preclinical PET-MRI Platform.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    High detection sensitivity with antibody-based PET radioligand for amyloid beta in brain2019In: NeuroImage, ISSN 1053-8119, E-ISSN 1095-9572, Vol. 184, p. 881-888Article in journal (Refereed)
    Abstract [en]

    PET imaging of amyloid-beta (A beta) deposits in brain has become an important aid in Alzheimer's disease diagnosis, and an inclusion criterion for patient enrolment into clinical trials of new anti-A beta treatments. Available PET radioligands visualizing A beta bind to insoluble fibrils, i.e. A beta plaques. Levels of prefibrillar A beta forms, e.g. soluble oligomers and protofibrils, correlate better than plaques with disease severity and these soluble species are the neurotoxic form of A beta leading to neurodegeneration. The goal was to create an antibody-based radioligand, recognizing not only fibrillary A beta , but also smaller and still soluble aggregates. We designed and expressed a small recombinant bispecific antibody construct, di-scFv 3D6-8D3, targeting the A beta N-terminus and the transferrin receptor (TfR). Natively expressed at the blood-brain barrier (BBB), TfR could thus be used as a brain-blood shuttle. Di-scFv 3D6-8D3 bound to A beta 1-40 with high affinity and to TfR with moderate affinity. Di-scFv [I-124] 3D6-8D3 was injected in two transgenic mouse models overexpressing human A beta and wild-type control mice and PET scanned at 14, 24 or 72 h after injection. Di-scFv [I-124] 3D6-8D3 was retained in brain of transgenic animals while it was cleared from wild-type lacking A beta . This difference was observed from 24 h onwards, and at 72 h, 18 months old transgenic animals, with high load of A beta pathology, displayed SUVR of 2.2-3.5 in brain while wildtype showed ratios close to unity. A subset of the mice were also scanned with [C-11] PIB. Again wt mice displayed ratios of unity while transgenes showed slightly, non-significantly, elevated SUVR of 1.2, indicating improved sensitivity with novel di-scFv [I-124] 3D6-8D3 compared with [C-11] PIB. Brain concentrations of di-scFv [I-124] 3D6-8D3 correlated with soluble A beta (p < 0.0001) but not with total A beta, i.e. plaque load (p = 0.34). We have successfully created a small bispecific antibody-based radioligand capable of crossing the BBB, subsequently binding to and visualizing intrabrain A beta in vivo. The radioligand displayed better sensitivity compared with [C-11] PIB, and brain concentrations correlated with soluble neurotoxic A beta aggregates.

  • 5.
    Fang, Xiaotian T.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells2017In: Biological Procedures Online, ISSN 1480-9222, E-ISSN 1480-9222, Vol. 19, article id 11Article in journal (Refereed)
    Abstract [en]

    Background: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.

    Methods: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost).

    Results: The protocol has been used to generate multiple variants of tetra-and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (A beta) aggregates in vivo.

    Conclusions: This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.

  • 6.
    Gustafsson, Sofia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gustavsson, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Roshanbin, Sahar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hammarlund-Udenaes, Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Blood-Brain Barrier Integrity in a Mouse Model of Alzheimer’s Disease With or Without Acute 3D6 Immunotherapy2018In: Neuropharmacology, ISSN 0028-3908, E-ISSN 1873-7064, Vol. 143, p. 1-9Article in journal (Refereed)
    Abstract [en]

    The blood-brain barrier (BBB) is suggested to be compromised in Alzheimer's disease (AD). The concomitant presence of vascular amyloid beta (AD) pathology, so called cerebral amyloid angiopathy (CAA), also predisposes impairment of vessel integrity. Additionally, immunotherapy against A beta may lead to further damage of the BBB. To what extent this affects the BBB passage of molecules is debated. The current study aimed to investigate BBB integrity to large molecules in transgenic mice displaying abundant A beta pathology and age matched wild type animals, with or without acute anti-A beta antibody treatment. Animals were administered a single i.v. injection of PBS or 3D6 (10 mg/kg), i.e. the murine version of the clinically investigated A beta antibody bapineuzumab, supplemented with [(125)]3D6. Three days post injections, a 4 kDa FITC and a 150 kDa Antonia Red dextran were administered i.v. to all animals. After termination, fluorescent detection in brain and serum was used for the calculation of dextran brain-to-blood concentration ratios. Further characterization of antibody fate and the presence of CAA were investigated using radioactivity measurements and Congo red staining. BBB passage of large molecules was equally low in wild type and transgenic mice, suggesting an intact BBB despite A beta pathology. Neither was the BBB integrity affected by acute 3D6 treatment. However, CAA was confirmed in the transgenes and local antibody accumulations were observed in the brain, indicating CAA-antibody interactions. The current study shows that independently of A beta pathology or acute 3D6 treatment, the BBB is intact, without extensive permeability to large molecules, including the 3D6 antibody.

  • 7. Haq, S. Raza
    et al.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bach, Anders
    Dogan, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Karlsson, O. Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lundstrom, Patrik
    Montemiglio, Linda C.
    Stromgaard, Kristian
    Gianni, Stefano
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Side-Chain Interactions Form Late and Cooperatively in the Binding Reaction between Disordered Peptides and PDZ Domains2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 1, p. 599-605Article in journal (Refereed)
    Abstract [en]

    Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. Here we have used short peptides as a model system for intrinsically disordered proteins. Linear free energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate-limiting barrier for binding and in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins toward their targets are generally governed by their association rate constants. Instead, we observed the opposite for peptide-PDZ interactions, namely, that changes in K-d correlate with changes in k(off).

  • 8.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Protein Folding, Binding and Evolution: PDZ domains and paralemmins as model systems2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins present at the synapse need to be multitasking in order to perform all vital functions in this limited space. In this thesis I have analyzed the function and evolution of such proteins, focusing on the PDZ domain and the paralemmin family. The PDZ domains bind to a wide variety of interaction partners. The affinity for each partner is regulated by residues at the binding site, but also through intradomain allostery. How this intradomain allostery is transferred to the binding site is not established. I here show that side chain interactions can explain all transfer of intradomain allostery in three analyzed PDZ domains. A circularly permuted PDZ domain has an identical set of amino acids as the original protein and a very similar structure with only a few perturbed side chains. By using the circular permutant I show that a slight alteration in the position of a side chain leads to a corresponding change in allosteric signal. I further study the folding of several PDZ domains and show that they all fold via a conserved folding mechanism, supporting the notion that the final structure has a part in deciding folding mechanism. The folding mechanism of the circularly permuted PDZ domain is conserved compared to the original protein illustrating how circular permutations can be tolerated through evolution. The multifunctionality of paralemmins probably lies in their highly flexible structures. I have studied the evolution of the paralemmins and found that the four mammalian paralemmins arose in the two whole-genome duplications that occurred early in the vertebrate evolution. The fact that all four paralemmins have survived evolution since the gene duplications suggests that they have important functions, possibly in the development of the nervous system. Synaptic proteins are crucial for many biological processes, and their misfolding implicated in many diseases. The results presented here shed light on the mechanisms of action of the synaptic proteins and will help us to understand how they generate disease.

    List of papers
    1. An expanded view of the protein folding landscape of PDZ domains
    Open this publication in new window or tab >>An expanded view of the protein folding landscape of PDZ domains
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    2012 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 421, no 3, p. 550-553Article in journal (Refereed) Published
    Abstract [en]

    Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting for the folding reaction under different conditions, as seen for the PDZ protein domain family. We have here analyzed the folding kinetics of two PDZ domains and found that a previously undetected third transition state is rate limiting under conditions that stabilize the native state relative to the denatured state. In light of these results, we have re-analyzed previous folding data on PDZ domains and present a unified folding mechanism with three distinct transition states separated by two high-energy intermediates. Our data show that sequence composition tunes the relative stabilities of folding transition states within the PDZ family, while the overall mechanism is determined by topology. This model captures the kinetic folding mechanism of all PDZ domains studied to date.

    Keywords
    PDZ folding transition state
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-185575 (URN)10.1016/j.bbrc.2012.04.042 (DOI)000306152000025 ()22521641 (PubMedID)
    Available from: 2012-11-26 Created: 2012-11-26 Last updated: 2017-10-16Bibliographically approved
    2. Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain
    Open this publication in new window or tab >>Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain
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    2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, p. e50055-Article in journal (Refereed) Published
    Abstract [en]

    Circular permutation is a common molecular mechanism for evolution of proteins. However, such re-arrangement of secondary structure connectivity may interfere with the folding mechanism causing accumulation of folding intermediates, which in turn can lead to misfolding. We solved the crystal structure and investigated the folding pathway of a circularly permuted variant of a PDZ domain, SAP97 PDZ2. Our data illustrate how well circular permutation may work as a mechanism for molecular evolution. The circular permutant retains the overall structure and function of the native protein domain. Further, unlike most examples in the literature, this circular permutant displays a folding mechanism that is virtually identical to that of the wild type. This observation contrasts with previous data on the circularly permuted PDZ2 domain from PTP-BL, for which the folding pathway was remarkably affected by the same mutation in sequence connectivity. The different effects of this circular permutation in two homologous proteins show the strong influence of sequence as compared to topology. Circular permutation, when peripheral to the major folding nucleus, may have little effect on folding pathways and could explain why, despite the dramatic change in primary structure, it is frequently tolerated by different protein folds.

    Keywords
    Circular permutant, protein folding, PDZ domain
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-185577 (URN)10.1371/journal.pone.0050055 (DOI)000311821000173 ()
    Available from: 2012-11-26 Created: 2012-11-26 Last updated: 2017-12-07Bibliographically approved
    3. Energetic pathway sampling in a protein interaction domain
    Open this publication in new window or tab >>Energetic pathway sampling in a protein interaction domain
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    2013 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, p. 1193-1202Article in journal (Other academic) Published
    Abstract [en]

    The affinity and specificity of protein-ligand interactions are influenced by energeticcrosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interactionpathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands. 

    Keywords
    intradomain allostery, PDZ domain, protein binding, circular permutant
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-185579 (URN)10.1016/j.str.2013.05.010 (DOI)000321681600016 ()
    Available from: 2012-12-10 Created: 2012-11-26 Last updated: 2017-10-16Bibliographically approved
    4. Evolution of the Vertebrate Paralemmin Gene Family: Ancient Origin of Gene Duplicates Suggests Distinct Functions
    Open this publication in new window or tab >>Evolution of the Vertebrate Paralemmin Gene Family: Ancient Origin of Gene Duplicates Suggests Distinct Functions
    2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 7, p. e41850-Article in journal (Refereed) Published
    Abstract [en]

    Paralemmin-1 is a protein implicated in plasma membrane dynamics, the development of filopodia, neurites and dendritic spines, as well as the invasiveness and metastatic potential of cancer cells. However, little is known about its mode of action, or about the biological functions of the other paralemmin isoforms: paralemmin-2, paralemmin-3 and palmdelphin. We describe here evolutionary analyses of the paralemmin gene family in a broad range of vertebrate species. Our results suggest that the four paralemmin isoform genes (PALM1, PALM2, PALM3 and PALMD) arose by quadruplication of an ancestral gene in the two early vertebrate genome duplications. Paralemmin-1 and palmdelphin were further duplicated in the teleost fish specific genome duplication. We identified a unique sequence motif common to all paralemmins, consisting of 11 highly conserved residues of which four are invariant. A single full-length paralemmin homolog with this motif was identified in the genome of the sea lamprey Petromyzon marinus and an isolated putative paralemmin motif could be detected in the genome of the lancelet Branchiostoma floridae. This allows us to conclude that the paralemmin gene family arose early and has been maintained throughout vertebrate evolution, suggesting functional diversification and specific biological roles of the paralemmin isoforms. The paralemmin genes have also maintained specific features of gene organisation and sequence. This includes the occurrence of closely linked downstream genes, initially identified as a readthrough fusion protein with mammalian paralemmin-2 (Palm2-AKAP2). We have found evidence for such an arrangement for paralemmin-1 and -2 in several vertebrate genomes, as well as for palmdelphin and paralemmin-3 in teleost fish genomes, and suggest the name paralemmin downstream genes (PDG) for this new gene family. Thus, our findings point to ancient roles for paralemmins and distinct biological functions of the gene duplicates.

    Keywords
    genome duplication, molecular characterization, chromosome duplications, membrane dynamics, amphioxus genome, teleost fishes, protein family, insights, reveals, identification
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-184483 (URN)10.1371/journal.pone.0041850 (DOI)000306806600134 ()
    Available from: 2012-11-08 Created: 2012-11-07 Last updated: 2019-01-03Bibliographically approved
  • 9.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Haq, Raza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Punekar, Avinash
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Chi, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bach, Anders
    Strømgaard, Kristian
    University of Copenhagen.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gianni, Stefano
    Sapienza Università di Roma.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Energetic pathway sampling in a protein interaction domain2013In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, p. 1193-1202Article in journal (Other academic)
    Abstract [en]

    The affinity and specificity of protein-ligand interactions are influenced by energeticcrosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interactionpathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands. 

  • 10.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Molecular Cell Biology.
    Ocampo Daza, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Larhammar, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Kilimann, Manfred W.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Molecular Cell Biology.
    Evolution of the Vertebrate Paralemmin Gene Family: Ancient Origin of Gene Duplicates Suggests Distinct Functions2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 7, p. e41850-Article in journal (Refereed)
    Abstract [en]

    Paralemmin-1 is a protein implicated in plasma membrane dynamics, the development of filopodia, neurites and dendritic spines, as well as the invasiveness and metastatic potential of cancer cells. However, little is known about its mode of action, or about the biological functions of the other paralemmin isoforms: paralemmin-2, paralemmin-3 and palmdelphin. We describe here evolutionary analyses of the paralemmin gene family in a broad range of vertebrate species. Our results suggest that the four paralemmin isoform genes (PALM1, PALM2, PALM3 and PALMD) arose by quadruplication of an ancestral gene in the two early vertebrate genome duplications. Paralemmin-1 and palmdelphin were further duplicated in the teleost fish specific genome duplication. We identified a unique sequence motif common to all paralemmins, consisting of 11 highly conserved residues of which four are invariant. A single full-length paralemmin homolog with this motif was identified in the genome of the sea lamprey Petromyzon marinus and an isolated putative paralemmin motif could be detected in the genome of the lancelet Branchiostoma floridae. This allows us to conclude that the paralemmin gene family arose early and has been maintained throughout vertebrate evolution, suggesting functional diversification and specific biological roles of the paralemmin isoforms. The paralemmin genes have also maintained specific features of gene organisation and sequence. This includes the occurrence of closely linked downstream genes, initially identified as a readthrough fusion protein with mammalian paralemmin-2 (Palm2-AKAP2). We have found evidence for such an arrangement for paralemmin-1 and -2 in several vertebrate genomes, as well as for palmdelphin and paralemmin-3 in teleost fish genomes, and suggest the name paralemmin downstream genes (PDG) for this new gene family. Thus, our findings point to ancient roles for paralemmins and distinct biological functions of the gene duplicates.

  • 11.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pedersen, Søren
    University of Copenhagen.
    Strømgaard, Kristian
    University of Copenhagen.
    Chi, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Gianni, Stefano
    Sapienza Università di Roma.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    An expanded view of the protein folding landscape of PDZ domains2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 421, no 3, p. 550-553Article in journal (Refereed)
    Abstract [en]

    Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting for the folding reaction under different conditions, as seen for the PDZ protein domain family. We have here analyzed the folding kinetics of two PDZ domains and found that a previously undetected third transition state is rate limiting under conditions that stabilize the native state relative to the denatured state. In light of these results, we have re-analyzed previous folding data on PDZ domains and present a unified folding mechanism with three distinct transition states separated by two high-energy intermediates. Our data show that sequence composition tunes the relative stabilities of folding transition states within the PDZ family, while the overall mechanism is determined by topology. This model captures the kinetic folding mechanism of all PDZ domains studied to date.

  • 12.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Punekar, Avinash
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Morrone, Angela
    Sapienza Università di Roma.
    Chi, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Gianni, Stefano
    Sapienza Università di Roma.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, p. e50055-Article in journal (Refereed)
    Abstract [en]

    Circular permutation is a common molecular mechanism for evolution of proteins. However, such re-arrangement of secondary structure connectivity may interfere with the folding mechanism causing accumulation of folding intermediates, which in turn can lead to misfolding. We solved the crystal structure and investigated the folding pathway of a circularly permuted variant of a PDZ domain, SAP97 PDZ2. Our data illustrate how well circular permutation may work as a mechanism for molecular evolution. The circular permutant retains the overall structure and function of the native protein domain. Further, unlike most examples in the literature, this circular permutant displays a folding mechanism that is virtually identical to that of the wild type. This observation contrasts with previous data on the circularly permuted PDZ2 domain from PTP-BL, for which the folding pathway was remarkably affected by the same mutation in sequence connectivity. The different effects of this circular permutation in two homologous proteins show the strong influence of sequence as compared to topology. Circular permutation, when peripheral to the major folding nucleus, may have little effect on folding pathways and could explain why, despite the dramatic change in primary structure, it is frequently tolerated by different protein folds.

  • 13.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Fang, Xiaotian T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Bivalent Brain Shuttle Increases Antibody Uptake by Monovalent Binding to the Transferrin Receptor2017In: Theranostics, ISSN 1838-7640, E-ISSN 1838-7640, Vol. 7, no 2, p. 308-318Article in journal (Refereed)
    Abstract [en]

    The blood-brain barrier (BBB) is an obstacle for antibody passage into the brain, impeding the development of immunotherapy and antibody-based diagnostics for brain disorders. In the present study, we have developed a brain shuttle for active transport of antibodies across the BBB by receptor-mediated transcytosis. We have thus recombinantly fused two single-chain variable fragments (scFv) of the transferrin receptor (TfR) antibody 8D3 to the light chains of mAb158, an antibody selectively binding to A beta protofibrils, which are involved in the pathogenesis of Alzheimer's disease (AD). Despite the two TfR binders, a monovalent interaction with TfR was achieved due to the short linkers that sterically hinder bivalent binding to the TfR dimer. The design enabled efficient receptor-mediated brain uptake of the fusion protein. Two hours after administration, brain concentrations were 2-3% of the injected dose per gram brain, comparable to small molecular drugs and 80-fold higher than unmodified mAb158. After three days, fusion protein concentrations in AD transgenic mouse brains were 9-fold higher than in wild type mice, demonstrating high in vivo specificity. Thus, our innovative recombinant design markedly increases mAb158 brain uptake, which makes it a strong candidate for improved Aa immunotherapy and as a PET radioligand for early diagnosis and evaluation of treatment effect in AD. Moreover, this approach could be applied to any target within the brain.

  • 14.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Åberg, Emma
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Camilloni, Carlo
    Univ Cambridge, Dept Chem, Cambridge, England.;Tech Univ Munich, Dept Chem, Munich, Germany.;Tech Univ Munich, Inst Adv Study, Munich, Germany..
    Sundell, Gustav
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Andersson, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dogan, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden..
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH, Lab Phys Chem, Zurich, Switzerland.
    Vendruscolo, Michele
    Univ Cambridge, Dept Chem, Cambridge, England.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, Dept Med Biochem & Microbiol, Uppsala, Sweden..
    Emergence and evolution of an interaction between intrinsically disordered proteins2017In: eLIFE, E-ISSN 2050-084X, Vol. 6, article id e16059Article in journal (Refereed)
    Abstract [en]

    Protein-protein interactions involving intrinsically disordered proteins are important for cellular function and common in all organisms. However, it is not clear how such interactions emerge and evolve on a molecular level. We performed phylogenetic reconstruction, resurrection and biophysical characterization of two interacting disordered protein domains, CID and NCBD. CID appeared after the divergence of protostomes and deuterostomes 450-600 million years ago, while NCBD was present in the protostome/deuterostome ancestor. The most ancient CID/NCBD formed a relatively weak complex (K(d similar to)5 mu M). At the time of the first vertebrate-specific whole genome duplication, the affinity had increased (K-d\similar to 200 nM) and was maintained in further speciation. Experiments together with molecular modeling using NMR chemical shifts suggest that new interactions involving intrinsically disordered proteins may evolve via a low-affinity complex which is optimized by modulating direct interactions as well as dynamics, while tolerating several potentially disruptive mutations.

  • 15.
    Jemth, Per
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Karlsson, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Vogeli, Beat
    Univ Colorado Denver, Dept Biochem & Mol Genet, 12801 East 17th Ave, Aurora, CO 80045 USA.
    Guzovsky, Brenda
    Univ Buenos Aires, IQUIBICEN CONICET, FCEyN, Prot Physiol Lab, Intendente Guiraldes 2160,Ciudad Univ,C1428EGA, Buenos Aires, DF, Argentina.
    Andersson, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Dogan, Jakob
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden.
    Guntert, Peter
    Swiss Fed Inst Technol, Lab Phys Chem, ETH Honggerberg, Zurich, Switzerland;Goethe Univ, Ctr Biomol Magnet Resonance, Inst Biophys Chem, D-60438 Frankfurt, Germany;Tokyo Metropolitan Univ, Grad Sch Sci, Tokyo 1920397, Japan.
    Riek, Roland
    Swiss Fed Inst Technol, Lab Phys Chem, ETH Honggerberg, Zurich, Switzerland.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Structure and dynamics conspire in the evolution of affinity between intrinsically disordered proteins2018In: Science Advances, E-ISSN 2375-2548, Vol. 4, no 10, article id eaau4130Article in journal (Refereed)
    Abstract [en]

    In every established species, protein-protein interactions have evolved such that they are fit for purpose. However, the molecular details of the evolution of new protein-protein interactions are poorly understood. We have used nuclear magnetic resonance spectroscopy to investigate the changes in structure and dynamics during the evolution of a protein-protein interaction involving the intrinsically disordered CREBBP (CREB-binding protein) interaction domain (CID) and nuclear coactivator binding domain (NCBD) from the transcriptional coregulators NCOA (nuclear receptor coactivator) and CREBBP/p300, respectively. The most ancient low-affinity "Cambrian-like" [540 to 600 million years (Ma) ago] CID/NCBD complex contained less secondary structure and was more dynamic than the complexes from an evolutionarily younger "Ordovician-Silurian" fish ancestor (ca. 440 Ma ago) and extant human. The most ancient Cambrian-like CID/NCBD complex lacked one helix and several interdomain interactions, resulting in a larger solvent-accessible surface area. Furthermore, the most ancient complex had a high degree of millisecond-to-microsecond dynamics distributed along the entire sequences of both CID and NCBD. These motions were reduced in the Ordovician-Silurian CID/NCBD complex and further redistributed in the extant human CID/NCBD complex. Isothermal calorimetry experiments show that complex formation is enthalpically favorable and that affinity is modulated by a largely unfavorable entropic contribution to binding. Our data demonstrate how changes in structure and motion conspire to shape affinity during the evolution of a protein-protein complex and provide direct evidence for the role of structural, dynamic, and frustrational plasticity in the evolution of interactions between intrinsically disordered proteins.

  • 16.
    Limbach, Christoph
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Laue, Michael M.
    Wang, Xiaolu
    Hu, Bin
    Thiede, Nadine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Molecular Cell Biology.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Kilimann, Manfred W.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Molecular in situ topology of Aczonin/Piccolo and associated proteins at the mammalian neurotransmitter release site2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 31, p. E392-E401Article in journal (Refereed)
    Abstract [en]

    The protein machinery of neurotransmitter exocytosis requires efficient orchestration in space and time, for speed and precision of neurotransmission and also for synaptic ontogeny and plasticity. However, its spatial organization in situ is virtually unknown. Aczonin/Piccolo is a putative organizer protein of mammalian active zones. We determined by immunogold electron microscopy (EM) (i) the spatial arrangement (i. e., topology) of 11 segments of the Aczonin polypeptide in situ, and correlated it to (ii) the positioning of Aczonin-interacting domains of Bassoon, CAST/ELKS, Munc13, and RIM and (iii) the ultrastructurally defined presynaptic macromolecular aggregates known as dense projections and synaptic ribbons. At conventional synapses, Aczonin assumes a compact molecular topology within a layer 35 to 80 nm parallel to the plasma membrane (PM), with a "trunk" sitting on the dense projection top and a C-terminal "arm" extending down toward the PM and sideward to the dense projection periphery. At ribbon synapses, Aczonin occupies the whole ribbon area. Bassoon colocalizes with Aczonin at conventional synapses but not at ribbon synapses. At both conventional and ribbon synapses, CAST, Munc13, and RIM are segregated from Aczonin, closer to the PM, and Aczonin is positioned such that it may control the access of neurotransmitter vesicles to the fusion site.

  • 17.
    Meier, Silvio R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fang, Xiaotian T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Roshanbin, Sahar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. BioArctic AB, Stockholm, Sweden.
    Neumann, Ulf
    Novartis Inst BioMed Res, Neurosci Res, Basel, Switzerland.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Antibody-Based In Vivo PET Imaging Detects Amyloid-beta Reduction in Alzheimer Transgenic Mice After BACE-1 Inhibition2018In: Journal of Nuclear Medicine, ISSN 0161-5505, E-ISSN 1535-5667, Vol. 59, no 12, p. 1885-1891Article in journal (Refereed)
    Abstract [en]

    Visualization of amyloid-beta (A beta) pathology with PET has become an important tool for making a specific clinical diagnosis of Alzheimer disease (AD). However, the available amyloid PET radioligands, such as C-11-Pittsburgh compound B, reflect levels of insoluble A beta plaques but do not capture soluble and protofibrillar A beta forms. Furthermore, the plaque load appears to be fairly static during clinical stages of AD and may not be affected by A beta-reducing treatments. The aim of the present study was to investigate whether a novel PET radioligand based on an antibody directed toward soluble aggregates of A beta can be used to detect changes in A beta levels during disease progression and after treatment with a beta-secretase (BACE-1) inhibitor. Methods: One set of transgenic mice (tg-ArcSwe, a model of A beta pathology) aged between 7 and 16 mo underwent PET with the A beta protofibril-selective radioligand I-124-RmAb158-scFv8D3 (where RmAb is recombinant mouse monoclonal antibody and scFv is single-chain variable fragment) to follow progression of A beta pathology in the brain. A second set of tg-ArcSwe mice, aged 10 mo, were treated with the BACE-1 inhibitor NB-360 for 3 mo and compared with an untreated control group. A third set of tg-ArcSwe mice, also aged 10 mo, underwent PET as a baseline group. Brain tissue was isolated after PET to determine levels of A beta by ELISA and immunohistochemistry. Results: The concentration of I-124-RmAb158-scFv8D3, as measured in vivo with PET, increased with age and corresponded well with the ex vivo autoradiography and A beta immunohistochemistry results. Mice treated with NB-360 showed significantly lower in vivo PET signals than untreated animals and were similar to the baseline animals. The decreased I-124-RmAb158-scFv8D3 concentrations in NB-360-treated mice, as quantified with PET, corresponded well with the decreased A beta levels measured in postmortem brain. Conclusion: Several treatments for AD are in phase 2 and 3 clinical trials, but the possibility of studying treatment effects in vivo on the important, nonfibrillar, forms of A beta is limited. This study demonstrated the ability of the A beta protofibril-selective radioligand I-124-RmAb158-scFv8D3 to follow disease progression and detect treatment effects with PET imaging in tg-ArcSwe mice.

  • 18.
    Pedersen, Soren W.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Stromgaard, Kristian
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 4, p. e95619-Article in journal (Refereed)
    Abstract [en]

    Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a b-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.

  • 19. Pedersen, Soren W.
    et al.
    Pedersen, Stine B.
    Anker, Louise
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kristensen, Anders S.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Stromgaard, Kristian
    Probing backbone hydrogen bonding in PDZ/ligand interactions by protein amide-to-ester mutations2014In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, p. 3215-Article in journal (Refereed)
    Abstract [en]

    PDZ domains are scaffolding modules in protein-protein interactions that mediate numerous physiological functions by interacting canonically with the C-terminus or non-canonically with an internal motif of protein ligands. A conserved carboxylate-binding site in the PDZ domain facilitates binding via backbone hydrogen bonds; however, little is known about the role of these hydrogen bonds due to experimental challenges with backbone mutations. Here we address this interaction by generating semisynthetic PDZ domains containing backbone amide-to-ester mutations and evaluating the importance of individual hydrogen bonds for ligand binding. We observe substantial and differential effects upon amide-to-ester mutation in PDZ2 of postsynaptic density protein 95 and other PDZ domains, suggesting that hydrogen bonding at the carboxylate-binding site contributes to both affinity and selectivity. In particular, the hydrogen-bonding pattern is surprisingly different between the non-canonical and canonical interaction. Our data provide a detailed understanding of the role of hydrogen bonds in protein-protein interactions.

  • 20.
    Sehlin, Dag
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Gumucio, Astrid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Laudon, H.
    BioArctic AB, Biochem, Stockholm, Sweden.
    Söderberg, L.
    BioArctic AB, Biochem, Stockholm, Sweden.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Efficient clearance of Amyloid-beta protofibrils in APP-transgenic mice treated with a brain penetrating bifunctional antibody2017In: Journal of Cerebral Blood Flow and Metabolism, ISSN 0271-678X, E-ISSN 1559-7016, Vol. 37, p. 415-416Article in journal (Other academic)
  • 21.
    Stenler, Sofia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Roshanbin, Sahar
    Yilmaz Ugur, Canan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Rostami, Jinar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Aguilar, Ximena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Over the BBB and into the cell: Pursuing intracellular targets for immunotherapy of Parkinson’s disease2019Conference paper (Other academic)
    Abstract [en]

    The aim of our research is to modify therapeutic antibodies so that they can reach their dementia target inside cells located on the other side of the blood brain barrier. While the aggregates associated with Alzheimer’s are located extracellularly and thus readily available for antibodies that have crossed the BBB barrier, this is not the case for Parkinson’s disease. In this study, we focus on developing a peptide shuttle that can deliver antibodies not only over the BBB but also into neuronal cells where the Tau and a-synuclein aggregates can be found.

    For this purpose, we have investigated the use of a peptide which binds to a receptor that co- localizes with the aggregates. Our in-house experience suggests that the peptide is not an efficient BBB transporter despite the fact that some groups have used it as such, but that it might be more suitable as a transporter for intracellular delivery.

    We have successfully expressed recombinant antibodies with the peptide on the N-terminal of an antibody targeting the aggregates associated with Parkinson’s disease. Our initial studies indicate that the tyrosine on the N-terminal of the peptide needs to be free and unmodified to be able to enhance uptake into neuronal cells. This hinders the use of the normal labelling method which attaches radiolabelled iodine to tyrosines where the affinity for peptide target would be destroyed. We have been pursuing alternative methods, such as using click chemistry to attach the peptide which will leave the antibody free to be radiolabelled, as well as methods to detect unlabelled antibodies in vivo and in vitro.

    We have assessed the peptide-assisted increase in uptake in appropriate neuronal cell line models. Furthermore, we have studied uptake and retention in brain in mouse models for Parkinson’s disease.

  • 22.
    Syvänen, Stina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Fang, Xiaotian T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Falting, J.
    BioArctic AB..
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Molecular Imaging. Uppsala Univ Hosp, PET Ctr..
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. Uppsala Univ, Uppsala, Sweden..
    Antibody-based PET radioligands for imaging of amyloid-beta protofibrils2017In: Journal of Cerebral Blood Flow and Metabolism, ISSN 0271-678X, E-ISSN 1559-7016, Vol. 37, p. 84-84Article in journal (Other academic)
  • 23.
    Syvänen, Stina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Fang, Xiaotian T.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Meier, Silvio R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    A bispecific Tribody PET radioligand for visualization of amyloid-beta protofibrils - a new concept for neuroimaging2017In: NeuroImage, ISSN 1053-8119, E-ISSN 1095-9572, Vol. 148, p. 55-63Article in journal (Refereed)
    Abstract [en]

    Antibodies are highly specific for their target molecules, but their poor brain penetrance has restricted their use as PET ligands for imaging of targets within the CNS. The aim of this study was to develop an antibody-based radioligand, using the Tribody(TM) format, for PET imaging of soluble amyloid-beta (All) protofibrils, which are suggested to cause neurodegeneration in Alzheimer's disease. Antibodies, even when expressed in smaller engineered formats, are large molecules that do not enter the brain in sufficient amounts for imaging purposes. Hence, their transport across the blood-brain barrier (BBB) needs to be facilitated, for example through interaction with the transferrin receptor (TfR). Thus, a Fab fragment of the TfR antibody 8D3 was fused with two single chain variable fragments (scFv) of the A beta protofibril selective antibody mAb158. Five Tribody proteins (A1-A5) were generated with different linkers between the Fab-8D3 and scFv-158. All proteins bound to TfR and All protofibrils in vitro. Three of the proteins (A1-A3) were radiolabeled with iodine-125 and studied ex vivo in wild-type (wt) and transgenic mice overexpressing human All. The systemic pharmacokinetics were similar with half-lives in blood of around 9 h for all three ligands. Brain concentrations at 2 h were around 1% of the injected dose per gram brain tissue, which is similar to what is observed for small molecular radioligands and at least 10-fold higher than antibodies in general. At 72 h, transgenic mice showed higher concentrations of radioactivity in the brain than wt mice (12, 15- and 16-fold for Al, A2 and A3 respectively), except in the cerebellum, an area largely devoid of A beta pathology. A3 was then labelled with iodine-124 for in vivo positron emission tomography (PET) imaging. Brain concentrations were quantified in six different regions showing a clear distinction both quantitatively and visually between wt and transgenic mice and a good correlation with A beta pathology. We have thus produced a recombinant, bispecific protein, actively transported into the brain, for PET imaging within the CNS. In a longer perspective, this technique may enable imaging of other proteins involved in neurodegenerative diseases for which imaging agents are completely lacking today.

  • 24.
    Syvänen, Stina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gustavsson, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Gumucio, Astrid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Laudon, Hanna
    BioArctic AB, Stockholm, Sweden.
    Söderberg, Linda
    BioArctic AB, Stockholm, Sweden.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. BioArctic AB, Stockholm, Sweden.
    Sehlin, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Efficient clearence of A beta protofibrils in A beta PP-transgenic mice treated with a brain-penetrating bifunctional antibody2018In: Alzheimer's Research & Therapy, E-ISSN 1758-9193, Vol. 10, article id 49Article in journal (Refereed)
    Abstract [en]

    Background: Amyloid-beta (A beta) immunotherapy is one of the most promising disease-modifying strategies for Alzheimer's disease (AD) Despite recent progress targeting aggregated forms of A beta, low antibody brain penetrance remains a challenge In the piesent study, we used transferrin receptor (TfR)-mediated transcytosis to facilitate brain uptake of our previously developed A beta protofibril-selective mAb158, with the aim of increasing the efficacy of immunotherapy directed toward soluble A beta protofibills. Methods: A beta protein precursor (A beta PP)-transgenic mice (tg-ArcSwe) were given a single dose of mAb158, modified for TfR-mediated transcytosis (RmAb158-scFvSDB), in companson with an equimolar dose or a tenfold higher dose of unmodified recombinant mAb158 (RmAb158) Soluble A beta protofibrills and total A beta in the brain were measured by enzyme-linked immunosorbent assay (ELISA) Brain distribution of radiolabeled antibodies was visualized by positron emission tomography (PET) and ex vivo autoiadiography. Results: ELISA analysis of Tris-buffered saline brain extracts demonstrated a 40% reduction of soluble A beta protofibrils in both RmAb158-scFv8D3- and high-dose RmAb158-treated mice, whereas there was no A beta protofibril reduction in mice treated with a low dose of RmAb158. Further, ex vivo autoradiography and PET imaging revealed diffeient brain distribution patterns of RmAb158-scFv8D3 and RmAb158, suggesting that these antibodies may affect A beta levels by different mechanisms. Conclusions: With a combination of biochemical and imaging analyses, this study demonstrates that antibodies engineered to be transported across the blood brain barrier can be used to increase the efficacy of A beta immunotherapy. This strategy may allow for decreased antibody doses and thereby reduced side effects and treatment costs.

  • 25.
    Åberg, Emma
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Saccoccia, Fulvio
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Grabherr, Manfred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ore, Wai Ying Josefin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Evolution of the p53-MDM2 pathway2017In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 17, article id 177Article in journal (Refereed)
    Abstract [en]

    Background: The p53 signalling pathway, which controls cell fate, has been extensively studied due to its prominent role in tumor development. The pathway includes the tumor supressor protein p53, its vertebrate paralogs p63 and p73, and their negative regulators MDM2 and MDM4. The p53/p63/p73-MDM system is ancient and can be traced in all extant animal phyla. Despite this, correct phylogenetic trees including both vertebrate and invertebrate species of the p53/p63/p73 and MDM families have not been published. Results: Here, we have examined the evolution of the p53/p63/p73 protein family with particular focus on the p53/ p63/p73 transactivation domain (TAD) and its co-evolution with the p53/p63/p73- binding domain (p53/p63/p73BD) of MDM2. We found that the TAD and p53/p63/p73BD share a strong evolutionary connection. If one of the domains of the protein is lost in a phylum, then it seems very likely to be followed by loss of function by the other domain as well, and due to the loss of function it is likely to eventually disappear. By focusing our phylogenetic analysis to p53/p63/ p73 and MDM proteins from phyla that retain the interaction domains TAD and p53/p63/p73BD, we built phylogenetic trees of p53/p63/p73 and MDM based on both vertebrate and invertebrate species. The trees follow species evolution and contain a total number of 183 and 98 species for p53/p63/p73 and MDM, respectively. We also demonstrate that the p53/p63/p73 and MDM families result from whole genome duplications. Conclusions: The signaling pathway of the TAD and p53/p63/p73BD in p53/p63/p73 and MDM, respectively, dates back to early metazoan time and has since then tightly co-evolved, or disappeared in distinct lineages.

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