35 results on '"Thirstrup K"'
Search Results
2. B17 Characterisation Of A Huntington's Disease Cellular Model For The Transcriptome-based Expression Analysis Of Deubiquitinating Enzymes
- Author
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Rauen, M., primary, Christensen, K., additional, Thirstrup, K., additional, Dantuma, N., additional, Norremolle, A., additional, Dachsel, J., additional, and Fog, K., additional
- Published
- 2014
- Full Text
- View/download PDF
3. Regulation of the Bcas1 and Baiap3 transcripts in the subthalamic nucleus in mice recovering from MPTP toxicity
- Author
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Lauridsen, J B, Johansen, J L, Rekling, J C, Thirstrup, K, Moerk, A, Sager, T N, Lauridsen, J B, Johansen, J L, Rekling, J C, Thirstrup, K, Moerk, A, and Sager, T N
- Abstract
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure leads to significant and irreversible damage to dopaminergic neurons in both mice and humans. While MPTP exposure in humans causes permanent symptoms of Parkinson's disease, MPTP treated mice will recover behaviorally over a 3-week period. This mouse specific recovery might be linked to transcriptional changes in the basal ganglia enabling mice to maintain normal motor function in spite of low striatal dopamine levels. Laser microdissection was used to isolate the subthalamic nucleus from mice 7 and 28 days following MPTP exposure. High quality RNA was recovered and expressional analysis was performed on whole mouse genome microarrays. Identified regulated transcripts were validated in a separate batch of animals using quantitative PCR. Two transcripts with a significant regulation from days 7 to 28 in the MPTP treated groups, were identified: The brain specific angiogenesis inhibitor associated protein 3 (Baiap3) and the breast carcinoma amplified sequence 1 (Bcas1). Further studies of the molecular pathways involving these two transcripts may uncover processes in the subthalamic nucleus associated with the behavioral recovery observed after MPTP exposure.
- Published
- 2011
4. Radioligand-dependent discrepancy in agonist affinities enhanced by mutations in the kappa-opioid receptor
- Author
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Sa, Hjorth, Thirstrup K, and Thue W. Schwartz
- Subjects
Pyrrolidines ,Narcotic Antagonists ,Receptors, Opioid, kappa ,Recombinant Fusion Proteins ,Receptors, Opioid, mu ,Diprenorphine ,Tritium ,Dynorphins ,Peptide Fragments ,Kinetics ,Radioligand Assay ,COS Cells ,Mutation ,Animals ,Benzofurans - Abstract
A series of kappa/mu receptor chimeras and a number of kappa receptors substituted in the second transmembrane segment (TM-II) were investigated using as radioligands, respectively, the kappa-selective agonist [3H]C1977 and the nonselective opioid antagonist [3H]diprenorphine (DIP). All of the receptor constructs bound [3H]DIP with similar and high affinity, whereas the apparent affinity of the nonpeptide agonist C1977, when estimated in competition binding with the antagonist [3H]DIP, was impaired between 42- and500-fold in the kappa/mu chimeras and between 64- and 153-fold in three of the kappa receptor mutants that had been substituted in the TM-II segment. However, homologous competition binding experiments, using [3H]C1977 as radioligand, showed that the high affinity binding of this nonpeptide agonist was in fact not impaired in four of the kappa/mu chimeras and in three TM-II substituted kappa receptors compared with the wild-type kappa receptor. In all cases in which mutations decreased the apparent affinity of C1977 without affecting its actual affinity, as determined in homologous assays using [3H]C1977, the calculated number of receptor sites (Bmax) was decreased. In three of the kappa/mu constructs, binding of [3H]C1977 was undetectable, indicating that in these chimeras the affinity of the nonpeptide agonist had actually been affected. Also, for the kappa-selective peptide agonist dynorphin A(1-8), the measured affinity for the receptor mutants was strongly dependent on whether it was determined using the antagonist [3H]DIP or the agonist [3H]C1977 in thator = 800-fold higher Ki values were determined in competition with the antagonist. It is concluded that mutations in the kappa-opioid receptor can cause large discrepancies between the affinity determined for agonists in homologous versus heterologous competition binding assays and that this pattern, which is compatible with a partial uncoupling of receptors, is observed in surprisingly many types of receptor mutations.
- Published
- 1996
5. Analysis of selective binding epitopes for the kappa-opioid receptor antagonist nor-binaltorphimine
- Author
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Sa, Hjorth, Thirstrup K, Dk, Grandy, and Thue W. Schwartz
- Subjects
Epitopes ,Glutamine ,Receptors, Opioid, kappa ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Receptors, Opioid, mu ,Animals ,Amino Acid Sequence ,Naltrexone ,Rats - Abstract
The structural determinants for the selective binding of the nonpeptide opioid receptor antagonist nor-binaltorphimine (nor-BNI) to the kappa-opioid receptor were characterized using a systematic series of chimeras between the kappa receptor and the homologous mu-opioid receptor. All 10 chimeric constructs bound the nonselective antagonists (-)-naloxone and diprenorphine with similar affinities, as did the two wild-type receptors. Introduction of amino-terminal segments of increasing length, extending to and including transmembrane segment VI, from the mu receptor into the kappa receptor did not impair the high affinity binding of nor-BNI, and neither did introduction of the intracellular carboxyl-terminal extension of the mu receptor. In contrast, nor-BNI binding was impairedor = 600-fold in constructs in which extracellular loop 3 and transmembrane segment VII originated from the mu receptor. The exchange of a single residue within this region, Glu297, for lysine, the corresponding residue from the mu receptor, reduced the binding affinity of nor-BNI 142-fold, without affecting the binding the nonselective compounds (-)-naloxone and diprenorphine. It is concluded that the selective binding of nor-BNI to the kappa-opioid receptor is determined by nonconserved residues located in extracellular loop 3 and transmembrane segment VII and that Glu297, located just outside transmembrane segment VI, plays a major role in the kappa-selective binding characteristics of nor-BNI.
- Published
- 1995
6. Restriction fragment differential display of pediocin-resistant Listeria monocytogenes 412 mutants shows consistent overexpression of a putative beta-glucoside-specific FTS system
- Author
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Gravesen, A., Warthoe, P., Knöchel, Susanne, Thirstrup, K., Gravesen, A., Warthoe, P., Knöchel, Susanne, and Thirstrup, K.
- Published
- 2000
7. Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor
- Author
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Elling, C E, Thirstrup, K, Holst, Birgitte, Schwartz, T W, Elling, C E, Thirstrup, K, Holst, Birgitte, and Schwartz, T W
- Abstract
Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the beta(2)-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III-or a His residue introduced at this position-and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu(2+), and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure-activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.
- Published
- 1999
8. Radioligand-dependent discrepancy in agonist affinities enhanced by mutations in the kappa-opioid receptor.
- Author
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Hjorth, S A, Thirstrup, K, and Schwartz, T W
- Abstract
A series of kappa/mu receptor chimeras and a number of kappa receptors substituted in the second transmembrane segment (TM-II) were investigated using as radioligands, respectively, the kappa-selective agonist [3H]C1977 and the nonselective opioid antagonist [3H]diprenorphine (DIP). All of the receptor constructs bound [3H]DIP with similar and high affinity, whereas the apparent affinity of the nonpeptide agonist C1977, when estimated in competition binding with the antagonist [3H]DIP, was impaired between 42- and > 500-fold in the kappa/mu chimeras and between 64- and 153-fold in three of the kappa receptor mutants that had been substituted in the TM-II segment. However, homologous competition binding experiments, using [3H]C1977 as radioligand, showed that the high affinity binding of this nonpeptide agonist was in fact not impaired in four of the kappa/mu chimeras and in three TM-II substituted kappa receptors compared with the wild-type kappa receptor. In all cases in which mutations decreased the apparent affinity of C1977 without affecting its actual affinity, as determined in homologous assays using [3H]C1977, the calculated number of receptor sites (Bmax) was decreased. In three of the kappa/mu constructs, binding of [3H]C1977 was undetectable, indicating that in these chimeras the affinity of the nonpeptide agonist had actually been affected. Also, for the kappa-selective peptide agonist dynorphin A(1-8), the measured affinity for the receptor mutants was strongly dependent on whether it was determined using the antagonist [3H]DIP or the agonist [3H]C1977 in that < or = 800-fold higher Ki values were determined in competition with the antagonist. It is concluded that mutations in the kappa-opioid receptor can cause large discrepancies between the affinity determined for agonists in homologous versus heterologous competition binding assays and that this pattern, which is compatible with a partial uncoupling of receptors, is observed in surprisingly many types of receptor mutations.
- Published
- 1996
9. Analysis of selective binding epitopes for the kappa-opioid receptor antagonist nor-binaltorphimine.
- Author
-
Hjorth, S A, Thirstrup, K, Grandy, D K, and Schwartz, T W
- Abstract
The structural determinants for the selective binding of the nonpeptide opioid receptor antagonist nor-binaltorphimine (nor-BNI) to the kappa-opioid receptor were characterized using a systematic series of chimeras between the kappa receptor and the homologous mu-opioid receptor. All 10 chimeric constructs bound the nonselective antagonists (-)-naloxone and diprenorphine with similar affinities, as did the two wild-type receptors. Introduction of amino-terminal segments of increasing length, extending to and including transmembrane segment VI, from the mu receptor into the kappa receptor did not impair the high affinity binding of nor-BNI, and neither did introduction of the intracellular carboxyl-terminal extension of the mu receptor. In contrast, nor-BNI binding was impaired > or = 600-fold in constructs in which extracellular loop 3 and transmembrane segment VII originated from the mu receptor. The exchange of a single residue within this region, Glu297, for lysine, the corresponding residue from the mu receptor, reduced the binding affinity of nor-BNI 142-fold, without affecting the binding the nonselective compounds (-)-naloxone and diprenorphine. It is concluded that the selective binding of nor-BNI to the kappa-opioid receptor is determined by nonconserved residues located in extracellular loop 3 and transmembrane segment VII and that Glu297, located just outside transmembrane segment VI, plays a major role in the kappa-selective binding characteristics of nor-BNI.
- Published
- 1995
10. Construction of a high affinity zinc switch in the kappa-opioid receptor.
- Author
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Thirstrup, K, Elling, C E, Hjorth, S A, and Schwartz, T W
- Abstract
Very limited structural information is available concerning the superfamily of G-protein-coupled receptors with their seven-transmembrane segments. Recently a non-peptide antagonist site was structurally and functionally replaced by a metal ion site in the tachykinin NK-1 receptor. Here, this Zn(II) site is transferred to the kappa-opioid receptor by substituting two residues at the outer portion of transmembrane V (TM-V), Asp223 and Lys227, and one residue at the top of TM-VI, Ala298, with histidyl residues. The histidyl residues had no direct effect on the binding of either the non-peptide antagonist [3H]diprenorphine or the non-peptide agonist, [3H]CI977, just as these mutations/substitutions did not affect the apparent affinity of a series of other peptide and non-peptide ligands when tested in competition binding experiments. However, zinc ions in a dose-dependent manner prevented binding of both agonist and antagonist ligands with an apparent affinity for the metal ion, which gradually was built up to 10(-6) M. This represents an increase in affinity for the metal ion of about 1000-fold as compared with the wild-type kappa receptor and is specific for Zn(II) as the affinity for e.g. Cu(II) was almost unaffected. The direct transfer of this high affinity metal ion switch between two only distantly related receptors indicates a common overall arrangement of the seven-helix bundle among receptors of the rhodopsin family.
- Published
- 1996
11. Cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2
- Author
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Carriere, F., Thirstrup, K., Hjorth, S., and Boel, E.
- Published
- 1994
- Full Text
- View/download PDF
12. Target-Mediated Brain Tissue Binding for Small Molecule Inhibitors of Heat Shock Protein 90.
- Author
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Badolo L, Thirstrup K, Nielsen SM, Püschl A, Jensen T, Watson S, and Bundgaard C
- Abstract
Drug distribution in the brain is generally associated with an affinity for fatty brain tissues and therefore known to be species- and concentration-independent. We report here the effect of target affinity on brain tissue binding for 10 small molecules designed to inhibit brain heat shock protein 90 (HSP90), a widespread protein whose expression is 1-2% of total cytosolic proteins in eucaryotes. Our results show that increasing the test item concentrations from 0.3 to 100 µM increased the unbound fraction 32-fold for the most potent molecules, with no change for the inactive one (1.1 fold change). Saturation of HSP90 led to normal concentration-independent brain tissue binding. In vivo pharmacokinetics performed in rats showed that the overall volume of distribution of compounds is correlated with their affinity for HSP90. The in vitro binding and in vivo pharmacokinetics (PK) performed in rats showed that small molecule HSP90 inhibitors followed the principle of target-mediated drug disposition. We demonstrate that assessing unbound fractions in brain homogenate was subject to HSP90 target interference; this may challenge the process of linking systemic-free drug concentrations to central nervous system unbound concentrations necessary to establish the proper pharmacokinetics/pharmacodynamics (PK/PD) relation needed for human dose prediction.
- Published
- 2020
- Full Text
- View/download PDF
13. Mass spectrometry analyses of normal and polyglutamine expanded ataxin-3 reveal novel interaction partners involved in mitochondrial function.
- Author
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Kristensen LV, Oppermann FS, Rauen MJ, Fog K, Schmidt T, Schmidt J, Harmuth T, Hartmann-Petersen R, and Thirstrup K
- Subjects
- Animals, Ataxin-3 genetics, HEK293 Cells, Humans, Machado-Joseph Disease genetics, Machado-Joseph Disease metabolism, Mass Spectrometry methods, Mice, Mice, Transgenic, Mitochondria genetics, Peptides genetics, Ataxin-3 analysis, Ataxin-3 metabolism, Mitochondria metabolism, Peptides analysis, Peptides metabolism, Protein Interaction Maps physiology
- Abstract
Deubiquitinating enzymes (DUBs) play important roles in a variety of cellular processes, including regulation of protein homeostasis. The DUB ataxin-3 is an enzyme implicated in protein quality control mechanisms. In the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3), ataxin-3 contains an expanded polyglutamine (polyQ) stretch that leads to aggregation of the protein and neuronal dysfunction. Increasing the understanding of ataxin-3 protein interaction partners could help to elucidate disease mechanisms. Hence, we analyzed the repertoire of proteins interacting with normal and polyQ expanded ataxin-3 by mass spectrometry. This showed that both normal and polyQ expanded ataxin-3 interacted with components of the protein quality control system and mitochondria. Five proteins showed increased interaction with polyQ expanded ataxin-3 relative to normal and three of these were mitochondrial proteins. The analyses underline the role of ataxin-3 in ubiquitin biology and point towards a role in mitochondrial biology., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
14. Design of Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitors Using a Crystallographic Surrogate Derived from Checkpoint Kinase 1 (CHK1).
- Author
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Williamson DS, Smith GP, Acheson-Dossang P, Bedford ST, Chell V, Chen IJ, Daechsel JCA, Daniels Z, David L, Dokurno P, Hentzer M, Herzig MC, Hubbard RE, Moore JD, Murray JB, Newland S, Ray SC, Shaw T, Surgenor AE, Terry L, Thirstrup K, Wang Y, and Christensen KV
- Subjects
- Animals, Brain metabolism, Checkpoint Kinase 1, Crystallography methods, HEK293 Cells, Humans, Kidney metabolism, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 genetics, Mice, Mutation, Parkinson Disease genetics, Protein Binding, Protein Domains, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors pharmacokinetics, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 antagonists & inhibitors, Protein Kinase Inhibitors therapeutic use
- Abstract
Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson's disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC
50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LRRK2-pSer935 IC50 values for 22 in mouse brain and kidney being 1.3 and 5 nM, respectively.- Published
- 2017
- Full Text
- View/download PDF
15. Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells.
- Author
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Thirstrup K, Dächsel JC, Oppermann FS, Williamson DS, Smith GP, Fog K, and Christensen KV
- Subjects
- Computational Biology methods, Dose-Response Relationship, Drug, Humans, Immunomodulation drug effects, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 antagonists & inhibitors, Leukocytes, Mononuclear immunology, Phosphoproteins metabolism, Phosphorylation, Protein Kinase Inhibitors pharmacology, Proteome, Proteomics, Reproducibility of Results, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 metabolism, Leukocytes, Mononuclear metabolism, rab GTP-Binding Proteins metabolism
- Abstract
Genetic variation in the leucine-rich repeat kinase 2 (LRRK2) gene is associated with risk of familial and sporadic Parkinson's disease (PD). To support clinical development of LRRK2 inhibitors as disease-modifying treatment in PD biomarkers for kinase activity, target engagement and kinase inhibition are prerequisite tools. In a combined proteomics and phosphoproteomics study on human peripheral mononuclear blood cells (PBMCs) treated with the LRRK2 inhibitor Lu AF58786 a number of putative biomarkers were identified. Among the phospho-site hits were known LRRK2 sites as well as two phospho-sites on human Rab10 and Rab12. LRRK2 dependent phosphorylation of human Rab10 and human Rab12 at positions Thr73 and Ser106, respectively, was confirmed in HEK293 and, more importantly, Rab10-pThr73 inhibition was validated in immune stimulated human PBMCs using two distinct LRRK2 inhibitors. In addition, in non-stimulated human PBMCs acute inhibition of LRRK2 with two distinct LRRK2 inhibitor compounds reduced Rab10-Thr73 phosphorylation in a concentration-dependent manner with apparent IC
50 's equivalent to IC50 's on LRRK2-pSer935. The identification of Rab10 phosphorylated at Thr73 as a LRRK2 inhibition marker in human PBMCs strongly support inclusion of assays quantifying Rab10-pThr73 levels in upcoming clinical trials evaluating LRRK2 kinase inhibition as a disease-modifying treatment principle in PD.- Published
- 2017
- Full Text
- View/download PDF
16. Polyglutamine expansion of ataxin-3 alters its degree of ubiquitination and phosphorylation at specific sites.
- Author
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Kristensen LV, Oppermann FS, Rauen MJ, Hartmann-Petersen R, and Thirstrup K
- Subjects
- Amino Acid Sequence, Binding Sites physiology, HEK293 Cells, Humans, Phosphorylation physiology, Ataxin-3 genetics, Ataxin-3 metabolism, Peptides genetics, Peptides metabolism, Repressor Proteins genetics, Repressor Proteins metabolism, Ubiquitination physiology
- Abstract
Ubiquitination and phosphorylation of proteins represent post translational modifications (PTMs) capable of regulating a variety of cellular processes. In the neurodegenerative disorder spinocerebellar ataxia type 3 (SCA3), the disease causing protein ataxin-3 carries an expanded polyglutamine (polyQ) stretch causing it to aggregate in nuclear inclusions. These inclusions are decorated with ubiquitin suggestive of a malfunction in the clearance of the mutant protein. Differences in ubiquitin chain topology between normal and polyQ expanded ataxin-3 could be involved in the differential clearance of the two proteins and play a role in SCA3 pathogenesis. Likewise, changes in phosphorylation patterns between the two variants could contribute to pathogenic processes involved in SCA3. We therefore determined the ubiquitination and phosphorylation patterns, together with the ubiquitin-linkage types associated with normal and polyQ expanded ataxin-3 by mass spectrometry (MS). This analysis revealed a similar ubiquitin linkage pattern on normal and expanded ataxin-3. However, the distribution of ubiquitinated lysine residues was altered in polyQ expanded ataxin-3, with increased ubiquitination at the new identified ubiquitination site lysine-8. MS analysis of phosphorylation also revealed novel phosphorylation sites in ataxin-3, and an increase in phosphorylation of expanded ataxin-3 at several positions. The study suggests that differences in clearance of normal and expanded ataxin-3 are not attributed to differences in ubiquitin-linkage pattern. However, the observed differences between the normal and polyQ expanded ataxin-3 with respect to the degree of ubiquitination and phosphorylation on specific sites may have an impact on ataxin-3 function and SCA3 pathogenesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
17. Linking HSP90 target occupancy to HSP70 induction and efficacy in mouse brain.
- Author
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Thirstrup K, Sotty F, Montezinho LC, Badolo L, Thougaard A, Kristjánsson M, Jensen T, Watson S, and Nielsen SM
- Subjects
- Alzheimer Disease drug therapy, Alzheimer Disease metabolism, Animals, Cell Line, HSP70 Heat-Shock Proteins metabolism, HSP90 Heat-Shock Proteins antagonists & inhibitors, Humans, Mice, Transgenic, tau Proteins genetics, Brain metabolism, HSP90 Heat-Shock Proteins metabolism
- Abstract
HSP90 (Heat shock protein 90) is a molecular chaperone protein ubiquitously expressed throughout all tissues in the body. HSP90 has been proposed as a target to increase turnover of pathological proteins leading to neurodegeneration in Huntington's disease, Parkinson's disease and Alzheimer's disease. The mechanism of how HSP90 inhibition leads to clearance of misfolded proteins is not fully understood. It may involve direct effects of inhibiting ATPase function, indirect effects by inducing the heat-shock-response resulting in upregulation of other chaperone proteins like HSP70 or a combination of both. In the current work we established a methodology to investigate the relationship between HSP90 target occupancy and HSP70 induction in vivo. We also characterized the acute effect of two different HSP90 inhibitors in the rTg4510 transgenic mouse model of Alzheimer's disease which displays a tau-mediated synaptic dysfunction. We show that reversal of synaptic impairments in this model can be obtained with a compound which has a high HSP70 induction capacity. The current developed assay methodologies may thus be of significant use in the further elucidation of the mechanism involved in the in vivo effect of HSP90 inhibition in models of neurodegeneration. Further on, the ability of HSP90 inhibitors to normalize synaptic dysfunction in an in vivo disease model of Alzheimer's disease could have therapeutic relevance and further strengthens the usefulness of this animal model to establish pharmacodynamic effect of HSP90 inhibition., (Copyright © 2016. Published by Elsevier Ltd.)
- Published
- 2016
- Full Text
- View/download PDF
18. Chronic treatment with 17-DMAG improves balance and coordination in a new mouse model of Machado-Joseph disease.
- Author
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Silva-Fernandes A, Duarte-Silva S, Neves-Carvalho A, Amorim M, Soares-Cunha C, Oliveira P, Thirstrup K, Teixeira-Castro A, and Maciel P
- Subjects
- Animals, Ataxin-3, Autophagy drug effects, Benzoquinones administration & dosage, Benzoquinones pharmacology, Disease Models, Animal, Female, HSP90 Heat-Shock Proteins antagonists & inhibitors, Humans, Lactams, Macrocyclic administration & dosage, Lactams, Macrocyclic pharmacology, Male, Mice, Mice, Transgenic, Motor Activity drug effects, Nerve Tissue Proteins metabolism, Nuclear Proteins metabolism, Repressor Proteins metabolism, Benzoquinones therapeutic use, Lactams, Macrocyclic therapeutic use, Machado-Joseph Disease drug therapy, Nerve Tissue Proteins genetics, Nuclear Proteins genetics, Postural Balance drug effects, Repressor Proteins genetics
- Abstract
Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease currently with no treatment. We describe a novel mouse model of MJD which expresses mutant human ataxin-3 at near endogenous levels and manifests MJD-like motor symptoms that appear gradually and progress over time. CMVMJD135 mice show ataxin-3 intranuclear inclusions in the CNS and neurodegenerative changes in key disease regions, such as the pontine and dentate nuclei. Hsp90 inhibition has shown promising outcomes in some neurodegenerative diseases, but nothing is known about its effects in MJD. Chronic treatment of CMVMJD mice with Hsp90 inhibitor 17-DMAG resulted in a delay in the progression of their motor coordination deficits and, at 22 and 24 weeks of age, was able to rescue the uncoordination phenotype to wild-type levels; in parallel, a reduction in neuropathology was observed in treated animals. We observed limited induction of heat-shock proteins with treatment, but found evidence that 17-DMAG may be acting through autophagy, as LC3-II (both at mRNA and protein levels) and beclin-1 were induced in the brain of treated animals. This resulted in decreased levels of the mutant ataxin-3 and reduced intranuclear aggregation of this protein. Our data validate this novel mouse model as a relevant tool for the study of MJD pathogenesis and for pre-clinical studies, and show that Hsp90 inhibition is a promising therapeutic strategy for MJD.
- Published
- 2014
- Full Text
- View/download PDF
19. Competitive HIF Prolyl Hydroxylase Inhibitors Show Protection against Oxidative Stress by a Mechanism Partially Dependent on Glycolysis.
- Author
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Bergström AL, Fog K, Sager TN, Bruun AT, and Thirstrup K
- Abstract
The hypoxia inducible factor 1 (HIF-1) is a central transcription factor involved in the cellular and molecular adaptation to hypoxia and low glucose supply. The level of HIF-1 is to a large degree regulated by the HIF prolyl hydroxylase enzymes (HPHs) belonging to the Fe(II) and 2-oxoglutarate-dependent dioxygenase superfamily. In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12). Although the competitive HPH-inhibitor compounds were found to be pharmacologically more potent than the non-competitive compounds at inhibiting HPH2 and HPH1, this was not translated into the cellular effects of the compounds, where the non-competitive inhibitors were actually more potent than the competitive in stabilizing and translocatingHIF1 α to the nucleus (quantified with Cellomics ArrayScan technology). This could be explained by the high cellular concentrations of the cofactor 2-oxoglutarate (2-OG) as the competitive inhibitors act by binding to the 2-OG site of the HPH enzymes. Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress. In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.
- Published
- 2013
- Full Text
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20. Endogenous 2-oxoglutarate levels impact potencies of competitive HIF prolyl hydroxylase inhibitors.
- Author
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Thirstrup K, Christensen S, Møller HA, Ritzén A, Bergström AL, Sager TN, and Jensen HS
- Subjects
- Cell Line, Dioxygenases antagonists & inhibitors, Dioxygenases genetics, Drug Evaluation, Preclinical methods, Humans, Hypoxia-Inducible Factor-Proline Dioxygenases, Nuclear Proteins antagonists & inhibitors, Nuclear Proteins genetics, Procollagen-Proline Dioxygenase antagonists & inhibitors, Procollagen-Proline Dioxygenase genetics, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins genetics, Recombinant Proteins metabolism, Dioxygenases metabolism, Enzyme Inhibitors pharmacology, Ketoglutaric Acids metabolism, Nuclear Proteins metabolism, Procollagen-Proline Dioxygenase metabolism
- Abstract
The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by oxygen-dependent hydroxylation that is catalyzed by three HIF prolyl 4-hydroxylases (HPHs). Use of HPH inhibition as a mean for HIF-upregulation has recently gained interest as a potential treatment paradigm against neurodegenerative diseases like ischemia and Parkinson's disease. In the present investigation we report the development of a new and robust assay to measure HPH activity. The assay is based on capture of hydroxylated peptide product by the von Hippel-Lindau protein which is directly measured in a scintillation proximity assay. In addition we describe the determination of HPH subtype potencies of HPH inhibitors which either directly or indirectly inhibit the HPH enzyme. The potencies of the HPH inhibitors displayed almost identical IC(50) values toward the HPH1 and HPH2 subtype while the potency against the HPH3 subtype was increased for several of the compounds. For the most potent compound, a hydroxyl thiazole derivative, the potency against HPH2 and HPH3 was 7nM and 0.49nM, respectively corresponding to a 14-fold difference. These results suggest that HPH subtype-selective compounds may be developed. In addition we determined the 2-oxoglutarate concentration in brain tissue and neuronal cell lines as 2-oxoglutarate is an important co-factor used by the HPH enzyme during the hydroxylation reaction. The high intracellular 2-oxoglutarate concentration provides an explanation for the diminished cellular HIF activating potency of a competitive HPH inhibitor compared to its orders of magnitude higher HPH inhibiting potency. The present reported data suggest that in the development of specific Hif prolyl hydroxylase inhibitors the high 2-oxoglutarate tissue level should be taken into account as this might affect the cellular potency. Thus to specifically inhibit the intracellular HPH enzymatic reaction a competitive inhibitor with a low Ki should be developed., (2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
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21. Discovery and SAR of a Series of Agonists at Orphan G Protein-Coupled Receptor 139.
- Author
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Shi F, Shen JK, Chen D, Fog K, Thirstrup K, Hentzer M, Karlsson JJ, Menon V, Jones KA, Smith KE, and Smith G
- Abstract
GPR139 is an orphan G-protein coupled receptor (GPCR) which is primarily expressed in the central nervous system (CNS). In order to explore the biological function of this receptor, selective tool compounds are required. A screening campaign identified compound 1a as a high potency GPR139 agonist with an EC50 = 39 nM in a calcium mobilization assay in CHO-K1 cells stably expressing the GPR139 receptor. In the absence of a known endogenous ligand, the maximum effect was set as 100% for 1a. Screening against 90 diverse targets revealed no cross-reactivity issues. Assessment of the pharmacokinetic properties showed limited utility as in vivo tool compound in rat with a poor whole brain exposure of 61 ng/g and a brain/plasma (b/p) ratio of 0.03. Attempts to identify a more suitable analogue identified the des-nitrogen analogue 1s with a reduced polar surface area of 76.7 Å(2) and an improved b/p ratio of 2.8. The whole brain exposure remained low at 95 ng/g due to a low plasma exposure.
- Published
- 2011
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22. HIF prolyl hydroxylase inhibition increases cell viability and potentiates dopamine release in dopaminergic cells.
- Author
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Johansen JL, Sager TN, Lotharius J, Witten L, Mørk A, Egebjerg J, and Thirstrup K
- Subjects
- Animals, Blotting, Western, Cell Line, Dopamine biosynthesis, Enzyme Inhibitors pharmacology, Genes, Reporter, Humans, Luciferases genetics, Membrane Potentials drug effects, Membrane Potentials physiology, Mitochondrial Membranes drug effects, Mitochondrial Membranes physiology, Neurons drug effects, PC12 Cells, Procollagen-Proline Dioxygenase metabolism, RNA Interference, Rats, Reverse Transcriptase Polymerase Chain Reaction, Tyrosine 3-Monooxygenase metabolism, Cell Survival drug effects, Dioxygenases antagonists & inhibitors, Dopamine metabolism, Dopamine physiology, Neurons metabolism, Procollagen-Proline Dioxygenase antagonists & inhibitors, Procollagen-Proline Dioxygenase genetics
- Abstract
Hypoxia-inducible factor (HIF) controls the expression of genes that adapts the cellular condition to accommodate oxidative stress. The potential beneficial effect of HIF up-regulation in ischemia has recently gained interest substantiated by the known HIF-regulation of erythropoietin and other hypoxia accommodating genes. So far the perspectives for HIF up-regulation has been focused on anemia and ischemia related diseases but little information is available about the relevance of HIF biology for neurodegenerative disease like Parkinson's disease. We therefore sought out to characterize the effect of HIF-up-regulation on survival and dopamine homeostasis in dopaminergic cells. We used a low molecular weight HIF prolyl hydroxylase (HPH) inhibitor and lentiviral based shRNA knockdown of HPH subtypes as molecular tools to increase HIF protein level and downstream HIF-regulated genes. We show that HIF induction results in protection against oxidative stress in cellular models based on PC12 cells and LUHMES cells. In addition, HPH inhibition elevates tyrosine hydroxylase expression and activity, which causes increased dopamine synthesis and release in both PC12 cells and a primary rat ventral mesencephalic cell culture. All together these findings suggest that prolyl hydroxylases may represent novel targets for therapeutic intervention in disorders characterized by dopamine homeostasis dysregulation like Parkinson's disease., (© 2010 H. Lundbeck A/S. Journal Compilation © 2010 International Society for Neurochemistry.)
- Published
- 2010
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23. The imprinted gene neuronatin is regulated by metabolic status and associated with obesity.
- Author
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Vrang N, Meyre D, Froguel P, Jelsing J, Tang-Christensen M, Vatin V, Mikkelsen JD, Thirstrup K, Larsen LK, Cullberg KB, Fahrenkrug J, Jacobson P, Sjöström L, Carlsson LM, Liu Y, Liu X, Deng HW, and Larsen PJ
- Subjects
- Adipose Tissue physiology, Animals, Energy Metabolism genetics, Gene Expression Profiling, Genotype, Homeostasis genetics, Hypothalamus physiology, Leptin genetics, Male, Membrane Proteins metabolism, Mice, Mice, Inbred C57BL, Mice, Obese, Nerve Tissue Proteins metabolism, PC12 Cells, Pancreas physiology, Pituitary Gland physiology, Polymorphism, Single Nucleotide, RNA, Messenger metabolism, Rats, Rats, Wistar, Signal Transduction physiology, Genomic Imprinting physiology, Leptin metabolism, Membrane Proteins genetics, Nerve Tissue Proteins genetics, Obesity genetics, Obesity physiopathology
- Abstract
Using restriction fragment differential display (RFDD) technology, we have identified the imprinted gene neuronatin (Nnat) as a hypothalamic target under the influence of leptin. Nnat mRNA expression is decreased in several key appetite regulatory hypothalamic nuclei in rodents with impaired leptin signaling and during fasting conditions. Furthermore, peripheral administration of leptin to ob/ob mice normalizes hypothalamic Nnat expression. Comparative immunohistochemical analysis of human and rat hypothalami demonstrates that NNAT protein is present in anatomically equivalent nuclei, suggesting human physiological relevance of the gene product(s). A putative role of Nnat in human energy homeostasis is further emphasized by a consistent association between single nucleotide polymorphisms (SNPs) in the human Nnat gene and severe childhood and adult obesity.
- Published
- 2010
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24. Nest building performance following MPTP toxicity in mice.
- Author
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Sager TN, Kirchhoff J, Mørk A, Van Beek J, Thirstrup K, Didriksen M, and Lauridsen JB
- Subjects
- 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine pharmacology, Animals, Behavior, Animal, Corpus Striatum metabolism, Corpus Striatum pathology, Disease Models, Animal, Dose-Response Relationship, Drug, MPTP Poisoning pathology, Male, Mice, Mice, Inbred C57BL, Neurotoxins toxicity, Recovery of Function physiology, Time Factors, Tyrosine 3-Monooxygenase metabolism, MPTP Poisoning physiopathology, Nesting Behavior physiology
- Abstract
Systemic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice is one of the primary models used to evaluate neuroprotective and symptomatic treatment strategies for Parkinson's disease. Many behavioral methods for evaluation of MPTP toxicity have been described, but they often involve challenging scenarios that require handling and transfer of animals to novel environments and in some cases prior animal training. These factors can profoundly influence animal behavior and potentially influence experimental outcome. Presented here is a new nest building scoring paradigm based on the animals' normal home cage behavior that is a simple, non-invasive, and reproducible measure for estimating neurological dysfunction in MPTP intoxicated mice. Nest building behavior requires orofacial and forelimb movement and has been shown to be dopamine-dependent making it a possible method for assessing parkinsonian-like symptoms. Significant deficits in nest building scores after 2x20 and 2x25 mg/kg MPTP coincided with a 90% reduction in striatal dopamine. Nest building deficits could be detected for more than a week after intoxication. However, after 28 days the change in behavior was no longer detected, which may reflect the plasticity of the tyrosine hydroxylase positive neurons in the dorsolateral part of striatum., (Copyright 2009 Elsevier B.V. All rights reserved.)
- Published
- 2010
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25. HIF prolyl hydroxylase inhibition augments dopamine release in the rat brain in vivo.
- Author
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Witten L, Sager T, Thirstrup K, Johansen JL, Larsen DB, Montezinho LP, and Mørk A
- Subjects
- Animals, Brain drug effects, Brain Stem drug effects, Brain Stem metabolism, Cobalt pharmacology, Cocaine pharmacology, Corpus Striatum drug effects, Corpus Striatum metabolism, Dopamine Uptake Inhibitors pharmacology, Extracellular Space metabolism, Gene Expression drug effects, Male, Motor Activity drug effects, Potassium metabolism, Procollagen-Proline Dioxygenase metabolism, Rats, Rats, Sprague-Dawley, Rats, Wistar, Tyrosine 3-Monooxygenase metabolism, Brain metabolism, Dopamine metabolism, Enzyme Inhibitors pharmacology, Phenanthrolines pharmacology, Procollagen-Proline Dioxygenase antagonists & inhibitors
- Abstract
The transcription factor hypoxia-inducible factor (HIF) is essential for the activation of several genes that promote the survival of cells exposed to oxidative stress. Expression of tyrosine hydroxylase (TH), which is the rate-limiting enzyme in the dopamine (DA) synthesis, is one of the genes that are positively regulated by HIF. Accordingly, HIF induction results in elevated DA release in various cell lines in vitro. HIF prolyl hydroxylase (HPH) is critically involved in the negative regulation of HIF levels. We investigated the in vivo effects of the HPH inhibitor FG0041 on brain DA function in rats by microdialysis in freely moving rats, locomotor activity, and Western blot analysis. Administration of FG0041 (10 mg/kg i.p.), as an acute (single injection), or as subchronic (once daily for 6 days) treatment and cobalt chloride (CoCl2) (60 mg/kg s.c.) potentiated potassium (K+) induced increases in extracellular levels of DA levels in the rat striatum. The increase in extracellular DA of freely moving rats was sought in relationship to locomotor activity in rats. A significant increase in locomotor activity was observed in FG0041-treated rats compared with vehicle on a cocaine challenge. In support of these findings, protein levels of TH in the rat brain stem were increased after treatment with FG0041. These data indicate that FG0041 augments DA function in the rat brain. Inhibition of HPH enhances DA function by increasing DA release, which has implications for the use of HIF induction in the treatment of neurodegenerative diseases., (Copyright 2009 Wiley-Liss, Inc.)
- Published
- 2009
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26. Multiprotein bridging factor 1 cooperates with c-Jun and is necessary for cardiac hypertrophy in vitro.
- Author
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Busk PK, Wulf-Andersen L, Strøm CC, Enevoldsen M, Thirstrup K, Haunsø S, and Sheikh SP
- Subjects
- 3T3 Cells, Amino Acid Sequence, Animals, Animals, Newborn, Calcineurin metabolism, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Calmodulin metabolism, Cardiotonic Agents pharmacology, Cells, Cultured, Gene Expression Profiling, Gene Expression Regulation, Genes, Reporter, Humans, Mice, Molecular Sequence Data, Myocytes, Cardiac cytology, Myocytes, Cardiac drug effects, Oligonucleotides, Antisense metabolism, Phenylephrine pharmacology, Proto-Oncogene Proteins c-jun genetics, Rats, Rats, Wistar, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Alignment, Trans-Activators genetics, Calmodulin-Binding Proteins, Cardiomegaly metabolism, Myocytes, Cardiac physiology, Proto-Oncogene Proteins c-jun metabolism, Trans-Activators metabolism
- Abstract
Cardiac hypertrophy is induced by a number of stimuli and can lead to cardiomyopathy and heart failure. Cardiomyocyte hypertrophy is characterized by increased cell size and altered gene expression. By differential-display polymerase chain reaction and Western blotting we found that the transcriptional coactivator MBF1 was upregulated during hypertrophy in cardiomyocyte cultures. Furthermore, MBF1 protein level increased in two animal models of hypertrophy, angiotensin II treatment and aortic banding. MBF1 antisense oligodeoxynuclotides blocked phenylephrine-induced hypertrophy, suggesting MBF1 plays a key role in hypertrophic growth. In contrast, overexpression of MBF1 potentiated the hormone-induced response of the atrial natriuretic peptide promoter. MBF1 overexpressed by transient transfection cooperated with the transcription factor c-Jun in activation of transcription but not with GATA4. MBF1 and c-Jun induced the activity of a transiently transfected atrial natriuretic peptide promoter, whereas neither MBF1 nor c-Jun could induce the promoter alone. Moreover, MBF1 bound to c-Jun in vitro. These data suggest that MBF1 is a transcriptional coactivator of c-Jun regulating hypertrophic gene expression. Inhibitor studies suggested that MBF1 activates the atrial natriuretic peptide promoter independently of the calcineurin and CaMK signaling pathways. Our results indicate that MBF1 participates in hormone-induced cardiomyocyte hypertrophy and activates hypertrophic gene expression as a coactivator of c-Jun.
- Published
- 2003
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27. Restriction fragment differential display of pediocin-resistant Listeria monocytogenes 412 mutants shows consistent overexpression of a putative beta-glucoside-specific PTS system.
- Author
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Gravesen A, Warthoe P, Knøchel S, and Thirstrup K
- Subjects
- Amino Acid Sequence, Base Sequence, DNA Primers genetics, Drug Resistance, Microbial genetics, Food Preservation, Gene Expression, Genes, Bacterial, Humans, Listeria monocytogenes pathogenicity, Molecular Sequence Data, Mutation, Pediocins, Phosphoenolpyruvate Sugar Phosphotransferase System genetics, Polymerase Chain Reaction methods, Sequence Homology, Amino Acid, Bacteriocins pharmacology, Listeria monocytogenes drug effects, Listeria monocytogenes genetics
- Abstract
Pediocin PA-1, which is a bacteriocin produced by lactic acid bacteria, has potential as a biopreservative of food. However, such use may lead to the development of resistance in the target organism. Gene expression in two independent pediocin-resistant mutants of Listeria monocytogenes 412 was compared to the original isolate by restriction fragment differential display PCR (RFDD-PCR). This method amplifies cDNA restriction fragments under stringent PCR conditions, enabled by the use of specific primers complementary to ligated adaptor sequences. RFDD-PCR was very well suited for analysis of listerial gene expression, giving reproducible PCR product profiles. Three gene fragments having increased expression in both resistant mutants were identified. All three had homology to components of beta-glucoside-specific phosphoenolpyruvate-dependent phosphotransferase systems (PTS), one fragment having homology to enzyme II permeases, and the two others to phospho-beta-glucosidases. Overexpression of the putative PTS system was consistently observed in 10 additional pediocin-resistant mutants, isolated at different pH, salt content and temperature. The results suggest that RFDD-PCR is a strong approach for the analysis of prokaryotic gene expression and that the putative beta-glucoside-specific PTS system is involved in mediating pediocin resistance.
- Published
- 2000
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28. Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor.
- Author
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Elling CE, Thirstrup K, Holst B, and Schwartz TW
- Subjects
- Amino Acid Sequence, Molecular Probes, Molecular Sequence Data, Mutagenesis, Site-Directed, Receptors, Adrenergic, beta-2 chemistry, Receptors, Adrenergic, beta-2 genetics, Signal Transduction, Adrenergic beta-Agonists metabolism, Chelating Agents metabolism, Metals metabolism, Receptors, Adrenergic, beta-2 metabolism
- Abstract
Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the beta(2)-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III-or a His residue introduced at this position-and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu(2+), and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure-activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.
- Published
- 1999
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- View/download PDF
29. Metal-ion sites as structural and functional probes of helix-helix interactions in 7TM receptors.
- Author
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Elling CE, Thirstrup K, Nielsen SM, Hjorth SA, and Schwartz TW
- Subjects
- Animals, Binding Sites, Kinetics, Ligands, Models, Molecular, Molecular Probes, Molecular Structure, Protein Structure, Secondary, Receptors, Neurokinin-1 chemistry, Receptors, Neurokinin-1 metabolism, Zinc metabolism, GTP-Binding Proteins metabolism, Metals metabolism, Receptors, Cell Surface chemistry, Receptors, Cell Surface metabolism
- Published
- 1997
- Full Text
- View/download PDF
30. Pancreatic lipase structure-function relationships by domain exchange.
- Author
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Carrière F, Thirstrup K, Hjorth S, Ferrato F, Nielsen PF, Withers-Martinez C, Cambillau C, Boel E, Thim L, and Verger R
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Bile Acids and Salts pharmacology, Colipases pharmacology, DNA Primers, Enzyme Activation, Glycosylation, Guinea Pigs, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed genetics, Mutation genetics, Pancreas enzymology, Phospholipids metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Sequence Analysis, Triglycerides metabolism, Lipase chemistry, Lipase metabolism, Structure-Activity Relationship
- Abstract
We designed chimeric mutants by exchanging the lid domains of the classical human pancreatic lipase (HPL) and the guinea pig pancreatic lipase related protein 2 (GPLRP2). This latter enzyme possesses naturally a large deletion within the lid domain and is not activated by lipid/water interfaces. Furthermore, GPLRP2 exhibits phospholipase A1 and lipase activities in the same order of magnitude, whereas HPL has no significant phospholipase activity and displays a clear interfacial activation. An HPL mutant [HPL(-lid)] with GPLRP2 mini-lid domain does not display interfacial activation. Its specific activity toward triglycerides is, however, dramatically reduced. A GPLRP2 mutant [GPLRP2(+lid)] with HPL full-length lid domain is not interfacially activated, and its lid domain probably exists under a permanent open conformation. Therefore, the phenomenon of interfacial activation in HPL is not only due to the presence of a full-length lid domain but also to other structural elements which probably allow the existence of stabilized closed and open conformations of the lid. GPLRP2(+lid) phospholipase activity is significantly reduced as compared to GPLRP2, whereas its lipase activity remains at the same level. Therefore, the lid domain plays a major role in substrate selectivity and can be considered as part of the active site. However, the presence of a full-length lid domain is not sufficient to explain the absence of phospholipase activity in HPL since HPL(-lid) does not display any phospholipase activity. We also produced a chimeric GPLRP2 mutant in which the C-terminal domain was substituted by the HPL C-terminal domain. The colipase effects, i.e., anchoring and stabilization of the lipase at the interface, are clearly observed with the chimera, whereas GPLRP2 is insensitive to colipase. The kinetic characterization of this chimera reveals for the first time that the interfacial stability of pancreatic lipases depends on the structure of the C-terminal domain.
- Published
- 1997
- Full Text
- View/download PDF
31. Engineering of metal-ion sites as distance constraints in structural and functional analysis of 7TM receptors.
- Author
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Elling CE, Thirstrup K, Nielsen SM, Hjorth SA, and Schwartz TW
- Subjects
- Binding Sites, Cations, Divalent, GTP-Binding Proteins metabolism, Receptors, Cell Surface metabolism, Structure-Activity Relationship, Zinc metabolism, Receptors, Cell Surface chemistry
- Abstract
G-protein-coupled receptors with their seven transmembrane (7TM) segments constitute the largest superfamily of proteins known. Unfortunately, still only relatively low resolution structures derived from electron cryo-microscopy analysis of 2D crystals are available for these proteins. We have used artificially designed Zn(II) metal-ion binding sites to probe 7TM receptors structurally and functionally and to define some basic distance constraints for molecular modeling. In this way, the relative helical rotation and vertical translocation of transmembrane helices TM-II, TM-III, TM-V, and TM-VI of the tachykinin NK-1 receptor have been restricted. Collectively, these zinc sites constitute a basic network of distance constraints that limit the degrees of freedom of the interhelical contact faces in molecular models of 7TM receptors. The construction of artificially designed metal-ion sites is discussed also in the context of probes for conformational changes occurring during receptor activation.
- Published
- 1997
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32. Cloning and expression in insect cells of two pancreatic lipases and a procolipase from Myocastor coypus.
- Author
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Thirstrup K, Carrière F, Hjorth SA, Rasmussen PB, Nielsen PF, Ladefoged C, Thim L, and Boel E
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, DNA, Enzyme Precursors, Molecular Sequence Data, Nucleopolyhedroviruses genetics, Phylogeny, Rodentia, Spodoptera, Colipases genetics, Lipase genetics, Pancreas enzymology, Protein Precursors genetics
- Abstract
The physiological role of pancreatic lipases has traditionally been assigned solely to triacylglyceride metabolism, while the digestion of phospholipids requires the presence of the pancreatic phospholipase A2, a 14-kDa enzyme unrelated to pancreatic lipases. However, in the guinea pig, it was observed that the pancreatic phospholipase A2 was absent and that a guinea pig pancreatic-lipase-related protein 2 (GPL-RP2) was responsible for phospholipase activity, in contrast to the situation observed in other mammalian species. As the guinea pig is a member of the hystricomorph rodents, it was of interest to investigate if other species within this evolutionary suborder display similar characteristics. The coypu (Myocastor coypus) also a member of the hystricomorph rodents, was chosen for further investigations. The cDNAs encoding two pancreatic lipases and a procolipase from the coypu were cloned, expressed and characterized. One lipase, CoPL-RP2, was identified as belonging to the RP2 subfamily, while the second, CoPL, was found to belong to the classical pancreatic lipase subfamily. Enzymic characterization and sequence data suggest a role for coypu colipase as a specific cofactor for CoPL, while this coypu colipase cannot be an important cofactor for CoPL-RP2 in vivo. Also, the new lipase cDNA sequences were used in a phylogentic analysis to reinvestigate the taxonomical position of the hystricomorph rodents (e.g. coypu and guinea pig) with respect to the myomorph rodents (e.g. rat and mouse).
- Published
- 1995
- Full Text
- View/download PDF
33. Structure-function relationships in naturally occurring mutants of pancreatic lipase.
- Author
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Carrière F, Thirstrup K, Boel E, Verger R, and Thim L
- Subjects
- Amino Acid Sequence, Animals, Aspergillus oryzae genetics, Colipases metabolism, Enzyme Activation, Guinea Pigs, Humans, Lipase genetics, Lipase metabolism, Lipase ultrastructure, Models, Biological, Models, Molecular, Molecular Sequence Data, Phospholipases analysis, Recombinant Proteins chemistry, Rodentia, Structure-Activity Relationship, Triglycerides metabolism, Lipase chemistry, Mutation
- Abstract
From primary structure comparison, the pancreatic lipase family is now divided into three subgroups: classical pancreatic lipases, pancreatic lipase-related proteins 1 (RPI) and pancreatic lipase-related proteins 2 (RP2). Among the RP2 subfamily, the guinea-pig and coypu enzymes share kinetic properties which differ from those of classical pancreatic lipases. Both enzymes display a high phospholipase activity and are not interfacially activated using a short chain triglyceride as substrate. Their activity towards insoluble triglycerides is inhibited by micellar concentrations of bile salts and is not restored by addition of colipase. These atypical kinetic properties are discussed in the light of amino acid sequence comparison between RP2 and classical pancreatic lipases, based on the closed and open conformations of the 3-D structure of human pancreatic lipase.
- Published
- 1994
- Full Text
- View/download PDF
34. Evidence for a pancreatic lipase subfamily with new kinetic properties.
- Author
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Thirstrup K, Verger R, and Carrière F
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, Consensus Sequence, DNA Primers, Gene Expression, Guinea Pigs, Humans, Kinetics, Lipase chemistry, Molecular Sequence Data, Phospholipases metabolism, Rodentia, Sequence Homology, Amino Acid, Substrate Specificity, Triglycerides metabolism, Lipase genetics, Lipase metabolism, Pancreas enzymology, Phylogeny
- Abstract
Several new members of the pancreatic lipase family have been reported recently, and amino acid sequence comparison reveals that this family can now be divided into three subgroups: (1) "classical" pancreatic lipases, (2) related proteins 1 (RP1), and (3) related proteins 2 (RP2) (Giller, T., et al. (1992) J. Biol. Chem. 267(23), 16509-16516). Whereas "classical" pancreatic lipases are well characterized with respect to kinetic properties, i.e., interfacial activation and dependence on colipase in the presence of bile salts, the two latter subfamilies have been poorly investigated so far. The kinetic behavior of a lipase from guinea pig pancreas differs, however, from that of "classical" lipases (Hjorth, A., et al. (1993) Biochemistry 32, 4702-4707). This enzyme is highly homologous to RP2 lipases with the exception of a deletion in the so-called lid domain that regulates access to the active center of pancreatic lipases. We have now characterized a novel lipase from coypu (Myocastor coypus) pancreas. This enzyme, also belonging to the RP2 subfamily, possesses a full-length lid domain, but its kinetic properties are very similar to those of the guinea pig enzyme: (1) a high phospholipase activity, (2) the absence of interfacial activation, and (3) the absence of a colipase effect at high bile salt concentrations. Since both guinea pig and coypu pancreas produce a classical pancreatic lipase and no measurable phospholipase A2 activity, it is suggested that RP2 enzymes act as real phospholipases under physiological conditions. In fact, all RP2 lipases from other species might share phospholipase activity and fulfill new biological functions.
- Published
- 1994
- Full Text
- View/download PDF
35. One-step purification and characterization of human pancreatic lipase expressed in insect cells.
- Author
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Thirstrup K, Carrière F, Hjorth S, Rasmussen PB, Wöldike H, Nielsen PF, and Thim L
- Subjects
- Amino Acid Sequence, Animals, Baculoviridae, Carbohydrate Sequence, Cell Line, Chromatography, Ion Exchange, Electrophoresis, Polyacrylamide Gel, Gene Expression Regulation, Enzymologic, Humans, Lipase genetics, Mass Spectrometry, Molecular Sequence Data, Moths, Pancreas enzymology, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Transfection, Lipase isolation & purification
- Abstract
A cDNA clone encoding the sequence of human pancreatic lipase (HPL) was subcloned into the baculovirus transfer vector pVL1392 and used in co-transfection of Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. A single recombinant protein (50 kDa) secreted by Sf9 cells was detectable in the culture medium 24 h post-infection using both anti-HPL polyclonal antibodies and potentiometric measurements of lipolytic activity. The expression level reached 40 mg/l of enzyme at 6 days. A single cation-exchange chromatography was sufficient to obtain a highly pure recombinant HPL as demonstrated by N-terminal sequencing, amino acid composition and carbohydrate analysis, as well as by mass spectrometry. These analyses revealed the production of mature protein with the correct processing of signal peptide and an homogenous glycosylation pattern. The kinetic properties of recombinant and native HPL were compared. Both enzymes showed similar profiles of interfacial activation, inhibition by bile salts and re-activation by colipase.
- Published
- 1993
- Full Text
- View/download PDF
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