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Revealing Secrets of Synaptic Protein Interactions: A Biosensor based Strategy
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein interactions are the basis of synaptic function, and studying these interactions on a molecular level is crucial for understanding basic brain function, as well as mechanisms underlying neurological disorders. In this thesis, kinetic and mechanistic characterization of synaptic protein interactions was performed by using surface plasmon resonance biosensor technology. Fragment library screening against the reverse transcriptase of HIV was included, as it served as an outlook for future drug discovery against ligand-gated ion channels.

The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealed caldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and compete at similar binding sites but their interactions display different mechanisms and kinetics. In contrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+ suggesting distinct in vivo functional properties of caldendrin and calmodulin.

Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51 histaminergic ligands were screened and a unique pharmacology was determined. A further requirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughput for screening for such ligands, as well as for being employed for fragment-based drug discovery.

AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity, and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secreted glycoprotein, interacts with the AMPA receptor. By using biochemical methods, it was shown that Noelin1 interacts directly with the receptor. The kinetics of the interaction were estimated by biosensor analysis, thereby confirming the interaction and suggesting low nanomolar affinity. The results provide a basis for functional characterization of a novel AMPA receptor protein interaction.

The results demonstrate how secrets of synaptic protein interactions and function were revealed by using a molecular based approach. Improving the understanding of such interactions is valuable for basic neuroscience. At the same time, the technical advancements that were achieved to study interactions of ligand-gated ion channels by surface plasmon resonance technology, provide an important tool for discovery of novel therapeutics against these important drug targets.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 73 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1133
Keyword [en]
Surface plasmon resonance, biosensor, AMPA receptor, GABAA receptor, ligand-gated ion channel, A-kinase anchoring protein, caldendrin, calmodulin, HIV, fragment based drug discovery
National Category
Biochemistry and Molecular Biology Neurosciences
Research subject
Biochemistry; Neuroscience
Identifiers
URN: urn:nbn:se:uu:diva-220879ISBN: 978-91-554-8916-8 (print)OAI: oai:DiVA.org:uu-220879DiVA: diva2:707873
Public defence
2014-05-16, B42, Husargatan 3, 751 23 Uppsala, 13:15 (English)
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework Programme
Available from: 2014-04-25 Created: 2014-03-22 Last updated: 2014-04-29
List of papers
1. AKAP79/150 interacts with the neuronal calcium-binding protein caldendrin
Open this publication in new window or tab >>AKAP79/150 interacts with the neuronal calcium-binding protein caldendrin
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2012 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 122, no 4, 714-726 p.Article in journal (Refereed) Published
Abstract [en]

The A kinase-anchoring protein AKAP79/150 is a postsynaptic scaffold molecule and a key regulator of signaling events. At the postsynapse it coordinates phosphorylation and dephosphorylation of receptors via anchoring kinases and phosphatases near their substrates. Interactions between AKAP79 and two Ca2+ -binding proteins caldendrin and calmodulin have been investigated here. Calmodulin is a known interaction partner of AKAP79/150 that has been shown to regulate activity of the kinase PKC in a Ca2+-dependent manner. Pull-down experiments and surface plasmon resonance biosensor analyses have been used here to demonstrate that AKAP79 can also interact with caldendrin, a neuronal calcium-binding protein implicated in regulation of Ca2+ -influx and release. We demonstrate that calmodulin and caldendrin compete for a partially overlapping binding site on AKAP79 and that their binding is differentially dependent on calcium. Therefore, this competition is regulated by calcium levels. Moreover, both proteins have different binding characteristics suggesting that the two proteins might play complementary roles. The postsynaptic enrichment, the complex binding mechanism, and the competition with calmodulin, makes caldendrin an interesting novel player in the signaling toolkit of the AKAP interactome.

Keyword
calcium sensor protein, calmodulin, postsynaptic density, surface plasmon resonance
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-179561 (URN)10.1111/j.1471-4159.2012.07828.x (DOI)000306475700007 ()
Available from: 2012-08-20 Created: 2012-08-20 Last updated: 2017-12-07Bibliographically approved
2. Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function
Open this publication in new window or tab >>Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function
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2012 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 25, no 10, 495-503 p.Article in journal (Refereed) Published
Abstract [en]

The kinetic and mechanistic details of the interaction between caldendrin, calmodulin and the B-domain of AKAP79 were determined using a biosensor-based approach. Caldendrin was found to compete with calmodulin for binding at AKAP79, indicating overlapping binding sites. Although the AKAP79 affinities were similar for caldendrin (KD?=?20?n m) and calmodulin (KD?=?30?n m), their interaction characteristics were different. The calmodulin interaction was well described by a reversible one-step model, but was only detected in the presence of Ca2+. Caldendrin interacted with a higher level of complexity, deduced to be an induced fit mechanism with a slow relaxation back to the initial encounter complex. It interacted with AKAP79 also in the absence of Ca2+, but with different kinetic rate constants. The data are consistent with a similar initial Ca2+-dependent binding step for the two proteins. For caldendrin, a second Ca2+-independent rearrangement step follows, resulting in a stable complex. The study shows the importance of establishing the mechanism and kinetics of proteinprotein interactions and that minor differences in the interaction of two homologous proteins can have major implications in their functional characteristics. These results are important for the further elucidation of the roles of caldendrin and calmodulin in synaptic function.

Keyword
postsynaptic density, A-kinase anchoring protein, caldendrin, calmodulin, surface plasmon resonance, induced fit
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-183563 (URN)10.1002/jmr.2215 (DOI)000309067200002 ()
Available from: 2012-12-06 Created: 2012-10-29 Last updated: 2017-12-07Bibliographically approved
3. Histaminergic pharmacology of homo-oligomeric beta 3 gamma-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology
Open this publication in new window or tab >>Histaminergic pharmacology of homo-oligomeric beta 3 gamma-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology
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2012 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 84, no 3, 341-351 p.Article in journal (Refereed) Published
Abstract [en]

A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric beta 3 GABA(A) receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to beta 3 GABA(A) receptors with micromolar affinity (K-D <300 mu M), thus extending previous evidence that beta 3 GABA(A) receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with beta 3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric beta 3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for beta 3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether beta 3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site. 

Keyword
Ligand-gated ion channel, gamma-Aminobutyric acid type A receptor, Histamine, Surface plasmon resonance, Competition analysis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-177842 (URN)10.1016/j.bcp.2012.04.008 (DOI)000305546200011 ()
Available from: 2012-07-20 Created: 2012-07-19 Last updated: 2017-12-07Bibliographically approved
4. Corrigendum to "Histaminergic pharmacology of homo-oligomeric β3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology" (Biochemical Pharmacology (2012) 84 (341-351))
Open this publication in new window or tab >>Corrigendum to "Histaminergic pharmacology of homo-oligomeric β3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology" (Biochemical Pharmacology (2012) 84 (341-351))
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2012 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 84, no 11, 1541- p.Article in journal (Refereed) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-187955 (URN)10.1016/j.bcp.2012.09.013 (DOI)000311465900017 ()
Available from: 2012-12-12 Created: 2012-12-12 Last updated: 2017-05-03Bibliographically approved
5. Extracellular matrix protein Noelin-1 interacts with the AMPA receptor
Open this publication in new window or tab >>Extracellular matrix protein Noelin-1 interacts with the AMPA receptor
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(English)Manuscript (preprint) (Other academic)
National Category
Neurosciences Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-221139 (URN)
Funder
EU, FP7, Seventh Framework Programme
Available from: 2014-03-25 Created: 2014-03-25 Last updated: 2014-04-29
6. Identification of a Novel Scaffold for Allosteric Inhibition of Wild Type and Drug Resistant HIV-1 Reverse Transcriptase by Fragment Library Screening
Open this publication in new window or tab >>Identification of a Novel Scaffold for Allosteric Inhibition of Wild Type and Drug Resistant HIV-1 Reverse Transcriptase by Fragment Library Screening
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2011 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 3, 699-708 p.Article in journal (Refereed) Published
Abstract [en]

A novel scaffold inhibiting wild type and drug resistant variants of human immunodeficiency virus type 1 reverse transcriptase (HIV-1RT) has been identified in a library consisting of 1040 fragments. The fragments were significantly different from already known non-nucleoside reverse transcriptase inhibitors (NNRTIs), as indicated by a Tversky similarity analysis. A screening strategy involving SPR biosensor-based interaction analysis and enzyme inhibition was used. Primary biosensor-based screening, using short concentration series, was followed by analysis of nevirapine competition and enzyme inhibition, thus identifying inhibitory fragments binding to the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Ten hits were discovered, and their affinities and resistance profiles were evaluated with wild type and three drug resistant enzyme variants (K103N, Y181C, and L100I). One fragment exhibited submillimolar K(D) and IC(50) values against all four tested enzyme variants. A substructure comparison between the fragment and 826 structurally diverse published NNRTIs confirmed that the scaffold was novel. The fragment is a bromoindanone with a ligand efficiency of 0.42 kcal/mol(-1).

National Category
Other Basic Medicine
Identifiers
urn:nbn:se:uu:diva-140680 (URN)10.1021/jm1010513 (DOI)000286798100002 ()21207961 (PubMedID)
Available from: 2011-01-07 Created: 2011-01-07 Last updated: 2017-12-11Bibliographically approved

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