title: Unexpected Heterodivalent Recruitment of NOS1AP to nNOS Reveals Multiple Sites for Pharmacological Intervention in Neuronal Disease Models.
abstract: The protein NOS1AP/CAPON mediates signaling from a protein complex of NMDA receptor, PSD95 and nNOS. The only stroke trial for neuroprotectants that showed benefit to patients targeted this ternary complex. NOS1AP/nNOS interaction regulates small GTPases, iron transport, p38MAPK-linked excitotoxicity, and anxiety. Moreover, the nos1ap gene is linked to disorders from schizophrenia, post-traumatic stress disorder, and autism to cardiovascular disorders and breast cancer. Understanding protein interactions required for NOS1AP function, therefore, has broad implications for numerous diseases. Here we show that the interaction of NOS1AP with nNOS differs radically from the classical PDZ docking assumed to be responsible. The NOS1AP PDZ motif does not bind nNOS as measured by multiple methods. In contrast, full-length NOS1AP forms an unusually stable interaction with nNOS. We mapped the discrepancy between full-length and C-terminal PDZ motif to a novel internal region we call the ExF motif. The C-terminal PDZ motif, although neither sufficient nor necessary for binding, nevertheless promotes the stability of the complex. It therefore potentially affects signal transduction and suggests that functional interaction of nNOS with NOS1AP might be targetable at two distinct sites. We demonstrate that excitotoxic pathways can be regulated, in cortical neuron and organotypic hippocampal slice cultures from rat, either by the previously described PDZ ligand TAT-GESV or by the ExF motif-bearing region of NOS1AP, even when lacking the critical PDZ residues as long as the ExF motif is intact and not mutated. This previously unrecognized heterodivalent interaction of nNOS with NOS1AP may therefore provide distinct opportunities for pharmacological intervention in NOS1AP-dependent signaling and excitotoxicity.
authors.affiliation: Department of Neurobiology, A. I. Virtanen Institute, University of Eastern Finland, Kuopio, FIN 70210, Finland.
authors.forename: Li-Li
authors.identifier: http://orcid.org/0000-0002-8888-153X
authors.initials: LL
authors.lastname: Li
authors.affiliation: Department of Neurobiology, A. I. Virtanen Institute, University of Eastern Finland, Kuopio, FIN 70210, Finland.
authors.forename: Raquel M
authors.identifier: http://orcid.org/0000-0003-2179-472X
authors.initials: RM
authors.lastname: Melero-Fernandez de Mera
authors.affiliation: Division of Life Science, Center for Systems Biology and Human Health, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
authors.forename: Jia
authors.initials: J
authors.lastname: Chen
authors.affiliation: Radboud University Medical Center, Donders Institute for Brain, Cognition and Behaviour, Department of Cognitive Neuroscience, Nijmegen 6500 HB, The Netherlands, and.
authors.forename: Wei
authors.initials: W
authors.lastname: Ba
authors.affiliation: Radboud University Medical Center, Donders Institute for Brain, Cognition and Behaviour, Department of Cognitive Neuroscience, Nijmegen 6500 HB, The Netherlands, and.
authors.forename: Nael Nadif
authors.identifier: http://orcid.org/0000-0002-7448-9289
authors.initials: NN
authors.lastname: Kasri
authors.affiliation: Division of Life Science, Center for Systems Biology and Human Health, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
authors.forename: Mingjie
authors.identifier: http://orcid.org/0000-0001-9404-0190
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Neurobiology, A. I. Virtanen Institute, University of Eastern Finland, Kuopio, FIN 70210, Finland, Turku Centre for Biotechnology, Åbo Akademi University and University of Turku, Turku, FIN 20521, Finland miccou@utu.fi.
authors.forename: Michael J
authors.identifier: http://orcid.org/0000-0001-8693-3933
authors.initials: MJ
authors.lastname: Courtney
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D019556:COS Cells; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D002522:Chlorocebus aethiops; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism*; D009924:Organ Culture Techniques; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018690:Excitatory Amino Acid Agonists; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; C488081:NOS1AP protein, human; D011994:Recombinant Proteins; C480591:SCRIB protein, human; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C087334:TIAM1 protein, human; D025521:Tumor Suppressor Proteins; D016202:N-Methylaspartate; D052248:Nitric Oxide Synthase Type I; D048051:p38 Mitogen-Activated Protein Kinases
keywords: NMDA receptor; NOS1AP/CAPON; excitotoxic lesion; nNOS/NOS1; protein interaction; target identification