title: Regulating levels of the neuromodulator d-serine in human brain: structural insight into pLG72 and d-amino acid oxidase interaction.
abstract: The human flavoenzyme d-amino acid oxidase (hDAAO) degrades the NMDA-receptor modulator d-serine in the brain. Although hDAAO has been extensively characterized, little is known about its main modulator pLG72, a small protein encoded by the primate-specific gene G72 that has been associated with schizophrenia susceptibility. pLG72 interacts with neosynthesized hDAAO, promoting its inactivation and degradation. In this work, we used low-resolution techniques to characterize the surface topology of the hDAAO-pLG72 complex. By using limited proteolysis coupled to mass spectrometry, we could map the exposed regions in the two proteins after complex formation and highlighted an increased sensitivity to proteolysis of hDAAO in complex with pLG72. Cross-linking experiments by using bis(sulfosuccinimidyl)suberate identified the single covalent bond between T182 in hDAAO and K62 in pLG72. In order to validate the designed mode of interaction, three pLG72 variants incrementally truncated at the C terminus, in addition to a form lacking the 71 N-terminal residues, were produced. All variants were dimeric, folded, and interacted with hDAAO. The strongest decrease in affinity for hDAAO (as well as for the hydrophobic drug chlorpromazine) was apparent for the N-terminally deleted pLG72(72-153) form, which lacked K62. On the other hand, eliminating the disordered C-terminal tail yielded a more stable pLG72 protein, improved the binding to hDAAO, although giving lower enzyme inhibition. Elucidation of the mode of hDAAO-pLG72 interaction now makes it possible to design novel molecules that, by targeting the protein complex, can be therapeutically advantageous for diseases related to impairment in d-serine metabolism.
authors.affiliation: Dipartimento di Scienze Chimiche, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Leila
authors.initials: L
authors.lastname: Birolo
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, Varese, Italy.|Centro Interuniversitario di Ricerca in Biotecnologie Proteiche "The Protein Factory", Politecnico di Milano and Università degli studi dell'Insubria, Milano, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Sacchi
authors.affiliation: IRCCS SDN, Napoli, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Smaldone
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, Varese, Italy.|Centro Interuniversitario di Ricerca in Biotecnologie Proteiche "The Protein Factory", Politecnico di Milano and Università degli studi dell'Insubria, Milano, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
authors.affiliation: Dipartimento di Scienze Chimiche, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Leo
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, Varese, Italy.|Centro Interuniversitario di Ricerca in Biotecnologie Proteiche "The Protein Factory", Politecnico di Milano and Università degli studi dell'Insubria, Milano, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Caldinelli
authors.affiliation: Italian Research National Council, Institute of Biostructures and Bioimaging, Napoli, Italy.
authors.forename: Luciano
authors.initials: L
authors.lastname: Pirone
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, Varese, Italy.
authors.forename: Patrick
authors.initials: P
authors.lastname: Eliometri
authors.affiliation: Italian Research National Council, Institute of Biostructures and Bioimaging, Napoli, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Di Gaetano
authors.affiliation: Dipartimento di Scienze Chimiche, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Ida
authors.initials: I
authors.lastname: Orefice
authors.affiliation: Italian Research National Council, Institute of Biostructures and Bioimaging, Napoli, Italy.
authors.forename: Emilia
authors.initials: E
authors.lastname: Pedone
authors.affiliation: Dipartimento di Scienze Chimiche, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Piero
authors.initials: P
authors.lastname: Pucci
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, Varese, Italy. loredano.pollegioni@uninsubria.it.|Centro Interuniversitario di Ricerca in Biotecnologie Proteiche "The Protein Factory", Politecnico di Milano and Università degli studi dell'Insubria, Milano, Italy. loredano.pollegioni@uninsubria.it.
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D000595:Amino Acid Sequence; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008958:Models, Molecular; D018377:Neurotransmitter Agents / Q000737:chemistry / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D059748:Proteolysis; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012559:Schizophrenia / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D040681:Structural Homology, Protein
chemical_list: D002352:Carrier Proteins; D003432:Cross-Linking Reagents; C475071:DAOA protein, human; D047908:Intracellular Signaling Peptides and Proteins; D018377:Neurotransmitter Agents; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D012694:Serine; C417983:DAO protein, human; D003605:D-Amino-Acid Oxidase
keywords: d-amino acid oxidase; d-serine; protein-protein interaction; regulation; schizophrenia