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D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone.

ABSTRACT: Schizophrenia is a highly heritable neuropsychiatric disorder affecting ?1% of the world's population. Linkage and association studies have identified multiple candidate schizophrenia susceptibility genes whose functions converge on the glutamatergic neurotransmitter system. One such susceptibility gene encoding D-amino acid oxidase (DAO), an enzyme that metabolizes the NMDA receptor (NMDAR) co-agonist D-serine, has the potential to modulate NMDAR function in the context of schizophrenia. To further investigate its cellular regulation, we sought to identify DAO-interacting proteins that participate in its functional regulation in rat cerebellum, where DAO expression is especially high. Immunoprecipitation with DAO-specific antibodies and subsequent mass spectrometric analysis of co-precipitated proteins yielded 24 putative DAO-interacting proteins. The most robust interactions occurred with known components of the presynaptic active zone, such as bassoon (BSN) and piccolo (PCLO). The interaction of DAO with BSN was confirmed through co-immunoprecipitation assays using DAO- and BSN-specific antibodies. Moreover, DAO and BSN colocalized with one another in cultured cerebellar granule cells and in synaptic junction membrane protein fractions derived from rat cerebellum. The functional consequences of this interaction were studied through enzyme assay experiments, where DAO enzymatic activity was significantly inhibited as a result of its interaction with BSN. Taking these results together, we hypothesize that synaptic D-serine concentrations may be under tight regulation by a BSN-DAO complex. We therefore predict that this mechanism plays a role in the modulation of glutamatergic signaling through NMDARs. It also furthers our understanding of the biology underlying this potential therapeutic entry point for schizophrenia and other psychiatric disorders.

SUBMITTER: Popiolek M

PROVIDER: S-EPMC3190694 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone.

Popiolek Michael M Ross John F JF Charych Erik E Chanda Pranab P Gundelfinger Eckart D ED Moss Stephen J SJ Brandon Nicholas J NJ Pausch Mark H MH

The Journal of biological chemistry 20110623 33

Schizophrenia is a highly heritable neuropsychiatric disorder affecting ∼1% of the world's population. Linkage and association studies have identified multiple candidate schizophrenia susceptibility genes whose functions converge on the glutamatergic neurotransmitter system. One such susceptibility gene encoding D-amino acid oxidase (DAO), an enzyme that metabolizes the NMDA receptor (NMDAR) co-agonist D-serine, has the potential to modulate NMDAR function in the context of schizophrenia. To fur ...[more]

PMID: 21700703

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Project description:Proteasomes are protein complexes that mediate controlled degradation of damaged or unneeded cellular proteins. In neurons, proteasome regulates synaptic function and its dysfunction has been linked to neurodegeneration and neuronal cell death. However, endogenous mechanisms controlling proteasomal activity are insufficiently understood. Here, we describe a novel interaction between presynaptic scaffolding protein bassoon and PSMB4, a β subunit of the 20S core proteasome. Expression of bassoon fragments that interact with PSMB4 in cell lines or in primary neurons attenuates all endopeptidase activities of cellular proteasome and induces accumulation of several classes of ubiquitinated and non-ubiquitinated substrates of the proteasome. Importantly, these effects are distinct from the previously reported impact of bassoon on ubiquitination and autophagy and might rely on a steric interference with the assembly of the 20S proteasome core. In line with a negative regulatory role of bassoon on endogenous proteasome we found increased proteasomal activity in the synaptic fractions prepared from brains of bassoon knock-out mice. Finally, increased activity of proteasome and lower expression levels of synaptic substrates of proteasome could be largely normalized upon expression of PSMB4-interacting fragments of bassoon in neurons derived from bassoon deficient mice. Collectively, we propose that bassoon interacts directly with proteasome to control its activity at presynapse and thereby it contributes to a compartment-specific regulation of neuronal protein homeostasis. These findings provide a mechanistic explanation for the recently described link of bassoon to human diseases associated with pathological protein aggregation. Presynaptic cytomatrix protein bassoon (Bsn) interacts with PSMB4, the β7 subunit of 20S core proteasome, via three independent interaction interfaces. Bsn inhibits proteasomal proteolytic activity and degradation of different classes of proteasomal substrates presumably due to steric interference with the assembly of 20S core of proteasome. Upon Bsn deletion in neurons, presynaptic substrates of the proteasome are depleted, which can be reversed upon expression of PSMB4-interacting interfaces of Bsn. Taken together, bsn controls the degree of proteasome degradation within the presynaptic compartment and thus, contributes to the regulation of synaptic proteome.

Project description:We used a 3-step protocol that separates lipid-associated proteins based on their buoyant density, size and charge. All steps were conducted either on ice or at 4C. Step 1 involved a sucrose density gradient centrifugation in which ca. 100 postnatal day 4 rat brains were homogenized in buffer A (5 mM MES, pH 7.0, 0.3 M sucrose, 1 mM EDTA) containing protease inhibitor cocktail. The homogenate was centrifuged at 1,000xg for 15 min. Post-nuclear supernatant (PNS) was lysed with 10 volumes of 5 mM MES pH 7.0, 1 mM EDTA and incubated for 30 min at 4C. The lysed PNS was then centrifuged at 100,000 x g for 1 h. The pellet was resuspended in buffer A and loaded on top of a discontinuous sucrose gradient of 0.8, 1.2 and 2.0 M. The gradient was spun for 3 h at 270,000 x g. The material between 0.3 and 0.8 M sucrose was removed, diluted with 5 mM MES, pH 7.0, 1 mM EDTA to a final sucrose concentration of 0.3 M, and centrifuged at 100,000 x g for 1 h. The pellet was further resuspended in a final volume of 2 mL of PBS with protease inhibitors. In step 2, this material was applied to a gel filtration column packed with Sephacryl S-1000 Superfine matrix. The dimensions of the column were 1.5 cm wide and approximately 1.5 m tall. Material flowed through the column by gravity with elution by 1x phosphate buffered saline (PBS, pH 7.4). The flow rate was ca. 0.5 mL/min, and a BioRad Model 2110 fraction collector was used to collect 4 mL per fraction. For the final step 3 of the protocol, the four Sephacryl S-1000 fractions most highly enriched in Piccolo and Bassoon as determined by WB were pooled and applied to a column packed with Q Sepharose Fast Flow. The dimensions of this column were diameter of 2.5 cm, height of approximately 12 cm with a bed volume of 60 mL packed Q Sepharose, and the flow rate using the 2110 fraction collector was 3 mL/min and 10 mL fractions were collected. The final elution profile (all buffers in 20 mM Tris pH 7.0) was 200 mL of 150 mM NaCl to wash, then stepwise elution of 120 mL buffer with the following NaCl concentrations: 210 mM, 290 mM, 350 mM, and 1000 mM. About 40 mL of the 210 mM, 290 mM (Piccolo and Bassoon enriched), and 350 mM NaCl fractions were concentrated to about 1 mL using Amicon Centricon Plus-20 30 kDa cutoff concentrators. The final recovery was 7.5 ug in the 210 mM fraction, and 16 ug in each of the 290 mM and 350 mM fractions. The fractions were precipitated with tricholoroacetic acid, and the protein pellet was stored at -20C until further use. The second approach to identify proteins associating with Piccolo and Bassoon was an anti-Piccolo IP. The starting material was 10 mg of the fraction between 0.3 and 0.8 M sucrose after density centrifugation (step 1 above). This material was diluted to 0.3 M sucrose with 5 mM MES, pH 7.0 and centrifuged at 100,000 x g for 1 h. The pellet was resuspended and incubated for 1 h with 4 mL of solubilization buffer (5 mM MES, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail). Lysate was cleared by centrifugation at 100,000 x g for 1 h. One mL of supernatant was incubated with 2.5 ug anti-Piccolo rabbit antibody or 2.5 ug rabbit IgG for 4 h. A 100 uL Protein A agarose slurry (Roche) was then added and further incubated overnight. Material was transferred to a small column and washed with 20 mL solubilization buffer. Proteins were eluted twice with 200 uL 50 mM Tris, pH 6.5, 100 mM DTT, 2% SDS. This material was then extracted with 4 volumes methanol and 1-volume chloroform. This mixture was combined with water, centrifuged for 90 sec at 16,000 x g. The aqueous phase was removed and extracted again with 3 volumes of methanol. This precipitated protein was collected by centrifugation for 90 sec at 16,000 x g, methanol removed, and the speed-vacuum was used to dry the protein pellet. The 210 mM, 290 mM, and 350 mM NaCl fractions from the Mono-Q column protocol and the eluates (both IgG alone and anti-Piccolo IPs) from two separate IP experiments were subjected to Multi-dimensional Protein Identification technology.

Project description:pLG72 is a small, primate-specific protein of 153 amino acids. It is the product of the G72 gene, expressed in testis, spinal cord, and brain. The presence of G72 transcript and pLG72 has recurrently been called into question, however G72 mRNA and pLG72 protein levels were higher in blood and brain of patients with schizophrenia than in healthy controls. On the one hand, the SNP rs2391191 corresponding to the R30K substitution in pLG72 was genetically linked to schizophrenia, reduced thickness of the brain cortex in schizophrenia-affected individuals, and altered memory function. Various lines of evidence indicated that pLG72 is a mitochondrial protein, specifically an extrinsic protein bound on the outer membrane. Over the years, pLG72 was proposed to be involved in different functions: (a) overexpression induces mitochondria fragmentation, increasing the numbers of shorter and more mobile ones which could be delivered faster to regions of intense growth and facilitating the dendritic complexity; (b) it might induce oxidative stress by interacting with methionine-R-sulfoxide reductase B2; and (c) it binds and modulates the activity of FMN-containing oxidoreductase of the respiratory complex I. The main role of this protein, however, is related to its binding to the human flavoenzyme D-amino acid oxidase (hDAAO), i.e., the main catabolic enzyme for D-enantiomer of serine. This D-amino acid is a main endogenous coagonist of the N-methyl-D-aspartate type glutamate receptor (NMDAR) involved in main functions such as synaptic plasticity, learning, memory, and excitotoxicity. For this work, we reviewed the recent literature concerning the hDAAO-pLG72 interaction, focusing on the molecular details of the interaction, the effect of hDAAO function and stability, and the cellular effects, especially on D-serine concentration. The main effects related to the pathological R30K substitution are also reported. We have highlighted the gaps in our knowledge of this human protein as well as the relevance of clarifying the molecular details of hDAAO-pLG72 interaction in order to design molecules to modulate hDAAO activity/stability and thus NMDAR function acting at the D-serine cellular level.

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D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone.

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D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone.

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