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Dysbindin-associated proteome in the p2 synaptosome fraction of mouse brain.

Han MH, Hu Z, Chen CY, Chen Y, Gucek M, Li Z, Markey SP - J. Proteome Res. (2014)

Bottom Line: However, little is known about the endogenous dysbindin-containing complex in the brain synaptosome.The interactions of several selected candidates, including WDR11, FAM91A1, snapin, muted, pallidin, and two proteasome subunits, PSMD9 and PSMA4, were verified by coimmunoprecipitation.Our data suggest that dysbindin is functionally interrelated to the ubiquitin-proteasome system and offer a molecular repertoire for future study of dysbindin functional networks in brain.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Mental Health , Bethesda, Maryland 20892, United States.

ABSTRACT
The gene DTNBP1 encodes the protein dysbindin and is among the most promising and highly investigated schizophrenia-risk genes. Accumulating evidence suggests that dysbindin plays an important role in the regulation of neuroplasticity. Dysbindin was reported to be a stable component of BLOC-1 complex in the cytosol. However, little is known about the endogenous dysbindin-containing complex in the brain synaptosome. In this study, we investigated the associated proteome of dysbindin in the P2 synaptosome fraction of mouse brain. Our data suggest that dysbindin has three isoforms associating with different complexes in the P2 fraction of mouse brain. To facilitate immunopurification, BAC transgenic mice expressing a tagged dysbindin were generated, and 47 putative dysbindin-associated proteins, including all components of BLOC-1, were identified by mass spectrometry in the dysbindin-containing complex purified from P2. The interactions of several selected candidates, including WDR11, FAM91A1, snapin, muted, pallidin, and two proteasome subunits, PSMD9 and PSMA4, were verified by coimmunoprecipitation. The specific proteasomal activity is significantly reduced in the P2 fraction of the brains of the dysbindin- mutant (sandy) mice. Our data suggest that dysbindin is functionally interrelated to the ubiquitin-proteasome system and offer a molecular repertoire for future study of dysbindin functional networks in brain.

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Characterization of anti-dysbindinantibody, and sucrose densitygradient ultracentrifugation (SG) analysis of dysbindin-containingcomplex(es) in the P2 synaptosome fraction of mouse brain. (A) Immunoblotanalysis of extract from HEK293T cells transfected with DTNBP1 expressionconstruct using the custom-made polyclonal anti-dysbindin antibody.The extract from untransfected HEK293T cells was used as a negativecontrol. (B) Immunoblot analysis of total extracts from wild-type(WT) and sandy (sdy) mouse brains using the same antibody as thatin panel A. (C) SG analysis of dysbindin-containing complex(es) inthe P2 synaptosome fraction of wild-type mouse brain with and withoutDSP cross-linking. Equal aliquots from individual fractions were resolvedby SDS-PAGE and analyzed by immunoblotting using the anti-dysbindinantibody. β-Tubulin served as a loading control in panels Aand B. Samples from sandy mice served as a negative control to showthe specificity of immunoblot analysis in panels B and C. (D) Structuresof the alternative splicing transcripts based on the annotated mousedysbindin isoforms in the UniProt database. The colored boxes indicatethe alternative splicing coding exons. Sizes of the structures arenot scaled precisely.
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fig1: Characterization of anti-dysbindinantibody, and sucrose densitygradient ultracentrifugation (SG) analysis of dysbindin-containingcomplex(es) in the P2 synaptosome fraction of mouse brain. (A) Immunoblotanalysis of extract from HEK293T cells transfected with DTNBP1 expressionconstruct using the custom-made polyclonal anti-dysbindin antibody.The extract from untransfected HEK293T cells was used as a negativecontrol. (B) Immunoblot analysis of total extracts from wild-type(WT) and sandy (sdy) mouse brains using the same antibody as thatin panel A. (C) SG analysis of dysbindin-containing complex(es) inthe P2 synaptosome fraction of wild-type mouse brain with and withoutDSP cross-linking. Equal aliquots from individual fractions were resolvedby SDS-PAGE and analyzed by immunoblotting using the anti-dysbindinantibody. β-Tubulin served as a loading control in panels Aand B. Samples from sandy mice served as a negative control to showthe specificity of immunoblot analysis in panels B and C. (D) Structuresof the alternative splicing transcripts based on the annotated mousedysbindin isoforms in the UniProt database. The colored boxes indicatethe alternative splicing coding exons. Sizes of the structures arenot scaled precisely.

Mentions: Toinvestigate the interactome of dysbindin in the synaptosome, we firstcharacterized dysbindin-containing protein complexes in the P2 synaptosomefraction. To this end, a polyclonal antibody against the C-terminalregion (amino acid 202–350) of dysbindin was generated in rabbitsand affinity purified. As shown in Figure 1A, this antibody specifically reacts with the recombinant dysbindinexpressed in HEK293T cells. When it was tested on the mouse braintissue extract, three specific dysbindin isoforms were detected withthe sizes of about 50, 35, and 32 kDa (Figure 1B). We found that this antibody also detects non-specific bands around40–50 kDa in the sample from sandy mouse, but they are muchweaker than the specific bands (Figure 1B,C).In human, there are three major isoforms, dysbindin-1A, -1B, and -1C,that can be detected by immunoblotting.24 In the UniProt database, three mouse dysbindin isoforms were annotated(Figure 1D), including 1A (352 amino acidsor aa) and 1C (271 aa) that are orthologues of human dysbindin isoforms,24 as well as a unique isoform (300 aa) that hasnot been named. According to their sizes, it is likely that the polyclonalantibody detects all these isoforms. Therefore, we designated theseisoforms as dysbindin-1A, -1B, and -1C, from large to small size,respectively. Using this antibody as a detection tool, we profileddysbindin-associated complexes in P2. The insoluble P2 fraction ofmouse brain was isolated, cross-linked by dithiobis(succinimidyl proprionate)(DSP, omitted for control), and then solubilized using 1% Triton X-100in TBS. DSP cross-linking can stabilize transient and/or weak protein–proteininteractions under harsh isolation conditions (i.e., with detergent)and has been applied successfully to isolate labile multiprotein complexes.49,50 The cleared P2 lysate was separated by ultracentrifugation on a10–40% linear sucrose gradient (SG). The sucrose gradientswere fractionated into 14 equal fractions from top to bottom, andan equal aliquot of each fraction was subjected to immunoblot analysisusing the anti-dysbindin antibody. As shown in Figure 1C, dysbindin-1A was concentrated close to BSA (66 kDa, 4.4S)without DSP cross-linking. The overall profile shifted to higher densityfractions with DSP cross-linking, indicating that dysbindin-1A ispresent in large protein complex(es) that are labile to Triton X-100extraction and DSP stabilizes these associations. There is no obviouspeak in the shifted fractions after fractions 3 and 4, suggestingthat dysbindin-1A may be present in multiple complexes that overlapin the sucrose gradient. Similarly, dysbindin-1B and -1C also shiftedto higher density fractions, and a distinct complex peak of dysbindin-1Bin fractions 5 and 6 can be observed after cross-linking (Figure 1C). Taken together, the SG analyses and DSP cross-linkingdemonstrate that dysbindin is present in macromolecular complexesin the P2 synaptosome fraction of brain. Consistent with previousstudy that dysbindin isoforms have distinct subsynaptic localization,23 our data suggest that dysbindin-1A, -1B, and-1C are present in different complexes. Dysbindin-1A is present incomplex(es) larger than that of BLOC-1, which was estimated to be∼230 kDa (5.2S).34,51


Dysbindin-associated proteome in the p2 synaptosome fraction of mouse brain.

Han MH, Hu Z, Chen CY, Chen Y, Gucek M, Li Z, Markey SP - J. Proteome Res. (2014)

Characterization of anti-dysbindinantibody, and sucrose densitygradient ultracentrifugation (SG) analysis of dysbindin-containingcomplex(es) in the P2 synaptosome fraction of mouse brain. (A) Immunoblotanalysis of extract from HEK293T cells transfected with DTNBP1 expressionconstruct using the custom-made polyclonal anti-dysbindin antibody.The extract from untransfected HEK293T cells was used as a negativecontrol. (B) Immunoblot analysis of total extracts from wild-type(WT) and sandy (sdy) mouse brains using the same antibody as thatin panel A. (C) SG analysis of dysbindin-containing complex(es) inthe P2 synaptosome fraction of wild-type mouse brain with and withoutDSP cross-linking. Equal aliquots from individual fractions were resolvedby SDS-PAGE and analyzed by immunoblotting using the anti-dysbindinantibody. β-Tubulin served as a loading control in panels Aand B. Samples from sandy mice served as a negative control to showthe specificity of immunoblot analysis in panels B and C. (D) Structuresof the alternative splicing transcripts based on the annotated mousedysbindin isoforms in the UniProt database. The colored boxes indicatethe alternative splicing coding exons. Sizes of the structures arenot scaled precisely.
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Related In: Results  -  Collection

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Show All Figures
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fig1: Characterization of anti-dysbindinantibody, and sucrose densitygradient ultracentrifugation (SG) analysis of dysbindin-containingcomplex(es) in the P2 synaptosome fraction of mouse brain. (A) Immunoblotanalysis of extract from HEK293T cells transfected with DTNBP1 expressionconstruct using the custom-made polyclonal anti-dysbindin antibody.The extract from untransfected HEK293T cells was used as a negativecontrol. (B) Immunoblot analysis of total extracts from wild-type(WT) and sandy (sdy) mouse brains using the same antibody as thatin panel A. (C) SG analysis of dysbindin-containing complex(es) inthe P2 synaptosome fraction of wild-type mouse brain with and withoutDSP cross-linking. Equal aliquots from individual fractions were resolvedby SDS-PAGE and analyzed by immunoblotting using the anti-dysbindinantibody. β-Tubulin served as a loading control in panels Aand B. Samples from sandy mice served as a negative control to showthe specificity of immunoblot analysis in panels B and C. (D) Structuresof the alternative splicing transcripts based on the annotated mousedysbindin isoforms in the UniProt database. The colored boxes indicatethe alternative splicing coding exons. Sizes of the structures arenot scaled precisely.
Mentions: Toinvestigate the interactome of dysbindin in the synaptosome, we firstcharacterized dysbindin-containing protein complexes in the P2 synaptosomefraction. To this end, a polyclonal antibody against the C-terminalregion (amino acid 202–350) of dysbindin was generated in rabbitsand affinity purified. As shown in Figure 1A, this antibody specifically reacts with the recombinant dysbindinexpressed in HEK293T cells. When it was tested on the mouse braintissue extract, three specific dysbindin isoforms were detected withthe sizes of about 50, 35, and 32 kDa (Figure 1B). We found that this antibody also detects non-specific bands around40–50 kDa in the sample from sandy mouse, but they are muchweaker than the specific bands (Figure 1B,C).In human, there are three major isoforms, dysbindin-1A, -1B, and -1C,that can be detected by immunoblotting.24 In the UniProt database, three mouse dysbindin isoforms were annotated(Figure 1D), including 1A (352 amino acidsor aa) and 1C (271 aa) that are orthologues of human dysbindin isoforms,24 as well as a unique isoform (300 aa) that hasnot been named. According to their sizes, it is likely that the polyclonalantibody detects all these isoforms. Therefore, we designated theseisoforms as dysbindin-1A, -1B, and -1C, from large to small size,respectively. Using this antibody as a detection tool, we profileddysbindin-associated complexes in P2. The insoluble P2 fraction ofmouse brain was isolated, cross-linked by dithiobis(succinimidyl proprionate)(DSP, omitted for control), and then solubilized using 1% Triton X-100in TBS. DSP cross-linking can stabilize transient and/or weak protein–proteininteractions under harsh isolation conditions (i.e., with detergent)and has been applied successfully to isolate labile multiprotein complexes.49,50 The cleared P2 lysate was separated by ultracentrifugation on a10–40% linear sucrose gradient (SG). The sucrose gradientswere fractionated into 14 equal fractions from top to bottom, andan equal aliquot of each fraction was subjected to immunoblot analysisusing the anti-dysbindin antibody. As shown in Figure 1C, dysbindin-1A was concentrated close to BSA (66 kDa, 4.4S)without DSP cross-linking. The overall profile shifted to higher densityfractions with DSP cross-linking, indicating that dysbindin-1A ispresent in large protein complex(es) that are labile to Triton X-100extraction and DSP stabilizes these associations. There is no obviouspeak in the shifted fractions after fractions 3 and 4, suggestingthat dysbindin-1A may be present in multiple complexes that overlapin the sucrose gradient. Similarly, dysbindin-1B and -1C also shiftedto higher density fractions, and a distinct complex peak of dysbindin-1Bin fractions 5 and 6 can be observed after cross-linking (Figure 1C). Taken together, the SG analyses and DSP cross-linkingdemonstrate that dysbindin is present in macromolecular complexesin the P2 synaptosome fraction of brain. Consistent with previousstudy that dysbindin isoforms have distinct subsynaptic localization,23 our data suggest that dysbindin-1A, -1B, and-1C are present in different complexes. Dysbindin-1A is present incomplex(es) larger than that of BLOC-1, which was estimated to be∼230 kDa (5.2S).34,51

Bottom Line: However, little is known about the endogenous dysbindin-containing complex in the brain synaptosome.The interactions of several selected candidates, including WDR11, FAM91A1, snapin, muted, pallidin, and two proteasome subunits, PSMD9 and PSMA4, were verified by coimmunoprecipitation.Our data suggest that dysbindin is functionally interrelated to the ubiquitin-proteasome system and offer a molecular repertoire for future study of dysbindin functional networks in brain.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Mental Health , Bethesda, Maryland 20892, United States.

ABSTRACT
The gene DTNBP1 encodes the protein dysbindin and is among the most promising and highly investigated schizophrenia-risk genes. Accumulating evidence suggests that dysbindin plays an important role in the regulation of neuroplasticity. Dysbindin was reported to be a stable component of BLOC-1 complex in the cytosol. However, little is known about the endogenous dysbindin-containing complex in the brain synaptosome. In this study, we investigated the associated proteome of dysbindin in the P2 synaptosome fraction of mouse brain. Our data suggest that dysbindin has three isoforms associating with different complexes in the P2 fraction of mouse brain. To facilitate immunopurification, BAC transgenic mice expressing a tagged dysbindin were generated, and 47 putative dysbindin-associated proteins, including all components of BLOC-1, were identified by mass spectrometry in the dysbindin-containing complex purified from P2. The interactions of several selected candidates, including WDR11, FAM91A1, snapin, muted, pallidin, and two proteasome subunits, PSMD9 and PSMA4, were verified by coimmunoprecipitation. The specific proteasomal activity is significantly reduced in the P2 fraction of the brains of the dysbindin- mutant (sandy) mice. Our data suggest that dysbindin is functionally interrelated to the ubiquitin-proteasome system and offer a molecular repertoire for future study of dysbindin functional networks in brain.

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