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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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Tagging cassettes for BAC recombineering and characterization oftagged dysbindin in the brains of BAC transgenic mouse lines. (A)The tag was inserted immediately after the ATG start codon and consistsof a 3×FLAG tag for immunopurification and detection, a 6×Hisspacer for TEV protease cleavage, a tobacco etch virus (TEV) proteasecleavage site, and a Strep tag for the second affinity purificationand detection. The reporter cassette was inserted immediately afterthe stop codon and consists of an internal ribosome entry site (IRES)and a bacterial promoter (GB2) in front of the gene encoding Venusfluorescent protein. (B) Immunoblot analysis of total extracts fromwild-type, sandy, and BAC transgenic mouse brains using anti-dysbindinand anti-FLAG antibodies. (C) SG analysis of dysbindin-containingcomplex(es) in the P2 synaptosome fraction of the BAC transgenic mousebrains with and without DSP cross-linking. Equal aliquots from individualfractions were resolved by SDS-PAGE and analyzed by immunoblottingusing the anti-dysbindin antibody. (D) The blots in panel C were reprobedwith the anti-FLAG antibody. * indicates nonspecific bands.
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fig2: Tagging cassettes for BAC recombineering and characterization oftagged dysbindin in the brains of BAC transgenic mouse lines. (A)The tag was inserted immediately after the ATG start codon and consistsof a 3×FLAG tag for immunopurification and detection, a 6×Hisspacer for TEV protease cleavage, a tobacco etch virus (TEV) proteasecleavage site, and a Strep tag for the second affinity purificationand detection. The reporter cassette was inserted immediately afterthe stop codon and consists of an internal ribosome entry site (IRES)and a bacterial promoter (GB2) in front of the gene encoding Venusfluorescent protein. (B) Immunoblot analysis of total extracts fromwild-type, sandy, and BAC transgenic mouse brains using anti-dysbindinand anti-FLAG antibodies. (C) SG analysis of dysbindin-containingcomplex(es) in the P2 synaptosome fraction of the BAC transgenic mousebrains with and without DSP cross-linking. Equal aliquots from individualfractions were resolved by SDS-PAGE and analyzed by immunoblottingusing the anti-dysbindin antibody. (D) The blots in panel C were reprobedwith the anti-FLAG antibody. * indicates nonspecific bands.

Mentions: The finding that dysbindin is associated with large complexes inP2 prompted us to further explore the components in the complexes.Although more than 100 dysbindin-binding partners have been describedin the literature,12 there are no reportedsystematic proteomics analysis of in vivo (endogenous) dysbindin complexesin the brain to date. The lack of reports on this topic is due likelyto the absence of suitable antibodies available for the immunopurificationof dysbindin from brain extracts, prohibiting isolation and characterizationof the dysbindin complex for proteomics analyses. We tested our anti-dysbindinantibody and found that it was also not suitable for immunopurification(data not shown). To overcome this issue, we generated bacterial artificialchromosome (BAC) transgenic mice in which a tagged dysbindin is expressedthrough its genomic DNA and under the control of its native regulatorysequences.52,53 The BAC clone possessing thedysbindin genomic DNA was epitope-tagged at the N-terminus by recombineeringin Escherichia coli (Figure 2A),43 and BAC transgenicmice were made by the pronuclear microinjection method.53 To this end, two BAC lines with stable expressionof tagged dysbindin were obtained. The BAC transgenic mice appearnormal and express both tagged and wild-type dysbindin (Figure 2B). However, only tagged dysbindin-1A could be detectedbecause either the 1B and 1C isoforms do not contain the N-terminalend of isoform 1A after splicing or normal splicing was disruptedby the inserted tag or reporter gene. On the basis of Figure 2B, the expression levels of the tagged dysbindin-1Aare about 2.3- and 1.7-fold of that of the untagged dysbindin-1A inlines 1 and 2, respectively. As multiple BACs are commonly insertedinto the mouse genome after microinjection,54 it is likely that both BAC lines possess multiple BAC transgenes(the actual BAC copy numbers were not determined in this study). Inthe SG analysis, the tagged dysbindin is cofractionated with wild-typedysbindin (Figure 2C using anti-dysbindin antibodyand 2D using anti-FLAG antibody), indicating that the tagged dysbindinprotein is functionally incorporated into the endogenous dysbindincomplexes. Therefore, the BAC transgenic lines provide us with anoptimal source for affinity purification of the endogenous dysbindin-associatedcomplexes.


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)

Tagging cassettes for BAC recombineering and characterization oftagged dysbindin in the brains of BAC transgenic mouse lines. (A)The tag was inserted immediately after the ATG start codon and consistsof a 3×FLAG tag for immunopurification and detection, a 6×Hisspacer for TEV protease cleavage, a tobacco etch virus (TEV) proteasecleavage site, and a Strep tag for the second affinity purificationand detection. The reporter cassette was inserted immediately afterthe stop codon and consists of an internal ribosome entry site (IRES)and a bacterial promoter (GB2) in front of the gene encoding Venusfluorescent protein. (B) Immunoblot analysis of total extracts fromwild-type, sandy, and BAC transgenic mouse brains using anti-dysbindinand anti-FLAG antibodies. (C) SG analysis of dysbindin-containingcomplex(es) in the P2 synaptosome fraction of the BAC transgenic mousebrains with and without DSP cross-linking. Equal aliquots from individualfractions were resolved by SDS-PAGE and analyzed by immunoblottingusing the anti-dysbindin antibody. (D) The blots in panel C were reprobedwith the anti-FLAG antibody. * indicates nonspecific bands.
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Related In: Results  -  Collection

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fig2: Tagging cassettes for BAC recombineering and characterization oftagged dysbindin in the brains of BAC transgenic mouse lines. (A)The tag was inserted immediately after the ATG start codon and consistsof a 3×FLAG tag for immunopurification and detection, a 6×Hisspacer for TEV protease cleavage, a tobacco etch virus (TEV) proteasecleavage site, and a Strep tag for the second affinity purificationand detection. The reporter cassette was inserted immediately afterthe stop codon and consists of an internal ribosome entry site (IRES)and a bacterial promoter (GB2) in front of the gene encoding Venusfluorescent protein. (B) Immunoblot analysis of total extracts fromwild-type, sandy, and BAC transgenic mouse brains using anti-dysbindinand anti-FLAG antibodies. (C) SG analysis of dysbindin-containingcomplex(es) in the P2 synaptosome fraction of the BAC transgenic mousebrains with and without DSP cross-linking. Equal aliquots from individualfractions were resolved by SDS-PAGE and analyzed by immunoblottingusing the anti-dysbindin antibody. (D) The blots in panel C were reprobedwith the anti-FLAG antibody. * indicates nonspecific bands.
Mentions: The finding that dysbindin is associated with large complexes inP2 prompted us to further explore the components in the complexes.Although more than 100 dysbindin-binding partners have been describedin the literature,12 there are no reportedsystematic proteomics analysis of in vivo (endogenous) dysbindin complexesin the brain to date. The lack of reports on this topic is due likelyto the absence of suitable antibodies available for the immunopurificationof dysbindin from brain extracts, prohibiting isolation and characterizationof the dysbindin complex for proteomics analyses. We tested our anti-dysbindinantibody and found that it was also not suitable for immunopurification(data not shown). To overcome this issue, we generated bacterial artificialchromosome (BAC) transgenic mice in which a tagged dysbindin is expressedthrough its genomic DNA and under the control of its native regulatorysequences.52,53 The BAC clone possessing thedysbindin genomic DNA was epitope-tagged at the N-terminus by recombineeringin Escherichia coli (Figure 2A),43 and BAC transgenicmice were made by the pronuclear microinjection method.53 To this end, two BAC lines with stable expressionof tagged dysbindin were obtained. The BAC transgenic mice appearnormal and express both tagged and wild-type dysbindin (Figure 2B). However, only tagged dysbindin-1A could be detectedbecause either the 1B and 1C isoforms do not contain the N-terminalend of isoform 1A after splicing or normal splicing was disruptedby the inserted tag or reporter gene. On the basis of Figure 2B, the expression levels of the tagged dysbindin-1Aare about 2.3- and 1.7-fold of that of the untagged dysbindin-1A inlines 1 and 2, respectively. As multiple BACs are commonly insertedinto the mouse genome after microinjection,54 it is likely that both BAC lines possess multiple BAC transgenes(the actual BAC copy numbers were not determined in this study). Inthe SG analysis, the tagged dysbindin is cofractionated with wild-typedysbindin (Figure 2C using anti-dysbindin antibodyand 2D using anti-FLAG antibody), indicating that the tagged dysbindinprotein is functionally incorporated into the endogenous dysbindincomplexes. Therefore, the BAC transgenic lines provide us with anoptimal source for affinity purification of the endogenous dysbindin-associatedcomplexes.

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.

Show MeSH
Related in: MedlinePlus