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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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Specific chymotrypsin-likeproteasomal activities in the extractsof soluble and insoluble fractions of brains of wild-type and sandymice. The proteasome activity assay was performed using the Proteasome-GloAssay System with the substrate for chymotrypsin-like activity (Promega).The soluble and insoluble fractions of wild-type and sdy mouse brains(P14–15) were extracted, quantified, and diluted in assay bufferwith equal concentration. Ten micrograms of each sample was used andincubated with Proteasome-Glo substrate mix with or without specificproteasome inhibitor (AdaAhx3L3VS) in a 96-well plate for 60 min atRT, followed by luminescence measurement with a plate reader. Thespecific proteasomal activity was calculated as follows: specificproteasomal activity = total peptidase activity (no inhibitor) –non-specific peptidase activity (with inhibitor). The activities inthe soluble or insoluble fraction are given as percentages of themean activity of the WT. Student’s two-tailed t test was used for statistical analysis. n = 3;assay performed in triplicate; ***p < 0.001, comparedwith wild-type.
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fig6: Specific chymotrypsin-likeproteasomal activities in the extractsof soluble and insoluble fractions of brains of wild-type and sandymice. The proteasome activity assay was performed using the Proteasome-GloAssay System with the substrate for chymotrypsin-like activity (Promega).The soluble and insoluble fractions of wild-type and sdy mouse brains(P14–15) were extracted, quantified, and diluted in assay bufferwith equal concentration. Ten micrograms of each sample was used andincubated with Proteasome-Glo substrate mix with or without specificproteasome inhibitor (AdaAhx3L3VS) in a 96-well plate for 60 min atRT, followed by luminescence measurement with a plate reader. Thespecific proteasomal activity was calculated as follows: specificproteasomal activity = total peptidase activity (no inhibitor) –non-specific peptidase activity (with inhibitor). The activities inthe soluble or insoluble fraction are given as percentages of themean activity of the WT. Student’s two-tailed t test was used for statistical analysis. n = 3;assay performed in triplicate; ***p < 0.001, comparedwith wild-type.

Mentions: Protein degradation via UPS is a universalmechanism in maintaining protein homeostasis in eukaryotic cells.In addition to de novo protein synthesis, growing evidence suggeststhat UPS also plays an essential role in the regulation of synapticconnectivity and plasticity.60−62 The association of dysbindinwith proteasome subunits suggests that it may have functional linkagesto UPS. To test this hypothesis, we investigated whether proteasomalactivity is affected in the brain of sandy mice. As proteasomes arelocalized to both the cytosol and synaptosome in the brain,63 we measured the proteasomal activities in theprotein extracts from both fractions. Interestingly, we found thatthe specific proteasomal activity (see Materialsand Methods for specific proteasomal activity measurement)is significantly decreased to ∼60% in the P2 fraction of sandymice compared with wild-type, whereas the activity is unchanged inthe cytosol (Figure 6). Taken together, ourdata suggest that dysbindin may regulate proteasomal activity in synapsesby interacting with proteasome subunits.


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)

Specific chymotrypsin-likeproteasomal activities in the extractsof soluble and insoluble fractions of brains of wild-type and sandymice. The proteasome activity assay was performed using the Proteasome-GloAssay System with the substrate for chymotrypsin-like activity (Promega).The soluble and insoluble fractions of wild-type and sdy mouse brains(P14–15) were extracted, quantified, and diluted in assay bufferwith equal concentration. Ten micrograms of each sample was used andincubated with Proteasome-Glo substrate mix with or without specificproteasome inhibitor (AdaAhx3L3VS) in a 96-well plate for 60 min atRT, followed by luminescence measurement with a plate reader. Thespecific proteasomal activity was calculated as follows: specificproteasomal activity = total peptidase activity (no inhibitor) –non-specific peptidase activity (with inhibitor). The activities inthe soluble or insoluble fraction are given as percentages of themean activity of the WT. Student’s two-tailed t test was used for statistical analysis. n = 3;assay performed in triplicate; ***p < 0.001, comparedwith wild-type.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4227559&req=5

fig6: Specific chymotrypsin-likeproteasomal activities in the extractsof soluble and insoluble fractions of brains of wild-type and sandymice. The proteasome activity assay was performed using the Proteasome-GloAssay System with the substrate for chymotrypsin-like activity (Promega).The soluble and insoluble fractions of wild-type and sdy mouse brains(P14–15) were extracted, quantified, and diluted in assay bufferwith equal concentration. Ten micrograms of each sample was used andincubated with Proteasome-Glo substrate mix with or without specificproteasome inhibitor (AdaAhx3L3VS) in a 96-well plate for 60 min atRT, followed by luminescence measurement with a plate reader. Thespecific proteasomal activity was calculated as follows: specificproteasomal activity = total peptidase activity (no inhibitor) –non-specific peptidase activity (with inhibitor). The activities inthe soluble or insoluble fraction are given as percentages of themean activity of the WT. Student’s two-tailed t test was used for statistical analysis. n = 3;assay performed in triplicate; ***p < 0.001, comparedwith wild-type.
Mentions: Protein degradation via UPS is a universalmechanism in maintaining protein homeostasis in eukaryotic cells.In addition to de novo protein synthesis, growing evidence suggeststhat UPS also plays an essential role in the regulation of synapticconnectivity and plasticity.60−62 The association of dysbindinwith proteasome subunits suggests that it may have functional linkagesto UPS. To test this hypothesis, we investigated whether proteasomalactivity is affected in the brain of sandy mice. As proteasomes arelocalized to both the cytosol and synaptosome in the brain,63 we measured the proteasomal activities in theprotein extracts from both fractions. Interestingly, we found thatthe specific proteasomal activity (see Materialsand Methods for specific proteasomal activity measurement)is significantly decreased to ∼60% in the P2 fraction of sandymice compared with wild-type, whereas the activity is unchanged inthe cytosol (Figure 6). Taken together, ourdata suggest that dysbindin may regulate proteasomal activity in synapsesby interacting with proteasome subunits.

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