We explored prevalent cellular roles of the BLOC-1 complex in neuronal cells by performing a large-scale SILAC proteomic analysis. We identified 105 proteins that after a stringent two-tier set of filters, zeroed in on 24 proteins that associate with the BLOC-1 subunit dysbindin – a schizophrenia susceptibility factor. Of these 24 proteins, 11 were novel BLOC-1 binding partners. Prominent among these 24 proteins are all the subunits of the BLOC-1 and AP-3 adaptor complexes. Each of these 24 proteins was independently confirmed to be a BLOC-1 interactor and many were altered in content/distribution in cells or tissues deficient in BLOC-1 complexes. Major new findings from our studies are BLOC-1 interactions with the COG complex Golgi tether, and biochemical and genetic evidence supporting interactions between BLOC-1 and the antioxidant enzymes peroxiredoxins I and II. Thus, our quantitative proteomic analysis expanded upon the functional repertoire of the BLOC-1 complex and provides insight into molecular pathways of schizophrenia susceptibility, which may now include endosome to TGN retrograde traffic (COG) as well as redox metabolism (peroxiredoxins).
A common way to elucidate the function of proteins or their complexes is to identify other proteins associated with it. However, there are pitfalls to the methods employed to define interactors, in particular those associated with BLOC-1 (Ghiani and Dell’angelica, 2011
). To address several of the problems associated with studying the BLOC-1 complex, such as the low expression levels of BLOC-1 subunits, we employed a multifold approach. We quantitatively estimated peptides specifically enriched by immunoaffinity chromatography of FLAG-tagged BLOC-1 subunits purified from cells labeled with non-radioactive isotope tagged amino acids (Mann, 2006
). This procedure was coupled with in vivo
crosslinking with DSP to stabilize BLOC-1 interactions (Zlatic et al., 2010
). Since DSP was used at a sub-stoichiometric level, we likely identified major BLOC-1 interacting proteins as attested by the enrichment of BLOC-1 and AP-3 subunits. The sub-stoichiometric use of DSP was intended to stabilize immediate or first order, interactors and to minimize formation of extended crosslinked higher order networks. For example, crosslinked complexes revealed that BLOC-1 co-isolated with either the COG complex or exocyst complex proteins and did not result in a large complex containing BLOC-1, COG and exocyst subunits altogether (Ungar et al., 2002
). An additional layer of stringency in protein identification was the independent verification of protein interactions in BLOC-1 deficiencies, either using neuroblastoma cells rendered deficient by shRNA or hippocampal tissue from Dtnbp1sdy/sdy
mice. We reasoned that if proteins interact with the BLOC-1 complex then they could reveal novel phenotypes in BLOC-1 deficiencies. One of these phenotypes is the co-down-regulation of BLOC-1 subunits in cellular lysates when one of the subunits is absent or reduced (Zhang et al., 2002
; Li et al., 2003
; Starcevic and Dell’Angelica, 2004
). Such a phenotype was observed with peroxiredoxins I and II, as well as one of the COG subunits, Cog7. For another group of markers, we observed that their immunoreactivity was modified in the dentate gyrus of Dtnbp1sdy/sdy
mice. Such is the case of AP-3 subunits, KCNQ5, peroxiredoxins I and II, αN-catenin, CRMP4, the exocyst subunit Sec8, and Snap29 (unpublished observations).
Dysbindin interactions and their function are frequently considered independent of the BLOC-1 complex. However, several lines of evidence summarized by Ghiani and Dell’Angelica emphasize dysbindin to be an integral component of the BLOC-1 complex (Ghiani and Dell’angelica, 2011
; Mullin et al., 2011
). Our data are in rapport with this paradigm. The BLOC-1 interactions reported here are reproducibly obtained with two independent BLOC-1 subunits – dysbindin and muted. Furthermore, phenotypes induced by down-regulation of a third BLOC-1 subunit, pallidin, are recapitulated in dysbindin null, Dtnbp1sdy/sdy
mice. This is particularly evident in the down-regulation of Cog7 both in pallidin and dysbindin-deficient cells or tissues, respectively. What is the nature of the association between the COG and the BLOC-1 complex? One way we addressed this question was analyzing one of the COG-sensitive integral membrane Golgi proteins, collectively called GEAR proteins. GEAR proteins are reduced in COG-deficient cells and therefore provide a phenotypic readout for COG functions (Oka et al., 2004
). CASP, the GEAR protein analyzed here, is an integral Golgi membrane protein (Gillingham et al., 2002
). Our analysis demonstrated that when BLOC-1 was down-regulated, it led to a decreased level of COG complex proteins and in turn led to a reduction of CASP. These data indicate that the BLOC-1-dependent reduction of a COG complex subunit, although moderate, is sufficient to trigger a COG-dependent phenotype. These findings suggest that the BLOC-1 complex participates in an endosome route back to the Golgi complex delivering membrane proteins residen to or transiting through the Golgi complex (Smith et al., 2009
). Since the CASP down-regulation observed in BLOC-1 deficiency is subtle, as expected from a moderate Cog7 reduction, we speculate that a subset of COG-dependent vesicles derived from endosomes and bound to the Golgi complex may be uniquely susceptible to BLOC-1 deficiency. These vesicles likely would use the Snap29 SNARE, another new associate of the BLOC-1 complex. In agreement with this model, we have recently identified Snap29 as a direct binding partner of Cog6 protein (V.L., unpublished data).
Another interesting family of proteins identified in our SILAC proteomic analysis is peroxiredoxins. Peroxiredoxins I and II are ubiquitously expressed enzymes that remove low level peroxides generated as a result of steady state cellular metabolism (Bell and Hardingham, 2011
). These two enzymes are down-regulated in pallidin shRNA-treated cells and its immunoreactivity is decreased in the dentate gyrus of the hippocampal formation from BLOC-1 deficient Dtnbp1sdy/sdy
mice. This decrease in the peroxiredoxins, in turn, resulted in a significant increase in the steady levels of hydrogen peroxide in the range of low micromolar level. Apart from being a byproduct of oxidative metabolism, hydrogen peroxide also participates in cell signaling (Finkel, 2011
). Consequently, peroxiredoxins play essential roles in mediating signaling cascades targeted by hydrogen peroxide (Neumann et al., 2009
; Finkel, 2011
). BLOC-1 may regulate the activity or subcellular location of peroxiredoxins, for example in signal transduction by tyrosine kinase receptors on endosomes. Alternatively, peroxiredoxins may modulate BLOC-1 function by a redox mechanism, such as by regulating the oxidation status of cysteines either in BLOC-1 subunits (all human BLOC-1 isoforms contain cysteine residues), or in membrane proteins in close proximity of BLOC-1. It is of interest that the levels of peroxiredoxin I are reduced in the frontal cortex of schizophrenia patients (Focking et al., 2011
; Martins-de-Souza et al., 2011
). Such a reduction is consistent with the low expression of dysbindin and other BLOC-1 subunits in cortical areas of patients with schizophrenia (Talbot et al., 2004
; Tang et al., 2009a
; Mullin et al., 2011
; Talbot et al., 2011
). This raises the possibility that schizophrenia pathogenesis hypotheses centered on redox alterations and those linked to dysbindin may converge on a common molecular mechanism.
Part of our interest in BLOC-1 and dysbindin biology stems from its correlation with schizophrenia risk (Talbot et al., 2009
; Mullin et al., 2011
). We hypothesized that if dysbindin is part of a molecular pathway contributing to or affected by schizophrenia, then genes encoding dysbindin interactors should be significantly represented among those genes with structural variants associated with schizophrenia risk, such as SNAP29
(Malhotra et al., 2011
). To test this prediction we analyzed The International Schizophrenia Consortium database of cases carrying rare chromosomal deletions and duplications that increase risk of schizophrenia (Consortium, 2008
). This database contains 3,391 schizophrenia cases and 3,181 controls. Our prediction is strongly supported by CNVs encompassing genes encoding dysbindin interactors (). Eight of the 24 proteins identified as dysbindin-BLOC-1 interactors are represented among genes within CNVs exclusively found in schizophrenia patients. Among those we found COG and AP-3 complex subunits. None of these eight loci are affected in two genome wide analyses of unaffected individuals totaling 3,853 subjects (Consortium, 2008
; Buizer-Voskamp et al., 2011
). Strikingly, the dysbindin interactors Snap29 and the clathrin heavy chain isoform CLTCL1 (CHC22) were among those proteins whose genes are most frequently affected in schizophrenia individuals ().) These two genes are located within the chromosome 22q11.2 region. Individuals with haploinsufficiency of this region have 22q11.2 deletion syndrome and develop schizophrenia at rate of ~30%. These deletions account for as many as ~2% of de novo
schizophrenia cases in the general population (Karayiorgou et al., 2010
). The 22q11.2 deletion syndrome also encompasses SEPT5
, the gene encoding septin 5, a protein that binds septin 8 and AP-3 complexes both found in our dysbindin proteome (Baust et al., 2008
; Nakahira et al., 2010
). Thus, 22q11.2 deletion syndrome combines up to three haploinsufficiencies that converge on a pathway defined by the schizophrenia susceptibility factor, dysbindin. The molecular associations between a clathrin heavy chain isoform (CHC22), Snap29, and septin 5 suggest that the 22q11.2 deletion syndrome may have a pronounced deficiency of this pathway. These findings support the concept that quantitative proteomes of a schizophrenia susceptibility factor, such as dysbindin, can define putative schizophrenia susceptibility pathways by revealing unsuspected connections between the disease associated genomic loci. This hypothesis is supported by the dysbindin interactor Snap29 and the clathrin heavy chain isoform CLTCL1 (CHC22), which are among those proteins whose genes are frequently affected in schizophrenia individuals carrying CNVs associated to disease ().
Candidate genes included in CNVs from schizophrenia cases and controls
Pathogenic hypotheses for schizophrenia have tended to emphasize individual genes of “interest” rather than cell-autonomous pathways defined by the molecular interactions of a schizophrenia susceptibility factor (Ross et al., 2006
; Tandon et al., 2008
). One of those putative pathways is an endosomal hub defined by dysbindin and its protein interactors many of which remained unknown or were not prioritized based on their abundance in dysbindin isolates (Mead et al., 2010
). Based on the work presented here we propose that defective endosome sorting mechanisms controlled by the BLOC-1 complex may contribute to the pathogenesis of schizophrenia and systemic disorders that characterize the 22q11.2 deletion syndrome.