Exposing the subunit diversity within protein complexes: a mass spectrometry approach.

Identifying the list of subunits that make up protein complexes constitutes an important step towards understanding their biological functions. However, such knowledge alone does not reveal the full complexity of protein assemblies, as each subunit can take on multiple forms. Proteins can be post-translationally modified or cleaved, multiple products of alternative splicing can exist, and a single subunit may be encoded by more than one gene. Thus, for a complete description of a protein complex, it is necessary to expose the diversity of its subunits. Adding this layer of information is an important step towards understanding the mechanisms that regulate the activity of protein assemblies. Here, we describe a mass spectrometry-based approach that exposes the array of protein variants that comprise protein complexes. Our method relies on denaturing the protein complex, and separating its constituent subunits on a monolithic column prepared in-house. Following the subunit elution from the column, the flow is split into two fractions, using a Triversa NanoMate robot. One fraction is directed straight into an on-line ESI-QToF mass spectrometer for intact protein mass measurements, while the rest of the flow is fractionated into a 96-well plate for subsequent proteomic analysis. The heterogeneity of subunit composition is then exposed by correlating the subunit sequence identity with the accurate mass. Below, we describe in detail the methodological setting of this approach, its application on the endogenous human COP9 signalosome complex, and the significance of the method for structural mass spectrometry analysis of intact protein complexes.

Regulation of the GPR40 locus: towards a molecular understanding.

GPR40 {FFAR1 [non-esterified ('free') fatty acid receptor 1]} is a G-protein-coupled receptor expressed preferentially in pancreatic beta-cells. GPR40 functions as a receptor for medium and long-chain fatty acids, and has been implicated in mediating both physiological and pathological effects of fatty acids on beta-cells. The GPR40 gene is encoded at an interesting chromosomal locus that contains several genes: at the 5'-end of the locus, located approximately 4 kb upstream of GPR40, is CD22, a gene encoding a receptor expressed selectively in lymphocytes and involved in B-lymphocyte maturation and function. At the 3'-end of the locus are the GPR41 (FFAR3) and GPR43 (FFAR2) genes encoding receptors activated by short-chain fatty acids. The intergenic region between CD22 and GPR40 contains several evolutionarily conserved sequence blocks, among them HR2 and HR3. beta-Cell-specific expression of GPR40 is controlled at the transcriptional level through HR2, a potent beta-cell-specific enhancer. The mechanisms controlling cell-specific expression of the remaining genes in the cluster are unknown. Given the divergent modes of expression of the genes within the locus and their demonstrated physiological significance, it is important to analyse further the locus with a view to fully understanding the basis for transcriptional regulation of the encoded genes.

Regulation of the gene encoding GPR40, a fatty acid receptor expressed selectively in pancreatic beta cells.

GPR40 is a G protein-coupled receptor expressed preferentially in pancreatic beta cells. It is activated by long-chain fatty acids and has been implicated in mediating physiological and pathological effects of long-chain fatty acids on beta cells. We mapped the GPR40 transcription start site to a location 1044 bp upstream of the translation start site. This permitted definition of the GPR40 core promoter and the organization of the gene, which comprises a 24-bp non-coding exon, a 698-bp intron and a 4402-bp second exon, containing the entire protein coding sequence. Sequence analysis of the GPR40 locus revealed three evolutionarily conserved regions upstream to the translation start site (HR1-HR3). DNase I-hypersensitive sites were present in the HR2 and HR3 regions in beta cells but not in non-beta cells. The 5'-flanking region of the GPR40 gene was capable of directing transcriptional activity selectively in beta cells. An important component of this is attributable to the HR2 region, which showed strong beta cell-specific enhancer activity. Systematic mutagenesis of HR2 revealed several important sub-regions. Mutagenesis of sub-regions 4-5, and 9 reduced transcriptional activity by approximately 60 and 40%, respectively. These sub-regions can bind the beta cell-specific transcription factors PDX1 and BETA2, respectively, both in vitro and in vivo. Thus, cell-specific expression of the GPR40 gene involves a characteristic chromatin organization of the locus and is controlled at the transcriptional level through HR2, a potent beta cell-specific enhancer.

Role of intrinsic DNA binding specificity in defining target genes of the mammalian transcription factor PDX1.

PDX1 is a homeodomain transcription factor essential for pancreatic development and mature beta cell function. Homeodomain proteins typically recognize short TAAT DNA motifs in vitro: this binding displays paradoxically low specificity and affinity, given the extremely high specificity of action of these proteins in vivo. To better understand how PDX1 selects target genes in vivo, we have examined the interaction of PDX1 with natural and artificial binding sites. Comparison of PDX1 binding sites in several target promoters revealed an evolutionarily conserved pattern of nucleotides flanking the TAAT core. Using competitive in vitro DNA binding assays, we defined three groups of binding sites displaying high, intermediate and low affinity. Transfection experiments revealed a striking correlation between the ability of each sequence to activate transcription in cultured beta cells, and its ability to bind PDX1 in vitro. Site selection from a pool of oligonucleotides (sequence NNNTAATNNN) revealed a non-random preference for particular nucleotides at the flanking locations, resembling natural PDX1 binding sites. Taken together, the data indicate that the intrinsic DNA binding specificity of PDX1, in particular the bases adjacent to TAAT, plays an important role in determining the spectrum of target genes.