Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40.

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40-MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Analysis of secreted proteins as an in vitro model for discovery of liver toxicity markers.

Despite the wealth of sequence data and new technologies that can scan large portions of the transcriptome or proteome in a single experiment, attempts to identify human biomarkers of toxicity have been met with limited success. We have adapted an in vitro model system to identify proteins secreted by a human hepatoma-derived cell line (HepG2/C3A) in response to toxicant exposure. Using quantitative proteomics, we can find alterations in the abundance of proteins at the source of damage-liver cells-that are likely to be present in blood samples of exposed animals. In a proof of concept experiment, conditioned medium from cells exposed to ethanol was subjected to quantitative mass spectral analysis after abundant proteins were immunodepleted. Eighty-seven proteins were identified with almost half changing in abundance. Some of these were only identified in the highest treatment condition and presumably result from the release of intracellular proteins into the medium when the cell membrane is disrupted upon cell death. However, the majority of the identified proteins reflect known consequences of ethanol exposure or alcoholism. The analysis of proteins found in conditioned medium after exposure to toxicants appears to be a useful system for the expedited discovery of potential human biomarkers.

Daxx inhibits muscle differentiation by repressing E2A-mediated transcription.

The basic helix-loop-helix (HLH) E2A transcription factors bind to DNA as homodimers or as heterodimers formed with other basic HLH factors, activate gene expression, and promote differentiation of muscle, lymphoid, neuronal, and other cell types. These E2A functions can be inhibited by the Id proteins, HLH factors that sequester E2A in non-DNA binding dimers. Here we describe the direct interaction of E2A with Daxx, a broadly expressed non-HLH protein previously associated with apoptosis and transcriptional repression. Daxx inhibits E2A function, but not via an Id-like mechanism; rather, it recruits histone deacetylase activity to E2A-dependent promoters. Increased Daxx expression during muscle differentiation inhibits E2A-dependent expression of key myogenic genes and reduces myotube formation, while decreased Daxx expression promotes myotube formation. These results identify a new mechanism for limiting E2A activity and establish a link between Daxx-mediated gene regulation and control of cellular differentiation.

Functional identity of genes detectable in expression profiling assays following globin mRNA reduction of peripheral blood samples.

OBJECTIVES: Whole-blood RNA for microarray analysis is easily accessible but contains a large proportion of globin mRNA that interferes with the accurate assessment of other genes. This study investigated the biological significance of genes whose expression was unmasked by globin mRNA reduction in peripheral blood. DESIGN AND METHODS: Samples were collected from healthy subjects using the PAXgene Blood RNA System, and globin mRNA was depleted using GLOBINclear. Genes exhibiting consistent changes in expression on Affymetrix HU133A 2.0 arrays were characterized in three main areas of gene ontology--molecular function, biological process, and cellular component. RESULTS: Globin reduction permitted detection of 2652+/-395 additional genes per assay. Genes unmasked by globin reduction include low abundance transcripts that function primarily as molecular binding proteins and catalytic enzymes in biological processes including transcription, replication, and intracellular transport and signalling. Protein products of these genes are preferentially associated with membranes and the nucleus. CONCLUSIONS: Additional genes detectable only after globin reduction in whole-blood RNA function in a variety of biological processes that may be important to diverse fields of study.