Characterization of TRIF selectivity in the AGP class of lipid A mimetics: role of secondary lipid chains.

TLR4 agonists that favor TRIF-dependent signaling and the induction of type 1 interferons may have potential as vaccine adjuvants with reduced toxicity. CRX-547 (4), a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of lipid A mimetics possessing three (R)-3-decanoyloxytetradecanoyl groups and d-relative configuration in the aglycon, selectively reduces MyD88-dependent signaling resulting in TRIF-selective signaling, whereas the corresponding secondary ether lipid 6a containing (R)-3-decyloxytetradecanoyl groups does not. In order to determine which secondary acyl groups are important for the reduction in MyD88-dependent signaling activity of 4, the six possible ester/ether hybrid derivatives of 4 and 6a were synthesized and evaluated for their ability to induce NF-κB in a HEK293 cell reporter assay. An (R)-3-decanoyloxytetradecanoyl group on the 3-position of the d-glucosamine unit was found to be indispensable for maintaining low NF-κB activity irrespective of the substitutions (decyl or decanoyl) on the other two secondary positions. These results suggest that the carbonyl group of the 3-secondary lipid chain may impede homodimerization and/or conformational changes in the TLR4-MD2 complex necessary for MyD88 binding and pro-inflammatory cytokine induction.

Changes in vitamin D target gene expression in adipose tissue monitor the vitamin D response of human individuals.

SCOPE: Vitamin D3, its biologically most active metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), and the vitamin D receptor (VDR) are important for adipose tissue biology. METHODS AND RESULTS: We extrapolated genomic VDR association loci in adipocytes from 55 conserved genome-wide VDR-binding sites in nonfat tissues. Taking the genes DUSP10, TRAK1, NRIP1, and THBD as examples, we confirmed the predicted VDR binding sites upstream of their transcription start sites and showed rapid mRNA up-regulation of all four genes in SGBS human pre-adipocytes. Using adipose tissue biopsy samples from 47 participants of a 5-month vitamin D3 intervention study, we demonstrated that all four primary VDR target genes can serve as biomarkers for the vitamin D3 responsiveness of human individuals. Changes in DUSP10 gene expression appear to be the most comprehensive marker, while THBD mRNA changes characterized a rather different group of study participants. CONCLUSION: We present a new approach to predict vitamin D target genes based on conserved genomic VDR-binding sites. Using human adipocytes as examples, we show that such ubiquitous VDR target genes can be used as markers for the individual's response to a supplementation with vitamin D3.

Small molecule targeting Cdc42-intersectin interaction disrupts Golgi organization and suppresses cell motility.

Signaling through the Rho family of small GTPases has been intensely investigated for its crucial roles in a wide variety of human diseases. Although RhoA and Rac1 signaling pathways are frequently exploited with the aid of effective small molecule modulators, studies of the Cdc42 subclass have lagged because of a lack of such means. We have applied high-throughput in silico screening and identified compounds that are able to fit into the surface groove of Cdc42, which is critical for guanine nucleotide exchange factor binding. Based on the interaction between Cdc42 and intersectin (ITSN), a specific Cdc42 guanine nucleotide exchange factor, we discovered compounds that rendered ITSN-like interactions in the binding pocket. By using in vitro binding and imaging as well as biochemical and cell-based assays, we demonstrated that ZCL278 has emerged as a selective Cdc42 small molecule modulator that directly binds to Cdc42 and inhibits its functions. In Swiss 3T3 fibroblast cultures, ZCL278 abolished microspike formation and disrupted GM130-docked Golgi structures, two of the most prominent Cdc42-mediated subcellular events. ZCL278 reduces the perinuclear accumulation of active Cdc42 in contrast to NSC23766, a selective Rac inhibitor. ZCL278 suppresses Cdc42-mediated neuronal branching and growth cone dynamics as well as actin-based motility and migration in a metastatic prostate cancer cell line (i.e., PC-3) without disrupting cell viability. Thus, ZCL278 is a small molecule that specifically targets Cdc42-ITSN interaction and inhibits Cdc42-mediated cellular processes, thus providing a powerful tool for research of Cdc42 subclass of Rho GTPases in human pathogenesis, such as those of cancer and neurological disorders.

A Cdc42 mutant specifically activated by intersectin.

The Rho family GTPase Cdc42 functions as a molecular switch and controls many fundamental cellular processes such as cytoskeletal regulation, cell polarity, and vesicular trafficking. Guanine nucleotide exchange factors of the Dbl family activate Cdc42 and other Rho GTPases by catalyzing the removal of bound GDP, allowing for GTP loading, and subsequent effector recognition ultimately leading to downstream signaling events. Analysis of existing structural data reveals that the Dbl exchange factor intersectin engages a strictly conserved GTPase residue of Cdc42 (tyrosine 32) in a unique mode with respect to all other visualized exchange factor-Rho GTPase interfaces. To investigate this differential binding architecture, we analyzed the role of tyrosine 32 of Cdc42 in binding, and stimulation by Dbl family exchange factors. Deletion of the hydroxyl side chain of tyrosine 32 substantially increases the affinity of Cdc42 for intersectin, yet severely cripples interaction with Dbs, a normally potent exchange factor of Cdc42. Moreover, Cdc42(Y32F) is exclusively activated by intersectin, while virtually unresponsive to other Cdc42-activating exchange factors in vitro and in vivo. Further, the structural determinants unique to intersectin, which permit selective recognition and concomitant stimulation of Cdc42(Y32F), have been defined. Cdc42 and other individual Rho GTPases receive input stimulatory signals from a multitude of Dbl exchange factors, and therefore, Cdc42(Y32F) could act as a valuable reagent for understanding the specific influence of ITSN on Cdc42-mediated signaling phenomena.

Asb6, an adipocyte-specific ankyrin and SOCS box protein, interacts with APS to enable recruitment of elongins B and C to the insulin receptor signaling complex.

The APS adapter protein plays a pivotal role in coupling the insulin receptor to CAP and c-Cbl in the phosphatidylinositol 3-kinase-independent pathway of insulin-stimulated glucose transport. Yeast two-hybrid screening of a 3T3-L1 adipocyte library using APS as a bait identified a 418-amino acid ankyrin and SOCS (suppressor of cytokine signaling) box protein Asb6 as an interactor. Asb6 is an orphan member of a larger family of Asb proteins that are ubiquitously expressed. However, Asb6 expression appears to be restricted to adipose tissue. Asb6 was specifically expressed in 3T3-L1 adipocytes as a 50-kDa protein but not in fibroblasts. In Chinese hamster ovary-insulin receptor (CHO-IR) cells Myc epitope-tagged APS interacted constitutively with FLAG-tagged Asb6 in the presence or absence of insulin stimulation and insulin stimulation did not alter the interaction. In 3T3-L1 adipocytes, insulin receptor activation was accompanied by the APS-dependent recruitment of Asb6. Asb6 did not appear to undergo tyrosine phosphorylation. Immunofluorescence and confocal microscopy studies revealed that Asb6 colocalized with APS in CHO cells and in 3T3-L1 adipocytes. In immunoprecipitation studies in CHO cells or 3T3-L1 adipocytes, the Elongin BC complex was found to be bound to Asb6, and activation of the insulin receptor was required to facilitate Asb6 recruitment along with Elongins B/C. Prolonged insulin stimulation resulted in the degradation of APS when Asb6 was co-expressed but not in the absence of Asb6. We conclude that Asb6 functions to regulate components of the insulin signaling pathway in adipocytes by facilitating degradation by the APS-dependent recruitment of Asb6 and Elongins BC.

Crystal structures of the Dab homology domains of mouse disabled 1 and 2.

Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3, the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2)). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes.