Human profilin is a 15-kDa protein that plays a major role in the signaling pathway leading to cytoskeletal rearrangement. Essentially complete assignment of the 1H, 13C, and 15N resonances of human profilin have been made by analysis of multidimensional, double- and triple-resonance nuclear magnetic resonance (NMR) experiments. The deviation of the 13C alpha and 13C beta chemical shifts from their respective random coil values were analyzed and correlate well with the secondary structure determined from the NMR data. Twenty structures of human profilin were refined in the program X-PLOR using a total of 1186 experimentally derived conformational restraints. The structures converged to a root mean squared distance deviation of 1.5 A for the backbone atoms. The resultant conformational ensemble indicates that human profilin is an alpha/beta protein comprised of a seven-stranded, antiparallel beta-sheet and three helices. The secondary structure elements for human profilin are quite similar to those found in Acanthamoeba profilin I [Archer, S. J., Vinson, V. K., Pollard, T. D., & Torchia, D. A. (1993), Biochemistry 32, 6680-6687], suggesting that the three-dimensional structure of Acanthamoeba profilin I should be analogous to that determined here for human profilin. The structure determination of human profilin has facilitated the sequence alignment of lower eukaryotic and human profilins and provides a framework upon which the various functionalities of profilin can be explored. At least one element of the actin-binding region of human profilin is an alpha-helix. Two mechanisms by which phosphatidylinositol 4,5-bisphosphate can interfere with actin-binding by human profilin are proposed.

Rational modulation of the immune response with biologics represents one of the most promising and active areas for the realization of new therapeutic strategies. In particular, the use of function blocking monoclonal antibodies targeting checkpoint inhibitors such as CTLA-4 and PD-1 have proven to be highly effective for the systemic activation of the human immune system to treat a wide range of cancers. Ipilimumab is a fully human antibody targeting CTLA-4 that received FDA approval for the treatment of metastatic melanoma in 2011. Ipilimumab is the first-in-class immunotherapeutic for blockade of CTLA-4 and significantly benefits overall survival of patients with metastatic melanoma. Understanding the chemical and physical determinants recognized by these mAbs provides direct insight into the mechanisms of pathway blockade, the organization of the antigen-antibody complexes at the cell surface, and opportunities to further engineer affinity and selectivity. Here, we report the 3.0 Å resolution X-ray crystal structure of the complex formed by ipilimumab with its human CTLA-4 target. This structure reveals that ipilimumab contacts the front β-sheet of CTLA-4 and intersects with the CTLA-4:Β7 recognition surface, indicating that direct steric overlap between ipilimumab and the B7 ligands is a major mechanistic contributor to ipilimumab function. The crystallographically observed binding interface was confirmed by a comprehensive cell-based binding assay against a library of CTLA-4 mutants and by direct biochemical approaches. This structure also highlights determinants responsible for the selectivity exhibited by ipilimumab toward CTLA-4 relative to the homologous and functionally related CD28.immunotherapy | X-ray crystallography | CTLA-4 | ipilimumab | cancer

Pregnane X receptor (PXR) transactivation and binding assays have been developed into high-throughput assays, which are robust and reproducible (Z′ > 0.5). For most compounds, there was a good correlation between the results of the transactivation and binding assays. EC 50 values of compounds in the transactivation assay correlated reasonably well with their IC 50 values in the binding assay. However, there were discrepancies with some compounds showing high binding affinity in the binding assay translated into low transactivation. The most likely cause for these discrepancies was an agonistdependent relationship between binding affinity and transactivation response. In general, compounds that bound to human PXR and transactivated PXR tended to be large hydrophobic molecules. (Journal of Biomolecular Screening 2004:533-540)

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We evaluated the role insulin plays in the regulation of hepatic S14 gene transcription using the streptozotocin-induced diabetic rat model. Nuclear run-on activity and mRNAS14 levels were reduced by more than 85% in diabetic rats compared to those in intact animals. After the administration of insulin, both S14 run-on activity and mRNAS14 levels were rapidly induced. Within 1 h of insulin administration, S14 run-on activity and mRNAS14 were induced 5- and 8-fold, respectively. S14 gene expression was restored to intact levels within 4 h, with overall increases in run-on activity and mRNAS14 of 7- and 20-fold, respectively. The full induction of mRNAS14 cannot be accounted for solely by activation of S14 gene transcription, implicating insulin effects at the posttranscriptional level. However, our results show that the principal target for insulin action on the S14 gene is transcriptional. Administration of dibutryl cAMP and theophylline fully blocked the insulin-mediated increase in S14 gene transcription, indicating that hepatic cAMP levels play a dominant negative role in regulating S14 gene transcription in vivo. Fructose administration to starved diabetic rats induced only a marginal 60% increase in mRNAS14 and S14 run-on activity within 4 h, while insulin plus fructose or insulin plus glucose fully restored S14 gene expression to intact levels within the same time period. Thus, dietary fructose or a metabolite generated from fructose alone cannot induce S14 gene transcription or mRNAS14 to intact levels in the starved diabetic rat. Acute effects of dietary carbohydrate on hepatic S14 gene transcription are insulin dependent.(ABSTRACT TRUNCATED AT 250 WORDS)