Proline utilization A (PutA) from Escherichia coli is a membrane-associated trifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate and moonlights as a transcriptional regulator. As a regulatory protein, PutA represses transcription of the put regulon, which contains the genes encoding PutA and the proline transporter PutP. The binding of proline to the proline dehydrogenase active site and the subsequent reduction of the flavin induces high affinity membrane association of PutA and relieves repression of the put regulon, thereby causing PutA to switch from its regulatory to its enzymatic role. Here, we present evidence suggesting that residues of the β3-α3 loop of the proline dehydrogenase domain (βα)8 barrel are involved in proline-mediated allosteric regulation of PutA-membrane binding. Mutation of the conserved residues Asp370 and Glu372 in the β3-α3 loop abrogates the ability of proline to induce functional membrane association. Both in vitro lipid/membrane binding assays and in vivo cell-based assays demonstrate that mutagenesis of Asp370 (D370N/A) or Glu372 (E372A) dramatically impedes PutA functional switching. The crystal structures of the proline dehydrogenase domain mutants PutA86-630D370N and PutA86-630D370A complexed with the proline analog L-tetrahydro-2-furoic acid show that the mutations cause only minor perturbations to the active site but no major structural changes, suggesting that the lack of proline response is not due to a failure of the mutated active sites to correctly bind the substrate. Rather, these results suggest that the β3-α3 loop may be involved in transmitting the status of the proline dehydrogenase active site and flavin redox state to the distal membrane association domain.

The insulin-like growth factor (IGF) system is a well-studied growth regulatory pathway implicated in breast cancer biology. Clinical trials testing monoclonal antibodies directed against the type I IGF receptor (IGF1R) in combination with estrogen receptor-α (ER) targeting have been completed, but failed to show benefits in patients with endocrine resistant tumors compared to ER targeting alone. We have previously shown that the closely related insulin receptor (InsR) is expressed in tamoxifen resistant breast cancer cells. Here we examined if inhibition of InsR affected tamoxifen-resistant (TamR) breast cancer cells. InsR function was inhibited by three different mechanisms: InsR shRNA, a small InsR blocking peptide, S961 and an InsR monoclonal antibody (mAb). Suppression of InsR function by these methods in TamR cells successfully blocked insulin-mediated signaling, monolayer proliferation, cell cycle progression and anchorage-independent growth. This strategy was not effective in parental cells likely due to the presence of IGFR/InsR hybrid receptors. Down-regulation of IGF1R in conjunction with InsR inhibition was more effective in blocking IGF- and insulin-mediated signaling and growth in parental cells compared to single receptor targeting alone. Our findings show TamR cells were stimulated by InsR and were not sensitive to IGF1R inhibition, whereas in tamoxifen-sensitive parental cancer cells, the presence of both receptors, especially hybrid receptors, allowed cross-reactivity of ligand-mediated activation and growth. To suppress the IGF system, targeting of both IGF1R and InsR is optimal in endocrine sensitive and resistant breast cancer.

Insulin receptor (InsR) and the type I insulin-like growth factor (IGF1R) are homologous receptors necessary for signal transduction by their cognate ligands insulin, insulin-like growth factor-I and –II (IGF-I and IGF-II). IGF1R monoclonal antibodies, intended to inhibit malignant phenotypic signaling, failed to show benefit in patients with endocrine-resistant tumors in phase III clinical trials. Our previous work showed that in tamoxifen-resistant cells IGF1R expression was lacking but InsR inhibition effectively blocked growth. In endocrine-sensitive breast cancer cells, insulin was not growth stimulatory, likely due to the presence of hybrid InsR/IGF1R, which has high affinity for IGF-I, but not insulin. Combination inhibition of InsR and IGF1R showed complete suppression of the system in endocrine-sensitive breast cancer cells. To develop InsR-binding agents, we employed a small protein scaffold, T7 phage Gene 2 Protein (Gp2) with the long-term goal of creating effective InsR inhibitors and diagnostics. Using yeast display and directed evolution, we identified three Gp2 variants (Gp2 #1, #5 and #10) with low nanomolar affinity and specific binding to cell-surface InsR. These Gp2 variants inhibited insulin-mediated monolayer proliferation in both endocrine-sensitive and -resistant breast cancer, but did not downregulate InsR expression. Gp2 #5 and Gp2#10 disrupted InsR function by inhibiting ligand-induced receptor activation. In contrast, Gp2 #1 did not block InsR phosphorylation. Notably, Gp2 #1 binding was enhanced by pre-treatment of cells with insulin suggesting a unique receptor-ligand binding mode. These Gp2 variants are the first non-immunoglobulin protein scaffolds to target insulin receptor and present compelling opportunity for modulation of InsR signaling.

Nanoscale-driven design of electron emission materials can significantly increase their overall efficiency as cathodes for field-induced electron emission by taking advantage of the field enhancement effect. The refractory nature and low work function (1.6 eV) of neodymium hexaboride (NdB 6 ) suggest that high aspect ratio NdB 6 nanostructures are potential candidates as efficient field emission cathodes. Here we report the morphological evolution of one-dimensional neodymium boride nanostructures synthesized using palladium-nanoparticlecatalyzed chemical vapor deposition as a function of reaction temperature. Scanning electron microscopy data show that judicious choices of reaction temperatures (795-940 °C) can lead to the preferential growth of curly nanowires or high aspect ratio nanowires. Transmission electron microscopy and selected area electron diffraction reveal that the crystallinity of these nanostructures changes from amorphous, to polycrystalline, to single crystalline as the reaction temperature increases. At reaction temperatures above 900 °C, single-crystalline NdB 6 nanowires with preferential [100] growth direction were successfully synthesized. Energy dispersive X-ray spectroscopic data suggest that this morphological evolution was strongly influenced by the solubility profiles of Nd and B in the Pd catalyst nanoparticles at different reaction temperatures. The implication of these results on the criteria of catalyst choices for the growth of binary metallic boride nanomaterials is also discussed.