LIN-32/Atonal Controls Oxygen Sensing Neuron Development in Caenorhabditis elegans

AbstractDevelopment of complex nervous systems requires precisely controlled neurogenesis. The generation and specification of neurons occur through the transcriptional and post-transcriptional control of complex regulatory networks. In vertebrates and invertebrates, the proneural basic-helix-loop-helix (bHLH) family of transcription factors has multiple functions in neurogenesis. Here, we identified the LIN-32/Atonal bHLH transcription factor as a key regulator of URXL/R oxygen-sensing neuron development in Caenorhabditis elegans. When LIN-32/Atonal expression is lost, the expression of URX specification and terminal differentiation genes is abrogated. As such, lin-32 mutant animals are unable to respond to increases in environmental oxygen. The URX neurons are generated from a branch of the cell lineage that also produces the CEPDL/R and URADL/R neurons. We found development of these neurons is also defective, suggesting that LIN-32/Atonal regulates neuronal development of the entire lineage. Finally, our results show that aspects of URX neuronal fate are partially restored in lin-32 mutant animals when the apoptosis pathway is inhibited. This suggests that, as in other organisms, LIN-32/Atonal regulates neuronal apoptosis.</jats:p

Additional file 2: Figure S2. of Relaxin deficiency results in increased expression of angiogenesis- and remodelling-related genes in the uterus of early pregnant mice but does not affect endometrial angiogenesis prior to implantation

Expression of (a) 18 s, (b) PpiA, (c) Sdha and (d) Tbp in the uterus of Rln +/+ mice on days 1 to 4 of pregnancy (n = 6–8). Horizontal bars indicate mean values. Groups that do not share a letter are significantly different from one another (p < 0.05). (PDF 37 kb

Additional file 3: Table S1. of Relaxin deficiency results in increased expression of angiogenesis- and remodelling-related genes in the uterus of early pregnant mice but does not affect endometrial angiogenesis prior to implantation

Primer and probe sequences for the quantitative amplification of murine genes. Table S2. Mean CT values and the range of CT values for each gene analyzed by qPCR in the uterus of wildtype (Rln +/+ ) and relaxin deficient (Rln -/- ) mice (n = 6–8). Table S3. Primer sequences for the RT-PCR amplification of murine genes. (DOC 111 kb

Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Aryl Sulfonamides

The insulin-regulated aminopeptidase (IRAP) is a membrane-bound zinc metallopeptidase with many important regulatory functions. It has been demonstrated that inhibition of IRAP by angiotensin IV (Ang IV) and other peptides, as well as more druglike inhibitors, improves cognition in several rodent models. We recently reported a series of aryl sulfonamides as small-molecule IRAP inhibitors and a promising scaffold for pharmacological intervention. We have now expanded with a number of derivatives, report their stability in liver microsomes, and characterize the activity of the whole series in a new assay performed on recombinant human IRAP. Several compounds, such as the new fluorinated derivative <b>29</b>, present submicromolar affinity and high metabolic stability. Starting from the two binding modes previously proposed for the sulfonamide scaffold, we systematically performed molecular dynamics simulations and binding affinity estimation with the linear interaction energy method for the full compound series. The significant agreement with experimental affinities suggests one of the binding modes, which was further confirmed by the excellent correlation for binding affinity differences between the selected pair of compounds obtained by rigorous free energy perturbation calculations. The new experimental data and the computationally derived structure–activity relationship of the sulfonamide series provide valuable information for further lead optimization of novel IRAP inhibitors

Phenylalanine-544 plays a key role in substrate and inhibitor binding by providing a hydrophobic packing point at the active site of insulin-regulated aminopeptidase

Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and site-directed mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn2+ ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn2+ ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the Ki in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis. <br /