Systems-level analysis of cell-specific AQP2 gene expression in renal collecting duct

We used a systems biology-based approach to investigate the basis of cell-specific expression of the water channel aquaporin-2 (AQP2) in the renal collecting duct. Computational analysis of the 5′-flanking region of the AQP2 gene (Genomatix) revealed 2 conserved clusters of putative transcriptional regulator (TR) binding elements (BEs) centered at −513 bp (corresponding to the SF1, NFAT, and FKHD TR families) and −224 bp (corresponding to the AP2, SRF, CREB, GATA, and HOX TR families). Three other conserved motifs corresponded to the ETS, EBOX, and RXR TR families. To identify TRs that potentially bind to these BEs, we carried out mRNA profiling (Affymetrix) in mouse mpkCCDc14 collecting duct cells, revealing expression of 25 TRs that are also expressed in native inner medullary collecting duct. One showed a significant positive correlation with AQP2 mRNA abundance among mpkCCD subclones (Ets1), and 2 showed a significant negative correlation (Elf1 and an orphan nuclear receptor Nr1h2). Transcriptomic profiling in native proximal tubules (PT), medullary thick ascending limbs (MTAL), and IMCDs from kidney identified 14 TRs (including Ets1 and HoxD3) expressed in the IMCD but not PT or MTAL (candidate AQP2 enhancer roles), and 5 TRs (including HoxA5, HoxA9 and HoxA10) expressed in PT and MTAL but not in IMCD (candidate AQP2 repressor roles). In luciferase reporter assays, overexpression of 3 ETS family TRs transactivated the mouse proximal AQP2 promoter. The results implicate ETS family TRs in cell-specific expression of AQP2 and point to HOX, RXR, CREB and GATA family TRs as playing likely additional roles

Structure and Glycolipid Binding Properties of the Nematicidal Protein Cry5B

Crystal (Cry) proteins are globally used in agriculture as proteinaceous insecticides. They have also been recently recognized to have great potential as anthelmintic agents in targeting parasitic roundworms (e.g. hookworms). The most extensively characterized of the anthelmintic Cry proteins is Cry5B. We report here the 2.3 Å resolution structure of the proteolytically activated form of Cry5B. This structure, which is the first for a nematicidal Cry protein, shows the familiar three-domain arrangement seen in insecticidal Cry proteins. However, domain II is unusual in that it more closely resembles a banana lectin than it does other Cry proteins. This result is consistent with the fact that the receptor for Cry5B consists of a set of invertebrate-specific glycans (attached to lipids), and also suggests that domain II is important for receptor binding. We found that not only is galactose an efficient competitor for binding between Cry5B and glycolipids, but so too is N-acetylgalactosamine (GalNAc). GalNAc is one of the core arthroseries tetrasaccharides of the Cry5B receptor, and galactose an antennary sugar that emanates from this core. These and prior data suggest that the minimal binding determinant for Cry5B consists of a core GalNAc and two antennary galactoses. Lastly, the protoxin form of Cry5B was found to bind nematode glycolipids with equal specificity as activated Cry5B, but with lower affinity. This suggests that the initial binding of Cry5B protoxin to glycolipids can be stabilized at the nematode cell surface by proteolysis. These results lay the groundwork for the design of effective Cry5B-based anthelmintics