The structure of AQP1 water channel was determined to 2.2 Å resolution. The channel consists of three topological elements, an extracellular and a cytoplasmic vestibule connected by an extended narrow pore or selectivity filter. At the extracellular end of the selectivity filter is the constriction region, which establishes the steric upper limit of the channel and has an effective solvent accessible diameter of ~2.8 Å. Within the selectivity filter, four bound waters are localized along three hydrophilic nodes which punctuate an otherwise extremely hydrophobic pore segment. This novel combination of a long hydrophobic pore and a minimal number of solute binding sites facilitates rapid water transport. Residues of the constriction region, in particular histidine 182 which is conserved among all known water specific channels, are critical in establishing water specificity. Analysis of the AQP1 pore also indicates that the transport of protons through this channel is highly energetically unfavorable.

SUMMARY MYCN amplification and overexpression are common in neuroendocrine prostate cancer (NEPC). However, the impact of aberrant N-Myc expression in prostate tumorigenesis and the cellular origin of NEPC have not been established. We define N-Myc and activated AKT1 as oncogenic components sufficient to transform human prostate epithelial cells to prostate adenocarcinoma and NEPC with phenotypic and molecular features of aggressive, late-stage human disease. We directly show that prostate adenocarcinoma and NEPC can arise from a common epithelial clone. Further, N-Myc is required for tumor maintenance and destabilization of N-Myc through Aurora A kinase inhibition reduces tumor burden. Our findings establish N-Myc as a driver of NEPC and a target for therapeutic intervention.

The use of potent therapies inhibiting critical oncogenic pathways active in epithelial cancers has led to multiple resistance mechanisms including the development of highly aggressive, small cell neuroendocrine carcinoma (SCNC). SCNC patients have a dismal prognosis due in part to a limited understanding of the molecular mechanisms driving this malignancy and the lack of effective treatments. Here we demonstrate that a common set of defined oncogenic drivers reproducibly reprograms normal human prostate and lung epithelial cells to small cell prostate cancer (SCPC) and small cell lung cancer (SCLC), respectively. We identify shared active transcription factor binding regions in the reprogrammed prostate and lung SCNCs by integrative analyses of epigenetic and transcriptional landscapes. These results suggest that neuroendocrine cancers arising from distinct epithelial tissues may share common vulnerabilities that could be exploited for the development of drugs targeting SCNCs.

Hydrogen sulfide (H2S) has emerged as a new and important member in the group of gaseous signaling molecules. However, the molecular transport mechanism has not yet been identified. Because of structural similarities with H 2O, it was hypothesized that aquaporins may facilitate H 2S transport across cell membranes. We tested this hypothesis by reconstituting the archeal aquaporin AfAQP from sulfide reducing bacteria Archaeoglobus fulgidus into planar membranes and by monitoring the resulting facilitation of osmotic water flow and H2S flux. To measure H2O and H2S fluxes, respectively, sodium ion dilution and buffer acidification by proton release (H 2S % H ؉ ؉ HS ؊ ) were recorded in the immediate membrane vicinity. Both sodium ion concentration and pH were measured by scanning ion-selective microelectrodes. A lower limit of lipid bilayer permeability to H 2S, P M,H 2 S > 0.5 ؎ 0.4 cm/s was calculated by numerically solving the complete system of differential reaction diffusion equations and fitting the theoretical pH distribution to experimental pH profiles. Even though reconstitution of AfAQP significantly increased water permeability through planar lipid bilayers, PM,H 2 S remained unchanged. These results indicate that lipid membranes may well act as a barrier to water transport although they do not oppose a significant resistance to H 2S diffusion. The fact that cholesterol and sphingomyelin reconstitution did not turn these membranes into an H2S barrier indicates that H 2S transport through epithelial barriers, endothelial barriers, and membrane rafts also occurs by simple diffusion and does not require facilitation by membrane channels. aquaporins ͉ gas transport ͉ membrane permeability ͉ unstirred layer ͉ signaling

SUMMARY We used clinical tissue from lethal metastatic castration resistant prostate cancer (CRPC) patients obtained at rapid autopsy to evaluate diverse genomic, transcriptomic, and phosphoproteomic datasets for pathway analysis. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues to synthesize a robust signaling network consisting of druggable kinase pathways. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Individual autopsy profiles developed using these hallmarks revealed clinically relevant pathway information potentially suitable for patient stratification and targeted therapies in late stage prostate cancer. Here we describe phosphorylation-based cancer hallmarks using integrated personalized signatures (pCHIPS) that sheds light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients.

Abstract. We show that the ot-spectrin gene is essential for larval survival and development by characterizing several ot-spectrin mutations in Drosophila.P-element minigene rescue and sequence analysis were used to identify the o~-spectrin gene as the l(3)dre3 complementatign group of the Dras-Roughenedecdysoneless region of chromosome 3 (Sliter et al., 1988). Germ line transformants carrying an c~-speetrin eDNA, whose expression is driven by the ubiquitin promoter, fully rescued the first to second instar lethality characteristic of the l(3)dre3 alleles. The molecular defects in two -y-ray-induced alleles were identified. One of these mutations, which resulted in second instar lethality, contained a 37-bp deletion in ot-spectrin segment 22 (starting at amino acid residue 2312), producing a premature stop codon between the two EF hands found in this segment. The second mntation, which resulted in first instar lethality, contained a 20 base pair deletion in the middle of segment 1 (at amino acid residue 92), resulting in a premature stop codon. Examination of the spectrin-deficient larvae revealed a loss of contact between epithelial cells of the gut and disruption of cell-substratum interactions. The most pronounced morphological change was seen in tissues of complex cellular architecture such as the middle midgut where a loss of cell contact between cup-shaped cuprophilic cells and neighboring interstitial cells was accompanied by disorganization of the cuprophilic cell brush borders. Our examination of spectrin deficient larvae suggests that an important role of non-erythroid spectrin is to stabilize cell to cell interactions that are critical for the maintenance of cell shape and subcellular organization within tissues.p HENOTYPIC analyses of spectrin deficiencies in mice and humans have provided strong evidence that the spectrin-based membrane skeleton of the erythrocyte contributes to the long range order and stability of the plasma membrane by enhancing mechanical integrity and deformability (Davies and Lux, 1989;Palek and Lambert, 1990). Despite a number of similarities in the structural properties of erythroid and non-erythroid spectrins, significant differences between the two suggest that non-erythroid spectrins may perform functions that are not predicted by the red blood cell model. In non-erythroid cells spectrin can and does interact with a far greater variety of proteins than exists in the erythrocyte (for a review see

To explore the structural basis of the unique selectivity spectrum and conductance of the transmembrane channel protein AqpM from the archaeon Methanothermobacter marburgensis, we determined the structure of AqpM to 1.68-Å resolution by x-ray crystallography. The structure establishes AqpM as being in a unique subdivision between the two major subdivisions of aquaporins, the water-selective aquaporins, and the water-plus-glycerol-conducting aquaglyceroporins. In AqpM, isoleucine replaces a key histidine residue found in the lumen of water channels, which becomes a glycine residue in aquaglyceroporins. As a result of this and other side-chain substituents in the walls of the channel, the channel is intermediate in size and exhibits differentially tuned electrostatics when compared with the other subfamilies.integral membrane protein ͉ x-ray crystallography ͉ water channel ͉ gas A quaporins are a large family of transmembrane channel proteins that facilitate the passive, but highly selective, movement of water, small neutral alditols, including glycerol, or CO 2 across cell membranes (1). These channels are found throughout all domains of life and are fundamental elements in the osmoregulation of many organisms. The selectivity and biophysical characteristics of several aquaporins have been described through atomic resolution structures. The atomic resolution structures of aquaporins GlpF (2), Aqp1 (3, 4), AqpZ (5), Aqp0 (6, 7), and now most recently, AqpM, show high similarity in their tertiary structures. However, despite the similarity in topology and quaternary structure across the family, in vivo and in vitro water and glycerol conductance assays (8-10) have shown that each member exhibits unique conductance rates and displays selectivity spectra that extend to the selective permeation of small neutral molecules that include glycerol, urea, and CO 2 .The structural congruity that spans this family points to the side-chain variations in the channels as the main basis for the divergence in the selectivity and conductance rates between different aquaporins. For example, the crystal structures revealed the integral role of the extended loop that leads into the central plane of the membrane bilayer from each side of the membrane to the conserved -NPA-motifs that initiate the half-membrane spanning helices. These features are a signature for the entire family. Each of these loops provides a ladder of four carbonyl oxygens from successive amino acids in the sequence that are presented into the lumen of the channel. Forming a helical set of hydrogen-bond acceptors from one side to the other along the axis of the channel, they generate a ladder of hydrogen-bond-accepting groups spaced Ϸ2.8 Å apart in distance through the channel. They determine a key element of the chemical environment for the coordination and conductance of the permeants through the lumen of the channel. Also, the side chains that form the selectivity filter influence the electrostatics and diameter of the channel at its narrowest juncture (2) and se...

Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gramnegative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn 2+ alone. The crystal structure of ZneB to 2.8 Å resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the β-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release.Cupriavidus metallidurans CH34 | heavy-metal resistance | resistance nodulation cell division | periplasmic adaptor protein M icroorganisms depend on protective mechanisms to survive the effects of toxic compounds in the environment. In Gramnegative bacteria, resistance nodulation cell division (RND) -driven efflux systems confer resistance to many drugs and heavy metals (1, 2). The canonical RND-based system is formed by the association of three components: one integral to the plasma membrane, one integral to the outer membrane, and a periplasmic connector. The plasma membrane protein (RND) is a substrate/ proton antiporter. The outer-membrane factor (OMF) spans a large part of the periplasm and provides the exit portal. The periplasmic membrane fusion protein (MFP) links these two components together. Based on the nature of their substrate, tripartite RND-driven efflux systems are divided into two subclasses: the hydrophobe/amphiphile efflux (HAE) and the heavy-metal efflux (HME) subclasses. The selectivity and function of many HAE-RND systems such as the Acr and Mex families have been determined, alongside crystal structures of the RND, OMF, and MFP components of various systems (3-13). However, knowledge of HME-RND systems, including substrate specificity and the basis for selectivity, is much more limited.The integral plasma membrane components of the RND-based efflux systems are thought of as the pump; however, the discovery of increasing functions of MFPs shows that these proteins also play a major role in substrate transport. Several MFPs bind their respective substrates (14-16), facilitate substrate transport (17, 18), and are essential for transport in vitro (17). MFPs are also involved in OMF recruitment (19), and are also found in Grampositive bacteria, where no OMF is pr...