BRCTx is a novel, highly conserved RAD18-interacting protein

The BRCT domain is a highly conserved module found in many proteins that participate in DNA damage checkpoint regulation, DNA repair, and cell cycle control. Here we describe the cloning, characterization, and targeted mutagenesis of Brctx, a novel gene with a BRCT motif. Brctx was found to be expressed ubiquitously in adult tissues and during development, with the highest levels found in testis. Brctx-deficient mice develop normally, show no pathological abnormalities, and are fertile. BRCTx binds to the C terminus of hRAD18 in yeast two-hybrid and immunoprecipitation assays and colocalizes with this protein in the nucleus. Despite this, Brctx-deficient murine embryonic fibroblasts (MEFs) do not show overt sensitivity to DNA-damaging agents. MEFs from Brctx-deficient embryos grow at a similar rate to wild-type MEF CD4/CD8 expressions, and the cell cycle parameters of thymocytes from wild-type and Brctx knockout animals are indistinguishable. Intriguingly, the BRCT domain of BRCTx is responsible for mediating its localization to the nucleus and centrosome in interphase cells. We conclude that, although highly conserved, Brctx is not essential for the above-mentioned processes and may be redundant.     

Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP

Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca²⁺ concentration ([Ca²⁺]_i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca²⁺]_i, the PE-induced rise of [Ca²⁺]_i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca²⁺]_i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca²⁺ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca²⁺ release from intracellular stores. Reduction of intracellular Ca²⁺ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor     

Discovery of an MIT-like atracotoxin family: spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66–68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX–Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX–Hvf17, at concentrations up to 1 μM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca²⁺ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX–Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed β-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.     

Central administration of peptide and small molecule MC4 receptor antagonists induce hyperphagia in mice and attenuate cytokine-induced anorexia

We investigated the effect of melanocortin 4 receptor (MC4) antagonists on food intake in mice. Food intake during the light phase was significantly increased by ICV administration of mixed MC3/MC4 antagonists (AgRP and SHU9119) or MC4 selective antagonist peptide [(Cyclo (1-5)[Suc-D-Nal-Arg-Trp-Lys]NH2] (MBP10) and the small molecule antagonists THP and NBI-30. Both mixed and selective antagonists significantly reversed anorexia induced by ICV administration of the MC4 agonist (c (1-6) HfRWK-NH2) and the cytokine IL-1beta. These findings provide pharmacological evidence that the MC4 receptor mediates the effects of melanocortin agonists and antagonists on food intake in mice, and support the idea that selective small molecule MC4 antagonists may be useful as therapeutics for cachexia.     

Integration of mechanical cell disruption and fluidised bed recovery of G3PDH from unclarified disrupted yeast: a comparative study of the performance of unshielded and polymer shielded dye-ligand chromatography systems

The development of a simplified process for the simultaneous disruption and direct selective purification of intracellular proteins from unclarified yeast disruptate has been investigated. The recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast was selected as a potential demonstration of the generic applicability and practical feasibility of this integrated technique. The application of an adsorbent characterised by high density (UpFront steel-agarose; rho=2.65g ml(-1)) facilitated the combining of cell disruption operation (bead milling of 50% ww/v of yeast suspension at 7.2 lh(-1)) with fluidised bed dye-ligand (Cibacron Blue 3GA) adsorption operated immediately downstream of the disrupter. The adoption of a polymer shielded, dye-ligand technique advanced recovery efficiency. It was demonstrated that G3PDH could be recovered with a yield of 67.5% bound activity and a specific activity of 40.2IU mg(-1), after a single step elution with 0.15M NaCl. The generic application of this approach has been evaluated.     

Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14

The ubiquitin-specific processing protease (UBP) family of deubiquitinating enzymes plays an essential role in numerous cellular processes. Mammalian USP14 (Ubp6 in yeast) is unique among known UBP enzymes in that it is activated catalytically upon specific association with the 26S proteasome. Here, we report the crystal structures of the 45-kDa catalytic domain of USP14 in isolation and in a complex with ubiquitin aldehyde, which reveal distinct structural features. In the absence of ubiquitin binding, the catalytic cleft leading to the active site of USP14 is blocked by two surface loops. Binding by ubiquitin induces a significant conformational change that translocates the two surface loops thereby allowing access of the ubiquitin C-terminus to the active site. These structural observations, in conjunction with biochemical characterization, identify important regulatory mechanisms for USP14.     

VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis

Vascular endothelial growth factor receptor-2 (VEGFR-2) activation by VEGF-A is essential in vasculogenesis and angiogenesis. We have generated a pan-phosphorylation site map of VEGFR-2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C-terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR-2-expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T-cell-specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF-A-induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd-deficient mice, indicating a critical role of Y951-TSAd signaling in pathological angiogenesis.     

Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarrays

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation.