Defining Components of the ?catenin Destruction Complex and Exploring Its Regulation and Mechanisms of Action during Development

Background

A subset of signaling pathways play exceptionally important roles in embryonic and post-embryonic development, and mis-regulation of these pathways occurs in most human cancers. One such pathway is the Wnt pathway. The primary mechanism keeping Wnt signaling off in the absence of ligand is regulated proteasomal destruction of the canonical Wnt effector ßcatenin (or its fly homolog Armadillo). A substantial body of evidence indicates that SCF^βTrCP mediates βcat destruction, however, an essential role for Roc1 has not been demonstrated in this process, as would be predicted. In addition, other E3 ligases have also been proposed to destroy βcat, suggesting that βcat destruction may be regulated differently in different tissues.

Methodology/Principal Findings

Here we used cultured Drosophila cells, human colon cancer cells, and Drosophila embryos and larvae to explore the machinery that targets Armadillo for destruction. Using RNAi in Drosophila S2 cells to examine which SCF components are essential for Armadillo destruction, we find that Roc1/Roc1a is essential for regulating Armadillo stability, and that in these cells the only F-box protein playing a detectable role is Slimb. Second, we find that while embryonic and larval Drosophila tissues use the same destruction complex proteins, the response of these tissues to destruction complex inactivation differs, with Armadillo levels more elevated in embryos. We provide evidence consistent with the possibility that this is due to differences in armadillo mRNA levels. Third, we find that there is no correlation between the ability of different APC2 mutant proteins to negatively regulate Armadillo levels, and their recently described function in positively-regulating Wnt signaling. Finally, we demonstrate that APC proteins lacking the N-terminal Armadillo-repeat domain cannot restore Armadillo destruction but retain residual function in negatively-regulating Wnt signaling.

Conclusions/Significance

We use these data to refine our model for how Wnt signaling is regulated during normal development.     

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments.     

Matrix metalloproteinase-9 regulates survival of neurons in newborn hippocampus

The number of neurons in the adult rodent brain is strongly influenced by events in early postnatal life that eliminate approximately half of the neurons. Recently, we reported that neurotrophins induced survival of neonatal rat hippocampal neurons by promoting neural activity and activation of the Ser/Thr kinase, Akt. The survival of neurons also depended on integrin signaling, but a role for the extracellular matrix (ECM) in this mechanism was yet to be explored. Here, we show that levels of the matrix metalloproteinase-9 (MMP9) decrease, and the level of the ECM protein laminin increases in rat hippocampus during the period of neuronal death. Hippocampi from MMP9 null mice showed higher levels of laminin expression than wild type at P1 and no further increase at P10. In vitro, the matrix metalloproteinase inhibitor FN-439 promoted survival of neurons in a laminin-integrin β1-dependent manner. Blocking laminin signaling attenuated activation of Akt by depolarization. In vivo, injecting FN-439 into the neonatal hippocampus increased the level of laminin and promoted neuronal survival through an integrin-dependent mechanism. These results show signals from the ECM are not simply permissive but rather actively regulated, and they interact with neuronal activity to control the number of hippocampal neurons. This work is the first to report a role for MMP9 in regulating neuronal survival through the developmental process that establishes the functional brain.     

A highly conserved cytoplasmic cysteine residue in the α4 nicotinic acetylcholine receptor is palmitoylated and regulates protein expression

Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4β2 nAChR.     

Indomethacin-induced translocation of bacteria across enteric epithelia is reactive oxygen species-dependent and reduced by vitamin C

The enteric epithelium must absorb nutrients and water and act as a barrier to the entry of luminal material into the body; this barrier function is a key component of innate immunity. Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy occurs via inhibition of prostaglandin synthesis and perturbed epithelial mitochondrial activity. Here, the direct effect of NSAIDs [indomethacin, piroxicam (cyclooxygenase 1 and 2 inhibitors), and SC-560 (a cyclooxygenase 1 inhibitor)] on the barrier function of human T84 epithelial cell line monolayers was assessed by transepithelial electrical resistance (TER) and internalization and translocation of a commensal Escherichia coli. Exposure to E. coli in the presence and absence of drugs for 16 h reduced TER; however, monolayers cotreated with E. coli and indomethacin, but not piroxicam or SC-560, displayed significant increases in internalization and translocation of the bacteria. This was accompanied by increased reactive oxygen species (ROS) production, which was also increased in epithelia treated with E. coli only. Colocalization revealed upregulation of superoxide synthesis by mitochondria in epithelia treated with E. coli + indomethacin. Addition of antioxidants (vitamin C or a green tea polyphenol, epigallocathechin gallate) quenched the ROS and prevented the increase in E. coli internalization and translocation evoked by indomethacin, but not the drop in TER. Evidence of increased apoptosis was not observed in this model. The data implicate epithelial-derived ROS in indomethacin-induced barrier dysfunction and show that a portion of the bacteria likely cross the epithelium via a transcellular pathway. We speculate that addition of antioxidants as dietary supplements to NSAID treatment regimens would reduce the magnitude of decreased barrier function, specifically the transepithelial passage of bacteria.     

Genome‐wide association analysis for quantitative trait loci influencing Warner–Bratzler shear force in five taurine cattle breeds

We performed a genome‐wide association study for Warner–Bratzler shear force (WBSF), a measure of meat tenderness, by genotyping 3360 animals from five breeds with 54 790 BovineSNP50 and 96 putative single‐nucleotide polymorphisms (SNPs) within μ‐calpain [HUGO nomenclature calpain 1, (mu/I) large subunit; CAPN1] and calpastatin (CAST). Within‐ and across‐breed analyses estimated SNP allele substitution effects (ASEs) by genomic best linear unbiased prediction (GBLUP) and variance components by restricted maximum likelihood under an animal model incorporating a genomic relationship matrix. GBLUP estimates of ASEs from the across‐breed analysis were moderately correlated (0.31–0.66) with those from the individual within‐breed analyses, indicating that prediction equations for molecular estimates of breeding value developed from across‐breed analyses should be effective for genomic selection within breeds. We identified 79 genomic regions associated with WBSF in at least three breeds, but only eight were detected in all five breeds, suggesting that the within‐breed analyses were underpowered, that different quantitative trait loci (QTL) underlie variation between breeds or that the BovineSNP50 SNP density is insufficient to detect common QTL among breeds. In the across‐breed analysis, CAPN1 was followed by CAST as the most strongly associated WBSF QTL genome‐wide, and associations with both were detected in all five breeds. We show that none of the four commercialized CAST and CAPN1 SNP diagnostics are causal for associations with WBSF, and we putatively fine‐map the CAPN1 causal mutation to a 4581‐bp region. We estimate that variation in CAST and CAPN1 explains 1.02 and 1.85% of the phenotypic variation in WBSF respectively.     

Elevated copy number of L-A virus in yeast mutant strains defective in ribosomal stalk

The eukaryotic ribosomal stalk, composed of the P-proteins, is a part of the GTPase-associated-center which is directly responsible for stimulation of translation-factor-dependent GTP hydrolysis. Here we report that yeast mutant strains lacking P1/P2-proteins show high propagation of the yeast L-A virus. Affinity-capture-MS analysis of a protein complex isolated from a yeast mutant strain lacking the P1A/P2B proteins using anti-P0 antibodies showed that the Gag protein, the major coat protein of the L-A capsid, is associated with the ribosomal stalk. Proteomic analysis revealed that the elongation factor eEF1A was also present in the isolated complex. Additionally, yeast strains lacking the P1/P2-proteins are hypersensitive to paromomycin and hygromycin B, underscoring the fact that structural perturbations in the stalk strongly influence the ribosome function, especially at the level of elongation.     

Worldwide Patterns of Ancestry,Divergence, and Admixture in Domesticated Cattle

The domestication and development of cattle has considerably impacted human societies, but the histories of cattle breeds and populations have been poorly understood especially for African, Asian, and American breeds. Using genotypes from 43,043 autosomal single nucleotide polymorphism markers scored in 1,543 animals, we evaluate the population structure of 134 domesticated bovid breeds. Regardless of the analytical method or sample subset, the three major groups of Asian indicine, Eurasian taurine, and African taurine were consistently observed. Patterns of geographic dispersal resulting from co-migration with humans and exportation are recognizable in phylogenetic networks. All analytical methods reveal patterns of hybridization which occurred after divergence. Using 19 breeds, we map the cline of indicine introgression into Africa. We infer that African taurine possess a large portion of wild African auroch ancestry, causing their divergence from Eurasian taurine. We detect exportation patterns in Asia and identify a cline of Eurasian taurine/indicine hybridization in Asia. We also identify the influence of species other than Bos taurus taurus and B. t. indicus in the formation of Asian breeds. We detect the pronounced influence of Shorthorn cattle in the formation of European breeds. Iberian and Italian cattle possess introgression from African taurine. American Criollo cattle originate from Iberia, and not directly from Africa with African ancestry inherited via Iberian ancestors. Indicine introgression into American cattle occurred in the Americas, and not Europe. We argue that cattle migration, movement and trading followed by admixture have been important forces in shaping modern bovine genomic variation.