Increased glutamine catabolism mediates bone anabolism in response to WNT signaling

WNT signaling stimulates bone formation by increasing both the number of osteoblasts and their protein-synthesis activity. It is not clear how WNT augments the capacity of osteoblast progenitors to meet the increased energetic and synthetic needs associated with mature osteoblasts. Here, in cultured osteoblast progenitors, we determined that WNT stimulates glutamine catabolism through the tricarboxylic acid (TCA) cycle and consequently lowers intracellular glutamine levels. The WNT-induced reduction of glutamine concentration triggered a general control nonderepressible 2–mediated (GCN2-mediated) integrated stress response (ISR) that stimulated expression of genes responsible for amino acid supply, transfer RNA (tRNA) aminoacylation, and protein folding. WNT-induced glutamine catabolism and ISR were β-catenin independent, but required mammalian target of rapamycin complex 1 (mTORC1) activation. In a hyperactive WNT signaling mouse model of human osteosclerosis, inhibition of glutamine catabolism or Gcn2 deletion suppressed excessive bone formation. Together, our data indicate that glutamine is both an energy source and a protein-translation rheostat that is responsive to WNT and suggest that manipulation of the glutamine/GCN2 signaling axis may provide a valuable approach for normalizing deranged protein anabolism associated with human diseases

The Cul3–KLHL21 E3 ubiquitin ligase targets Aurora B to midzone microtubules in anaphase and is required for cytokinesis

Selective ubiquitination of Aurora B by different Cul3 adaptors targets it at the correct time to the correct place during mitosis

GPR80/99, proposed to be the P2Y15 receptor activated by adenosine and AMP, is not a P2Y receptor

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y15 receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca2+ mobilization (Inbe et al. J Biol Chem 2004; 279: 19790–9[12]). However, the cell line (HEK293) used to carry out those studies endogenously expresses A2A and A2B adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188–93 [15]) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, α-ketoglutarate. Consistent with this report, α-ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts α-ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by α-ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family

Transcriptome, Methylome and Genomic Variations Analysis of Ectopic Thyroid Glands

Congenital hypothyroidism from thyroid dysgenesis (CHTD) is predominantly a sporadic disease characterized by defects in the differentiation, migration or growth of thyroid tissue. Of these defects, incomplete migration resulting in ectopic thyroid tissue is the most common (up to 80%). Germinal mutations in the thyroid-related transcription factors NKX2.1, FOXE1, PAX-8, and NKX2.5 have been identified in only 3% of patients with sporadic CHTD. Moreover, a survey of monozygotic twins yielded a discordance rate of 92%, suggesting that somatic events, genetic or epigenetic, probably play an important role in the etiology of CHTD.Journal ArticleResearch Support, Non-U.S. Gov'tValidation StudiesSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Distinct Molecular Evolutionary Mechanisms Underlie the Functional Diversification of the Wnt and TGFβ Signaling Pathways

The canonical Wnt pathway is one of the oldest and most functionally diverse of animal intercellular signaling pathways. Though much is known about loss-of-function phenotypes for Wnt pathway components in several model organisms, the question of how this pathway achieved its current repertoire of functions has not been addressed. Our phylogenetic analyses of 11 multigene families from five species belonging to distinct phyla, as well as additional analyses employing the 12 Drosophila genomes, suggest frequent gene duplications affecting ligands and receptors as well as co-evolution of new ligand–receptor pairs likely facilitated the expansion of this pathway’s capabilities. Further, several examples of recent gene loss are visible in Drosophila when compared to family members in other phyla. By comparison the TGFβ signaling pathway is characterized by ancient gene duplications of ligands, receptors, and signal transducers with recent duplication events restricted to the vertebrate lineage. Overall, the data suggest that two distinct molecular evolutionary mechanisms can create a functionally diverse developmental signaling pathway. These are the recent dynamic generation of new genes and ligand–receptor interactions as seen in the Wnt pathway and the conservative adaptation of ancient pre-existing genes to new roles as seen in the TGFβ pathway. From a practical perspective, the former mechanism limits the investigator’s ability to transfer knowledge of specific pathway functions across species while the latter facilitates knowledge transfer

Specific oligomerization of the 5-HT1A receptor in the plasma membrane

In the present study we analyze the oligomerization of the 5-HT1A receptor within living cells at the sub-cellular level. Using a 2-excitation Förster Resonance Energy Transfer (FRET) method combined with spectral microscopy we are able to estimate the efficiency of energy transfer based on donor quenching as well as acceptor sensitization between CFP-and YFP-tagged 5-HT1A receptors at the plasma membrane. Through the analysis of the level of apparent FRET efficiency over the various relative amounts of donor and acceptor, as well as over a range of total surface expressions of the receptor, we verify the specific interaction of these receptors. Furthermore we study the role of acylation in this interaction through measurements of a palmitoylation-deficient 5-HT1A receptor mutant. Palmitoylation increases the tendency of a receptor to localize in lipid rich microdomains of the plasma membrane. This increases the effective surface density of the receptor and provides for a higher level of stochastic interaction

A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption

Intraepithelial and Interstitial Deposition of Pathological Prion Protein in Kidneys of Scrapie-Affected Sheep

Prions have been documented in extra-neuronal and extra-lymphatic tissues of humans and various ruminants affected by Transmissible Spongiform Encephalopathy (TSE). The presence of prion infectivity detected in cervid and ovine blood tempted us to reason that kidney, the organ filtrating blood derived proteins, may accumulate disease associated PrPSc. We collected and screened kidneys of experimentally, naturally scrapie-affected and control sheep for renal deposition of PrPSc from distinct, geographically separated flocks. By performing Western blot, PET blot analysis and immunohistochemistry we found intraepithelial (cortex, medulla and papilla) and occasional interstitial (papilla) deposition of PrPSc in kidneys of scrapie-affected sheep. Interestingly, glomerula lacked detectable signals indicative of PrPSc. PrPSc was also detected in kidneys of subclinical sheep, but to significantly lower degree. Depending on the stage of the disease the incidence of PrPSc in kidney varied from approximately 27% (subclinical) to 73.6% (clinical) in naturally scrapie-affected sheep. Kidneys from flocks without scrapie outbreak were devoid of PrPSc. Here we demonstrate unexpectedly frequent deposition of high levels of PrPSc in ovine kidneys of various flocks. Renal deposition of PrPSc is likely to be a pre-requisite enabling prionuria, a possible co-factor of horizontal prion-transmission in sheep

Identification of DNA-Damage DNA-Binding Protein 1 as a Conditional Essential Factor for Cytomegalovirus Replication in Interferon-γ-Stimulated Cells

The mouse cytomegaloviral (MCMV) protein pM27 represents an indispensable factor for viral fitness in vivo selectively, antagonizing signal transducer and activator of transcription 2 (STAT2)-mediated interferon signal transduction. We wished to explore by which molecular mechanism pM27 accomplishes this effect. We demonstrate that pM27 is essential and sufficient to curtail the protein half-life of STAT2 molecules. Pharmacologic inhibition of the proteasome restored STAT2 amounts, leading to poly-ubiquitin-conjugated STAT2 forms. PM27 was found in complexes with an essential host ubiquitin ligase complex adaptor protein, DNA-damage DNA-binding protein (DDB) 1. Truncation mutants of pM27 showed a strict correlation between DDB1 interaction and their ability to degrade STAT2. SiRNA-mediated knock-down of DDB1 restored STAT2 in the presence of pM27 and strongly impaired viral replication in interferon conditioned cells, thus phenocopying the growth attenuation of M27-deficient virus. In a constructive process, pM27 recruits DDB1 to exploit ubiquitin ligase complexes catalyzing the obstruction of the STAT2-dependent antiviral state of cells to permit viral replication

Genetic Structure and Demographic History Should Inform Conservation: Chinese Cobras Currently Treated as Homogenous Show Population Divergence

An understanding of population structure and genetic diversity is crucial for wildlife conservation and for determining the integrity of wildlife populations. The vulnerable Chinese cobra (Naja atra) has a distribution from the mouth of the Yangtze River down to northern Vietnam and Laos, within which several large mountain ranges and water bodies may influence population structure. We combined 12 microsatellite loci and 1117 bp of the mitochondrial cytochrome b gene to explore genetic structure and demographic history in this species, using 269 individuals from various localities in Mainland China and Vietnam. High levels of genetic variation were identified for both mtDNA and microsatellites. mtDNA data revealed two main (Vietnam + southern China + southwestern China; eastern + southeastern China) and one minor (comprising only two individuals from the westernmost site) clades. Microsatellite data divided the eastern + southeastern China clade further into two genetic clusters, which include individuals from the eastern and southeastern regions, respectively. The Luoxiao and Nanling Mountains may be important barriers affecting the diversification of lineages. In the haplotype network of cytchrome b, many haplotypes were represented within a “star” cluster and this and other tests suggest recent expansion. However, microsatellite analyses did not yield strong evidence for a recent bottleneck for any population or genetic cluster. The three main clusters identified here should be considered as independent management units for conservation purposes. The release of Chinese cobras into the wild should cease unless their origin can be determined, and this will avoid problems arising from unnatural homogenization