Wnt Pathway Activation Increases Hypoxia Tolerance during Development

Adaptation to hypoxia, defined as a condition of inadequate oxygen supply, has enabled humans to successfully colonize high altitude regions. The mechanisms attempted by organisms to cope with short-term hypoxia include increased ATP production via anaerobic respiration and stabilization of Hypoxia Inducible Factor 1α (HIF-1α). However, less is known about the means through which populations adapt to chronic hypoxia during the process of development within a life time or over generations. Here we show that signaling via the highly conserved Wnt pathway impacts the ability of Drosophila melanogaster to complete its life cycle under hypoxia. We identify this pathway through analyses of genome sequencing and gene expression of a Drosophila melanogaster population adapted over >180 generations to tolerate a concentration of 3.5–4% O₂ in air. We then show that genetic activation of the Wnt canonical pathway leads to increased rates of adult eclosion in low O₂. Our results indicate that a previously unsuspected major developmental pathway, Wnt, plays a significant role in hypoxia tolerance.     

The feruloyl esterase gene family of Fusarium graminearum is differentially regulated by aromatic compounds and hosts

Feruloyl esterases can liberate ferulic acid (FA) from plant cell wall polymers. They are expressed by plant pathogenic fungi and could play a role in pathogenicity, although this question has not been addressed yet. The fungus Fusarium graminearum is the principal causal agent of fusarium head blight (FHB) and gibberella ear rot (GER), major diseases of wheat, barley, and maize in all temperate regions of the world. The F. graminearum genome contains seven genes with strong homology to feruloyl esterase (FAE) sequences. Phylogenetic analysis showed that these included three type B, three type C, and one type D FAE genes. Expression profiling of the seven FAE genes showed complex regulation patterns unique to each gene. In F. graminearum-infected plant tissues, the FAE genes exhibited host-specific gene expression. On wheat, FAEB1 and FAED1 were strongly expressed while FAEB2, FAEB3, and FAEC1 were expressed at more modest levels. On maize, only FAEB3, FAEC1, and FAED1 were expressed and at low levels. When growing F. graminearum in liquid culture, only FAEB1 and FAEC1 were expressed. Both genes were induced by a small group of related aromatic compounds including FA, caffeic acid, and p-coumaric acid. FAEB1 was induced by xylose, while repressed by glucose and galactose. FAEC1 was constitutively expressed at low levels in the presence of those sugars. Expression of the other five FAE genes was not detected in the culture conditions used. To determine if FAE genes were important for pathogenicity of F. graminearum, mutant strains inactivated for faeB1?, faeD1? or both genes were constructed and tested on wheat plants. No statistically significant change in pathogenicity and no compensatory expression of the other FAE genes were observed in the fae gene mutants. Our results show that FAEB1 and FAED1 are not required for pathogenicity of F. graminearum on wheat.     

Crystal structure of a putative CN hydrolase from yeast

The crystal structure of a yeast hypothetical protein with sequence similarity to CN hydrolases has been determined to 2.4 A resolution by the multiwavelength anomalous dispersion (MAD) method. The protein folds as a four-layer alphabetabetaalpha sandwich and exists as a dimer in the crystal and in solution. It was selected in a structural genomics project as representative of CN hydrolases at a time when no structures had been determined for members of this family. Structures for two other members of the family have since been reported and the three proteins have similar topology and dimerization modes, which are distinct from those of other alphabetabetaalpha proteins whose structures are known. The dimers form an unusual eight-layer alphabetabetaalpha:alphabetabetaalpha structure. Although the precise enzymatic reactions catalyzed by the yeast protein are not known, considerable information about the active site may be deduced from conserved sequence motifs, comparative biochemical information, and comparison with known structures of hydrolase active sites. As with serine hydrolases, the active-site nucleophile (cysteine in this case) is positioned on a nucleophile elbow.     

Dose-dependent proteomic analysis of glioblastoma cancer stem cells upon treatment with γ-secretase inhibitor

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation, a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation, as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data, possibly due to their crosstalk with Notch Signaling. Overall, this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs.     

The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.     

Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.     

A novel mechanism for Clostridium botulinum neurotoxin inhibition

Clostridium botulinum neurotoxins are zinc endopeptidase proteins responsible for cleaving specific peptide bonds of proteins of neuroexocytosis apparatus. The ability of drugs to interfere with toxin's catalytic activity is being evaluated with zinc chelators and metalloprotease inhibitors. It is important to develop effective pharmacological treatment for the intact holotoxin before the catalytic domain separates and enters the cytosol. We present here evidence for a novel mechanism of an inhibitor binding to the holotoxin and for the chelation of zinc from our structural studies on Clostridium botulinum neurotoxin type B in complex with a potential metalloprotease inhibitor, bis(5-amidino-2-benzimidazolyl)methane, and provide snapshots of the reaction as it progresses. The binding and inhibition mechanism of this inhibitor to the neurotoxin seems to be unique for intact botulinum neurotoxins. The environment of the active site rearranges in the presence of the inhibitor, and the zinc ion is gradually removed from the active site and transported to a different site in the protein, probably causing loss of catalytic activity.     

Role of cytosol in the stimulation of RNA transport in vitro during cardiac hypertrophy in rats.

The 100,000 g supernatant isolated from hypertrophic hearts on fractionation by (NH4)2SO4 and DEAE-cellulose chromatography showed an enhanced RNA-transport activity when incubated with isolated nuclei from sham-operated hearts in vitro. Proteins of Mr 73,000, 68,000, 43,000 and 32,000 are enriched in the DEAE-cellulose fractions exhibiting maximal transport activity, and they are phosphorylatable. Pretreatment of the cytosol with antibodies to the Mr-68,000 and -32,000 proteins decreases the transport activity of the cytosol from 14% to 4.25%. Proteins of Mr 73,000, 68,000, 43,000 and 32,000 are translocated from the cytosol to the nuclear envelope under conditions of RNA transport in vitro. Our results here suggest that at least two of these proteins, those of Mr 68,000 and 32,000, play an indispensible role in the nucleocytoplasmic RNA transport in vitro. By making use of a specific myosin heavy-chain B-gene probe and hybridization, we have also shown the effect of cytosol on the transport of myosin heavy-chain mRNA from nucleus to cytosol.