Lys48-linked TAK1 polyubiquitination at lysine-72 downregulates TNFα-induced NF-κB activation via mediating TAK1 degradation

Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular processes. TAK1, a member of the MAPKKK family, is essential for TNFα-induced NF-κB activation. Phosphorylation and Lys(63)-linked polyubiquitination (polyUb) of TAK1 are critical for its activation. However, whether TAK1 is regulated by polyubiquitination-mediated protein degradation after its activation remains unknown. Here we report that TNFα induces TAK1 Lys(48) linked polyubiquitination and degradation at the later time course. Furthermore, we provide direct evidence that TAK1 is modified by Lys(48)-linked polyubiquitination at lysine-72 by mass spectrometry. A K72R point mutation on TAK1 abolishes TAK1 Lys(48)-linked polyubiquitination and enhances TAK1/TAB1 co-overexpression-induced NF-κB activation. As expected, TAK1 K72R mutation inhibits TNFα-induced Lys(48)-linked TAK1 polyubiquitination and degradation. TAK1 K72R mutant prolongs TNFα-induced NF-κB activation and enhances TNFα-induced IL-6 gene expression. Our findings demonstrate that TNFα induces Lys(48)-linked polyubiquitination of TAK1 at lysine-72 and this polyubiquitination-mediated TAK1 degradation plays a critical role in the downregulation of TNFα-induced NF-κB activation.     

Prediction of trabecular bone principal structural orientation using quantitative ultrasound scanning

Bone has the ability to adapt its structure in response to the mechanical environment as defined as Wolff's Law. The alignment of trabecular structure is intended to adapt to the particular mechanical milieu applied to it. Due to the absence of normal mechanical loading, it will be extremely important to assess the anisotropic deterioration of bone during the extreme conditions, i.e., long term space mission and disease orientated disuse, to predict risk of fractures. The propagation of ultrasound wave in trabecular bone is substantially influenced by the anisotropy of the trabecular structure. Previous studies have shown that both ultrasound velocity and amplitude is dependent on the incident angle of the ultrasound signal into the bone sample. In this work, seven bovine trabecular bone balls were used for rotational ultrasound measurement around three anatomical axes to elucidate the ability of ultrasound to identify trabecular orientation. Both ultrasound attenuation (ATT) and fast wave velocity (UV) were used to calculate the principal orientation of the trabecular bone. By comparing to the mean intercept length (MIL) tensor obtained from μCT, the angle difference of the prediction by UV was 4.45°, while it resulted in 11.67° angle difference between direction predicted by μCT and the prediction by ATT. This result demonstrates the ability of ultrasound as a non-invasive measurement tool for the principal structural orientation of the trabecular bone.     

Small molecule structure correctors abolish detrimental effects of apolipoprotein E4 in cultured neurons

Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC(50) of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.     

The effects of domestication on the scaling of below- vs. aboveground biomass in four selected wheat (Triticum; Poaceae) genotypes

? Premise of the study: Theory and empirical studies have shown that, on average, belowground biomass (M(B)) scales one-to-one (isometrically) with aboveground biomass (M(A)) within and across plant species both at the individual and population level, i.e., M(B) ∝ M(A)(α=1), where α is the scaling exponent. However, little is known about how domestication affects this relationship. ? Methods: To examine the effects of domestication, we investigated the root vs. shoot biomass relationship during the first 30 d of growth of four wheat genotypes: two older genotypes, MO4 (T. monococcum, a diploid) and DM31 (T. dicoccum, a tetraploid) and two more recent genotypes, DX24 and L8275 (T. aestivum, both hexaploids). ? Results: Biomass allocation to roots scaled more or less isometrically with respect to shoot biomass allocation during the first 30 d of growth for both of the older genotypes, whereas shoot biomass allocation exceeded root allocation for the two more recent genotypes. This difference was attributable to the first 15 d of growth. Although root biomass allocation exceeded shoot biomass allocation during the first 15 d of growth for the two older genotypes, shoot biomass exceeded root biomass allocation during this critical phase of development for the two more recent genotypes. ? Conclusions: Based on a very limited sample of wheat genotypes, these results indicate that domestication has resulted in an increased biomass allocation to shoots compared to root biomass allocation. This shift possibly reflects artificial selection under agricultural conditions (for which water and nutrients are not limiting) favoring higher crop yields.     

Extraction of lignins from aqueous-ionic liquid mixtures by organic solvents

The commercial development of ionic liquids (ILs) to pretreat lignocellulose by dissolution of whole biomass and cellulose precipitation by addition of water is hindered by the absence of an effective technique to recover the lignin content of the biomass from the IL. Three organic solvents [ethyl acetate, 1,4-dioxane, and tetrahydrofuran (THF)] were studied for their ability to form a two-liquid-phase system with water and 1-ethyl-3-methylimidazolium acetate ([C(2)mim][OAc]), and for partitioning model lignins and lignin monomers between the two liquid phases. Ternary diagrams were obtained for three [C(2)mim][OAc]/organic solvent/water systems at 22°C. Partition coefficients were measured for several types of lignin in these three systems. Partition coefficients increase with rising water content in the IL phase, and depend strongly on the type of lignin and on the organic solvent. Partition coefficients rise as the pH of the ionic-liquid-rich phase falls. Small molecule model lignin monomer compounds (guaiacol, syringaldehyde) are also readily extracted from the IL/water system by THF.     

Inhibitory mechanism of caspase-6 phosphorylation revealed by crystal structures, molecular dynamics simulations, and biochemical assays

The apoptotic effector caspase-6 (CASP6) has been clearly identified as a drug target due to its strong association with neurodegeneration and axonal pruning events as well as its crucial roles in Huntington disease and Alzheimer disease. CASP6 activity is suppressed by ARK5-mediated phosphorylation at Ser(257) with an unclear mechanism. In this work, we solved crystal structures of ΔproCASP6S257E and p20/p10S257E, which mimicked the phosphorylated CASP6 zymogen and activated CASP6, respectively. The structural investigation combined with extensive biochemical assay and molecular dynamics simulation studies revealed that phosphorylation on Ser(257) inhibited self-activation of CASP6 zymogen by "locking" the enzyme in the TEVD(193)-bound "inhibited state." The structural and biochemical results also showed that phosphorylation on Ser(257) inhibited the CASP6 activity by steric hindrance. These results disclosed the inhibition mechanism of CASP6 phosphorylation and laid the foundation for a new strategy of rational CASP6 drug design.     

Characteristic morphology of intracellular microcolonies of Legionella oakridgensis OR-10

Legionella oakridgensis occasionally causes pneumonia in humans. We report here the characteristic morphology of intracellular microcolonies of L. oakridgensis OR-10 in infected epithelial cells. By light microscopy after Gimenez staining, the bacteria showed serpentine-like chain, disk-like conglomerate, and granular forms when they grew intracellularly in Vero cells, HeLa cells, and A549 cells. In a time-lapse study, we observed the progressive change from a serpentine-like chain form to a conglomerate form in Vero cells. Transmission electron microscopy showed that L. oakridgensis OR-10 proliferated both inside membrane structures and in the cytoplasm. Such highly serpentine chain growth has not been reported in any intracellular bacteria. Furthermore, these results imply that L. oakridgensis OR-10 may be proliferating inside the endoplasmic reticulum.     

Increased IL-17-producing CD4(+) T cells in patients with esophageal cancer

Increased interleukin-17 (IL-17)-producing Th (Th17) cells have been described in a variety of human carcinoma cases, however, the mechanism of Th17 cells' accumulation in a tumor microenvironment remains elusive. This study was designed to investigate whether Th17 cells were involved in the development of esophageal cancer. We found that the proportion of Th17 cells increased within the peripheral blood and tumor tissues of esophageal cancer patients. Furthermore, the proportion of circulating Th17 cells was higher in advanced esophageal cancer patients than that in early esophageal cancer patients. In addition, the Th17 cells differentiation-related cytokines (IL-23, IL-1β, and IL-6) and accumulation-related chemokines (CCL22 and CCL20) were present in a tumor microenvironment. Therefore, the findings may partly explain the cause for the increased proportion of Th17 cells and indicate a potential prognostic marker of Th17 cells in esophageal cancer.