mTOR drives its own activation via SCF(βTrCP)-dependent degradation of the mTOR inhibitor DEPTOR

The activities of both mTORC1 and mTORC2 are negatively regulated by their endogenous inhibitor, DEPTOR. As such, the abundance of DEPTOR is a critical determinant in the activity status of the mTOR network. DEPTOR stability is governed by the 26S-proteasome through a largely unknown mechanism. Here we describe an mTOR-dependent phosphorylation-driven pathway for DEPTOR destruction via SCF(βTrCP). DEPTOR phosphorylation by mTOR in response to growth signals, and in collaboration with casein kinase I (CKI), generates a phosphodegron that binds βTrCP. Failure to degrade DEPTOR through either degron mutation or βTrCP depletion leads to reduced mTOR activity, reduced S6 kinase activity, and activation of autophagy to reduce cell growth. This work expands the current understanding of mTOR regulation by revealing a positive feedback loop involving mTOR and CKI-dependent turnover of its inhibitor, DEPTOR, suggesting that misregulation of the DEPTOR destruction pathway might contribute to aberrant activation of mTOR in disease.     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

Ascidian Wnt-5 gene is involved in the morphogenetic movement of notochord cells

Wnt proteins play important roles in many developmental events. Wnts are divided into two groups according to biological function. The Wnt-5a class proteins function in morphogenetic movement during embryogenesis. Previously, a Wnt-5 homolog has been isolated from the ascidian, Halocynthia roretzi. HrWnt-5 is expressed in the notochord until the tail-bud stage, implying a role in the notochord. In this study, the function of HrWnt-5 was investigated. When HrWnt-5 mRNA was injected into fertilized eggs, the embryos showed morphologic defects at around the neurula stage. The anterior-posterior axis was shorter than in control embryos. These defects were caused by the abnormal movement of notochord cells. However, the overexpression of HrWnt-5 mRNA did not affect the differentiation of tissues, suggesting that HrWnt-5 solely regulates the morphogenetic movement. Although endogenous HrWnt-5 is expressed in the notochord, the overexpression of HrWnt-5 mRNA caused the defects, suggesting that the amount of HrWnt-5 mRNA in the notochord is strictly regulated. These results suggest that HrWnt-5 regulates the morphogenetic movement of notochord cells during ascidian embryogenesis.     

Functional immobilization of plant receptor-like kinase onto microbeads towards receptor array construction and receptor-based ligand fishing

Despite the large number of receptor-like kinases (RLKs) in plants, few of their specific ligands are known. Ligand fishing is one of the most challenging post-genomic technologies. Here, we report a strategy for functional immobilization of plant RLKs on microbeads via covalent linkage. Because of the high density of immobilized RLKs, ligand-receptor interaction can be visualized at high sensitivity using fluorescent-labeled ligands under the confocal laser scanning microscope. Moreover, using a receptor-based affinity chromatography system with RLK microbeads, the ligand of the receptor was directly retrieved at high purity from complex natural samples. Our RLK microbeads and receptor-based ligand fishing approach is a feasible alternative to conventional forward genetics and bioassay-based biochemical purification for identification of novel ligand-receptor pairs in plants.     

Optineurin suppression causes neuronal cell death via NF‐κB pathway

Mutations in more than 10 genes are reported to cause familial amyotrophic lateral sclerosis (ALS). Among these genes, optineurin (OPTN) is virtually the only gene that is considered to cause classical ALS by a loss‐of‐function mutation. Wild‐type optineurin (OPTN^WT) suppresses nuclear factor‐kappa B (NF‐κB) activity, but the ALS‐causing mutant OPTN is unable to suppress NF‐κB activity. Therefore, we knocked down OPTN in neuronal cells and examined the resulting NF‐κB activity and phenotype. First, we confirmed the loss of the endogenous OPTN expression after siRNA treatment and found that NF‐κB activity was increased in OPTN‐knockdown cells. Next, we found that OPTN knockdown caused neuronal cell death. Then, overexpression of OPTN^WT or OPTN^E50K with intact NF‐κB‐suppressive activity, but not overexpression of ALS‐related OPTN mutants, suppressed the neuronal death induced by OPTN knockdown. This neuronal cell death was inhibited by withaferin A, which selectively inhibits NF‐κB activation. Lastly, involvement of the mitochondrial proapoptotic pathway was suggested for neuronal death induced by OPTN knockdown. Taken together, these results indicate that inappropriate NF‐κB activation is the pathogenic mechanism underlying OPTN mutation‐related ALS.

     

Mechanism of asymmetric ovarian development in chick embryos

In most animals, the gonads develop symmetrically, but most birds develop only a left ovary. A possible role for estrogen in this asymmetric ovarian development has been proposed in the chick, but the mechanism underlying this process is largely unknown. Here, we identify the molecular mechanism responsible for this ovarian asymmetry. Asymmetric PITX2 expression in the left presumptive gonad leads to the asymmetric expression of the retinoic-acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptors Ad4BP/SF-1 and estrogen receptor alpha in the right ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These data suggest that early asymmetric expression of PITX2 leads to asymmetric ovarian development through up- or downregulation of RALDH2, Ad4BP/SF-1, estrogen receptor alpha and cyclin D1.     

Synthesis and mitochondrial complex I inhibition of dihydroxy-cohibin A, non-THF annonaceous acetogenin analogue

To elucidate the inhibitory action of acetogenins, we synthesized an acetogenin derivative which possesses tetraol in place of the tetrahydrofuran ring and examined its inhibitory activity against bovine heart mitochondrial complex I. Our results indicate that these hydroxy groups are an essential structural factor though it is not effective as bis-THF hydroxy groups combination.     

Cutting edge: hypoxia-inducible factor 1alpha and its activation-inducible short isoform I.1 negatively regulate functions of CD4+ and CD8+ T lymphocytes

To evaluate the role of hypoxia-inducible factor 1alpha (HIF-1alpha) and its TCR activation-inducible short isoform I.1 in T cell functions, we genetically engineered unique mice with: 1) knockout of I.1 isoform of HIF-1alpha; 2) T cell-targeted HIF-1alpha knockdown; and 3) chimeric mice with HIF-1alpha gene deletion in T and B lymphocytes. In all three types of mice, the HIF-1alpha-deficient T lymphocytes, which were TCR-activated in vitro, produced more proinflammatory cytokines compared with HIF-1alpha-expressing control T cells. Surprisingly, deletion of the I.1 isoform, which represents < 30% of total HIF-1alpha mRNA in activated T cells, was sufficient to markedly enhance TCR-triggered cytokine secretion. These data suggest that HIF-1alpha not only plays a critical role in oxygen homeostasis but also may serve as a negative regulator of T cells.     

Native-State Heterogeneity of β2-Microglobulin as Revealed by Kinetic Folding and Real-Time NMR Experiments

The kinetic folding of β₂-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s^? 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s^? 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C).     

Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.