Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

Role of polyamines in the binding of initiator tRNA to the 70S ribosomes of extreme thermophilic bacterium Calderobacterium hydrogenophilum

Slowly cooled cells of an extreme thermophilic eubacterium Calderobacterium hydrogenophilum possess ribosomes with weakly associated subunits. These ribosomal subunits are capable of association to 70S ribosomes either at higher Mg²⁺ concentrations (30–40 mM) or at 4–10 mM Mg²⁺ and in the presence of polyamines. The contribution of 30S and 50S subunits to the hydrodynamic stability of ribosomes was examined by forming hybrid 30S–50S couples from C. hydrogenophilum and Escherichia coli. At lower Mg²⁺ (4–10 mM) heterogeneous subunits containing 30S E. coli and 50S C. hydrogenophilum and homogeneous subunits of the thermophilic bacterium associated only in the presence of polyamines. Ribosomal subunits associated at 30 mM Mg²⁺ lose thermal stability and activity concerning poly(AUG)-dependent binding of f[³H]Met-tRNA to the P-site on 70S ribosomes or translation of poly(UG). Poly(AUG), deacylated-tRNA or initiator-tRNA have no valuable effect on association of 30S and 50S subunits. Protein synthesis initiation factor IF3 of C. hydrogenophilum prevents association of ribosomal subunits to 70S ribosomes at physiological temperature (70°C). The factor also stimulates dissociation of 70S ribosomes of E. coli at 37°C. The codon-specific binding of f[³H]Met-tRNA to homogeneous 70S ribosomes of C. hydrogenophilum at 70°C is dependent on the presence of initiation factors and concentrations of tri-pentaamines. However, excess of polyamines inhibited the reaction. Our results indicate that tri-pentaamines enhance conformational stability of 70S initiation complex at elevated temperatures.     

New method to stimulate the onset of Bombus terrestris (Hymenoptera: Apidae) rearing: Using worker helpers in the presence of frozen pupae

The failure of bumblebee queens to enthusiastically start a colony under laboratory conditions may be due to lack of oviposition during an experiment, a long delay in oviposition from the termination of hibernation, and failure to rear a large first brood. In the present study, the use of frozen male pupae to start the colony with the assistance of bumblebee workers rather than fresh, young, male pupae was investigated under controlled room temperature and humidity conditions. The period of initiation of the colony decreased with an increase in the number of worker helpers from one to six. The period was as short as 3.9 days in the presence of six worker helpers. Second and third broods also started earlier with the help of workers. The rate of first worker production per egg cup was double that of the normal method (i.e. without worker helpers). Egg eating behavior of the queen was not found in the first brood if more than two workers helped the development of the larvae. Four worker helpers were found to be sufficient, as they could produce 100% colony foundation and 91.46% first worker production colonies. This study showed that using frozen old pupae is a good stimulator for colony foundation in the presence of four bumblebee worker helpers.     

Minor Secondary-Structure Variation in the 5"-Untranslated Region of the β-Globin mRNA Changes the Concentration Requirements for eIF2

Nucleotide sequence changes increasing the number of paired bases without producing stable secondary structure in the 5"-untranslated region (5"-UTR) of the -globin mRNA had a slight effect on its translation in rabbit reticulocyte lysate at low mRNA concentration and dramatically decreased translation efficiency at a high concentration. The removal of paired regions restored translation. Addition of purified eIF2 to the lysate resulted in equal translation efficiencies of templates differing in structure of 5"-UTR. A similar effect was observed for p50, a major mRNP protein. Other mRNA-binding initiation factors, eIF4F and eIF4B, had no effect on the dependence of translation efficiency on mRNA concentration. Analysis of the assembly of the 48S initiation complex from its purified components showed that less eIF2 is required for translation initiation on the -globin mRNA than on its derivative containing minor secondary structure elements in 5"-UTR. According to a model proposed, eIF2 not only delivers Met-tRNA, but it also stabilizes the interaction of the 40S ribosome subunit with 5"-UTR, which is of particular importance for translation initiation on templates with structured 5"-UTR.     

Biological Relevance and Therapeutic Potential of the Hypusine Modification System

Hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is emerging as a crucial regulator in cancer, infections, and inflammation. Although its contribution in translational regulation of proline repeat-rich proteins has been sufficiently demonstrated, its biological role in higher eukaryotes remains poorly understood. To establish the hypusine modification system as a novel platform for therapeutic strategies, we aimed to investigate its functional relevance in mammals by generating and using a range of new knock-out mouse models for the hypusine-modifying enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase as well as for the cancer-related isoform eIF-5A2. We discovered that homozygous depletion of deoxyhypusine synthase and/or deoxyhypusine hydroxylase causes lethality in adult mice with different penetrance compared with haploinsufficiency. Network-based bioinformatic analysis of proline repeat-rich proteins, which are putative eIF-5A targets, revealed that these proteins are organized in highly connected protein-protein interaction networks. Hypusine-dependent translational control of essential proteins (hubs) and protein complexes inside these networks might explain the lethal phenotype observed after deletion of hypusine-modifying enzymes. Remarkably, our results also demonstrate that the cancer-associated isoform eIF-5A2 is dispensable for normal development and viability. Together, our results provide the first genetic evidence that the hypusine modification in eIF-5A is crucial for homeostasis in mammals. Moreover, these findings highlight functional diversity of the hypusine system compared with lower eukaryotes and indicate eIF-5A2 as a valuable and safe target for therapeutic intervention in cancer.     

原核基因翻译起始位点预测的新方法

翻译起始位点（TIS,即基因5’端）的精确定位是原核生物基因预测的一个关键问题,而基因组GC含量和翻译起始机制的多样性是影响当前TIS预测水平的重要因素．结合基因组结构的复杂信息（包括GC含量、TIS邻近序列及上游调控信号、序列编码潜能、操纵子结构等）,发展刻画翻译起始机制的数学统计模型,据此设计TIS预测的新算法MED．StartPlus．并将MED．StartPlus与同类方法RBSfinder、GS．Finder、MED-Start、TiCo和Hon-yaku等进行系统地比较和评价．测试针对两种数据集进行：当前14个已知的TIS被确认的基因数据集,以及300个物种中功能已知的基因数据集．测试结果表明,MED-StartPlus的预测精度在总体上超过同类方法．尤其是对高GC含量基因组以及具有复杂翻译起始机制的基因组,MED-StartPlus具有明显的优势．