Characterization of the Raptor/4E-BP1 Interaction by Chemical Cross-linking Coupled with Mass Spectrometry Analysis

mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56–72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo.     

Leucine Signals to mTORC1 via Its Metabolite Acetyl-Coenzyme A

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mTOR-S6K1 pathway mediates cytoophidium assembly

CTP synthase (CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and mammalian cells. However, the regulation and function of the cytoophidium remain elusive. Here, we provide evidence that the mechanistic target of rapamycin (mTOR) pathway controls cytoophidium assembly in mammalian and Drosophila cells. In mammalian cells, we find that inhibition of mTOR pathway attenuates cytoophidium formation. Moreover, CTPS cytoophidium assembly appears to be dependent on the mTOR complex 1 (mTORC1) mainly. In addition, knockdown of the mTORC1 downstream target S6K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing mTOR protein expression results in a decrease of the length of cytoophidium in Drosophila follicle cells. Therefore, our study connects CTPS cytoophidium formation with the mTOR signaling pathway.     

Games as experiments to understand why wildlife devaluation may not reduce the number killed

Illegal hunting and trade might be reduced by devaluing the commodities of live animals. However, theory and anecdote suggest that the tactic’s success depends on the proportion devalued and may also generate unintuitive and counter-productive outcomes. Unfortunately, the difficulty of researching criminals limits empirical and experimental tests of theory and practice. We overcame those constraints by designing and conducting a competitive game; simulating criminal hunting of protected wildlife where the proportion of animals devalued across a meta-population increases at a cost to investment in security. We conducted 33 games each with 10 different volunteers from the public visiting five public events (fairs, gala, and carnivals) in different communities of the Wellington metropolitan area, New Zealand. Games confirmed the relative effectiveness of security over devaluation tactics. Even moderate security protected the majority of commodities, but only when devaluation reached extraordinary levels (≥90%) was it protective, and even then only partially. When the risk of hunting is low, reduced rewards were enough to still motivate hunting. Games also revealed that when security and devaluation tactics are mixed across a meta-population, elevated risk caused hunters to revise their valuation of devalued commodities upwards and hunt and harvest them anyway. Counter-intuitively, more valuable devalued commodities were preserved but those of lesser value harvested, then sometimes discarded but at other times their value claimed. And lastly, hunters choose to take greater risks, shifting their activities to adjacent populations where the returns could be greater even though known to be better protected. The survival of wildlife in those populations deteriorated, even as the number of hunters caught increased. In all these ways, our games demonstrated how the addition of devaluation tactics to strategies for protecting wildlife can incentivise and exacerbate, rather than mitigate, the illegal hunting and trade of wildlife, consistent with theory and anecdote.     

北京发现凤头鹰和黑翅鸢

报道了凤头鹰(Accipiter trivirgatus)和黑翅鸢(Elanus caeruleus)2个北京鸟类分布新记录。以上鸟类未见于《北京鸟类志》、《北京脊椎动物检索表》等文献记载。《中国鸟类分布与分类名录》上所列其分布区中也不包括北京。以上鸟类是北京地区近年来新记录到的猛禽。     

Reciprocal Regulation of the TOR Kinase and ABA Receptor Balances Plant Growth and Stress Response

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Discrete functions of rictor and raptor in cell growth regulation in Drosophila

The TOR signaling pathway regulates cell growth and metabolism in response to various nutrient signals by forming complexes with either rictor or raptor. To distinguish the physiological roles of the complexes formed by the two different TOR partners, we compared the in vivo functions of rictor and raptor in Drosophila. In rictor-null mutants, Akt-induced tissue hyperplasia was reduced and Akt-Ser-505 phosphorylation was decreased. Furthermore, FOXO-dependent apoptosis, which is inhibited by Akt, was augmented in the rictor-null background, indicating that rictor is essential for the Akt-FOXO signaling module. We found that neither S6K-dependent cell growth nor S6K-Thr-398 phosphorylation was affected in rictor-null mutants. However, the knockdown of another TOR binding partner, raptor, decreased S6K-Thr-398 phosphorylation and inhibited S6K-induced cell overgrowth. Collectively, our findings strongly support that the association of TOR with rictor or raptor plays pivotal roles in TOR-mediated cell apoptosis and growth control by differentially regulating Akt- and S6K-dependent signaling pathways, respectively.     

Mechanisms involved in the coordinate regulation of mTORC1 by insulin and amino acids

In this study, we explored the coordinate regulation of mTORC1 by insulin and amino acids. Rat livers were perfused with medium containing various concentrations of insulin and/or amino acids. At fasting (1×) or 2× (2×AA) concentrations of amino acids, insulin maximally stimulated Akt phosphorylation but had no effect on global rates of protein synthesis. In the absence of insulin, 4×AA produced a moderate stimulation of protein synthesis and activation of mTORC1. The combination of 4×AA and insulin produced a maximal stimulation of protein synthesis and activation of mTORC1. These effects were accompanied by decreases in raptor and PRAS40 and an increase in RagC associated with mTOR (mammalian target of rapamycin). The studies were extended to a cell culture model in which mTORC1 activity was repressed by deprivation of leucine and serum, and resupplementation with the amino acid and insulin acted in an additive manner to restore mTORC1 activation. In deprived cells, mTORC1 was activated by expressing either constitutively active (ca) Rheb or a caRagB·caRagC complex, and coexpression of the constructs had an additive effect. Notably, resupplementation with leucine in cells expressing caRheb or with insulin in cells expressing the caRagB·caRagC complex was as effective as resupplementation with both leucine and insulin in non-transfected cells. Moreover, changes in mTORC1 activity correlated directly with altered association of mTOR with RagB/RagC, Rheb, raptor, and PRAS40. Overall, the results suggest that amino acids signal through the Rag complex and insulin through Rheb to achieve coordinate activation of mTORC1.     

The conservation significance of the proposed Mbaéré-Bodingué national park,Central African Republic,with special emphasis on its primate community

The proposed Mbaéré-Bodingué National Park (872km²) is located in the southwestern part of the Central African Republic and consists of terra firma rain forests and seasonally flooded forests. We assessed the conservation significance of this site using data on wildlife and habitat conditions collected during a comprehensive primate survey. Scores were attributed to a system of variables and sub-variables to assess the conservation value of the proposed park at the national level. We identified a total of 10 diurnal primate species (eight monkeys, two apes) and flooded forests were considered as a key habitat for primates as they hold a greater number of species than terra firma forests. We recorded the presence of 12 mammal species listed under the 2000 IUCN Red List of Threatened Species, including four classified as Endangered (two Primates, one Proboscidae and one Insectivora). We found that the conservation value of the proposed park ranked second out of the 15 existing protected areas of the country. Primate abundance was found to be generally higher than in the nearby Dzanga-Ndoki National Park (the sole other park located in the same vegetation unit), in particular for the species occurring in flooded forests. This highlights the fact that the proposed Mbaéré-Bodingué National Park is an important area for the conservation of species associated with flooded forests, a habitat which is currently under-represented in the national protected area system. Poaching pressure was identified as the main current threat to the integrity of the proposed park.     

Population census of the white-headed langur (Trachypithecus leucocephalus) at Longrui Karst Hills, Guangxi, China

A population census of white-headed langurs (Trachypithecus leucocephalus) was conducted using sleeping-site counts and the line-transect method from July to August 2002 at the Longrui Karst Hills, Guangxi, China. This area had been the largest habitat of the langurs across their range before the 1990s. While our survey revealed 146 old sleeping-sites, no white-headed langurs or fresh sleeping-sites were found in this area. Our study indicated that there had previously been a large population of langurs at Longrui Karst Hills, but now the langurs are possibly locally extinct in this area. If langurs still exist within the area, the population density must be very low. Interviews with local people confirmed that the number of white-headed langurs has been decreasing since the 1980s. Poaching was very common in the past and continues to be a problem at present, and it is likely that this has caused the decrease in the white-headed langur population at Longrui Karst Hills.