Inhibition of mTORC1 by astrin and stress granules prevents apoptosis in cancer cells.

Mammalian target of rapamycin complex 1 (mTORC1) controls growth and survival in response to metabolic cues. Oxidative stress affects mTORC1 via inhibitory and stimulatory inputs. Whereas downregulation of TSC1-TSC2 activates mTORC1 upon oxidative stress, the molecular mechanism of mTORC1 inhibition remains unknown. Here, we identify astrin as an essential negative mTORC1 regulator in the cellular stress response. Upon stress, astrin inhibits mTORC1 association and recruits the mTORC1 component raptor to stress granules (SGs), thereby preventing mTORC1-hyperactivation-induced apoptosis. In turn, balanced mTORC1 activity enables expression of stress factors. By identifying astrin as a direct molecular link between mTORC1, SG assembly, and the stress response, we establish a unifying model of mTORC1 inhibition and activation upon stress. Importantly, we show that in cancer cells, apoptosis suppression during stress depends on astrin. Being frequently upregulated in tumors, astrin is a potential clinically relevant target to sensitize tumors to apoptosis.

Proteomic profiling of VCP substrates links VCP to K6-linked ubiquitylation and c-Myc function.

Valosin-containing protein (VCP) is an evolutionarily conserved ubiquitin-dependent ATPase that mediates the degradation of proteins through the ubiquitin-proteasome pathway. Despite the central role of VCP in the regulation of protein homeostasis, identity and nature of its cellular substrates remain poorly defined. Here, we combined chemical inhibition of VCP and quantitative ubiquitin remnant profiling to assess the effect of VCP inhibition on the ubiquitin-modified proteome and to probe the substrate spectrum of VCP in human cells. We demonstrate that inhibition of VCP perturbs cellular ubiquitylation and increases ubiquitylation of a different subset of proteins compared to proteasome inhibition. VCP inhibition globally upregulates K6-linked ubiquitylation that is dependent on the HECT-type ubiquitin E3 ligase HUWE1. We report ~450 putative VCP substrates, many of which function in nuclear processes, including gene expression, DNA repair and cell cycle. Moreover, we identify that VCP regulates the level and activity of the transcription factor c-Myc.

Stuck in a (literal) tight spot: Cycling between tram rails, sharrows and parked cars.

PROBLEM: Cyclists riding next to parked vehicles are at risk of crashes with opening vehicle doors. A central position, out of this dooring zone, decreases such a risk but comes with other problems like potentially smaller passing distances kept by overtaking motorists or having to cross tram rails. METHOD: Factors influencing cyclists' choice of position were investigated by showing a total of 3,444 German cyclists different traffic situations in two online surveys. In the first study (N = 1,850), parked cars, the position of a cyclist riding ahead in the presented images (towards the curb/center of the lane), and presence and kind of sharrows were varied. As the variation in results for the different sharrow types was negligible, in Study 2 (N = 1,594), only the most common type was used. Whether cyclists prefer to accept the risk of falling while crossing tram rails or the risk of being too close to the curb or parked cars was investigated, varying the presence of tram rails, which has not been previously researched. In both studies, respondents indicated which position on the road they would choose in the depicted situations and answered questions about subjective safety, a factor closely related to cyclists' choice of position. RESULTS: Cyclists chose positions farther towards the center of the road if there were parked cars and they chose an even more central position with tram rails. Respondents felt safer with sharrows on the road as well as in situations without parked cars and in situations without tram rails. Discussion and practical implications: The results indicate that, in addition to infrastructure characteristics, other cyclists' behavior (descriptive norm) influences cyclists' position on the road as well as their perceived safety. Implications for infrastructure design, especially regarding (the removal of) parked cars, are discussed.

Safe cycling in winter: Results of a case study on the role of de-icing in the city of Hamburg, Germany.

INTRODUCTION: In parts of Europe and North America, cycling volumes decrease in winter due to a reduction in subjective safety. To counter this, high-quality winter maintenance is required on cycle paths. At the moment, grit and sodium chloride are considered state-of-the-art gritting/de-icing materials in Germany. However, grit has to be removed after winter because it poses a serious injury risk on dry streets, and, in various German cities, using sodium chloride is prohibited on segregated bike paths due to the harmful impact on surrounding trees. Therefore, there is a need for alternative gritting/de-icing materials. METHOD: We used a mixed-methods approach consisting of qualitative and quantitative surveys together with laboratory investigations and a life cycle assessment to find suitable alternatives to sodium chloride and grit for use on segregated bike paths, and tested four de-icing materials (sodium chloride as a reference, sodium formate, calcium magnesium acetate or CMA, and potassium acetate) at two sites in Hamburg, Germany. The tests were accompanied by on-site cyclist surveys. RESULTS: The results show that the use of alternative (non-sodium chloride) de-icing materials either reduces or eliminates negative impacts on the environment at a local level, for example on trees along the cycle path. However, this reduction goes hand in hand with increasing negative environmental impacts at a global level due to higher overall emissions associated with the tested alternative de-icing materials. Regarding cyclists' safety, sodium formate was the only de-icing material which delivered comparable results to sodium chloride and should therefore be tested in extended conditions. CONCLUSIONS: Further research is needed on the large-scale application of the investigated de-icing agents on cycle paths in different cities, along with a survey of a larger number of cyclists. A final evaluation of the environmental aspects can only be made when the supply and production conditions for large-scale use are clear. PRACTICAL APPLICATIONS: Basically there are two options for the practical application of de-icing materials: The usage of the costlier alternatives with better properties at a local level in the hope of fast development towards a better global GHG footprint in their manufacturing processes, or sticking to the use of grit and sodium chloride (where it is possible) while committing to improving clean-up after the snow and ice melt to prevent unsafe road conditions in spring.

The long-term effect of four hours of hyperventilation on neurocognitive performance and lesion size after controlled cortical impact in rats.

BACKGROUND: We investigated the effects of 4 h of posttraumatic hyperventilation on neurocognitive performance, motor function, and coordination as well as lesion volume in rats subjected to focal traumatic brain injury. METHODS: After a 14-day training period with various neurocognitive tests including hole-board, beam walk, and beam balance, 21 male Sprague-Dawley rats (369 +/- 15 g) were anesthetized with halothane, tracheally intubated, their lungs mechanically ventilated, and subjected to controlled cortical impact (1.75 mm depth, diameter 5 mm, 4 m/s). They were then randomized to either normoventilation (n = 10; PaCO(2) = 38-42 mm Hg) or hyperventilation (n = 11; PaCO(2) = 28-32 mm Hg) and ventilated for 4 h, respectively. Posttraumatic performance in the behavioral and motor tests was evaluated for 20 days. Rats were then decapitated under deep anesthesia, and their brains frozen and sliced to evaluate lesion volume. RESULTS: Hyperventilated animals performed significantly worse in explicit memory tests compared with normoventilated rats over time. Both groups showed deficits in advanced motor function and coordination (evaluated by beam walk and beam balance) initially, with a significantly worse performance of hyperventilated compared with normoventilated animals. However, there was no difference between groups by the end of the study. On Day 20 after injury, lesion volume was significantly larger with hyperventilated (69.7 +/- 13.0 mm(3)) compared with normoventilated animals (48.3 +/- 15.6 mm(3)). CONCLUSIONS: Although hyperventilation enhanced histologic damage, there was no long-term adverse neurocognitive effect from 4 h of posttraumatic hyperventilation (PaCO(2) = 28-32 mm Hg) in rats.

PLK1 (polo like kinase 1) inhibits MTOR complex 1 and promotes autophagy.

Mechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1's and PLK1's functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy.

A systems study reveals concurrent activation of AMPK and mTOR by amino acids.

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational-experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.

TSC1 activates TGF-ß-Smad2/3 signaling in growth arrest and epithelial-to-mesenchymal transition.

The tuberous sclerosis proteins TSC1 and TSC2 are key integrators of growth factor signaling. They suppress cell growth and proliferation by acting in a heteromeric complex to inhibit the mammalian target of rapamycin complex 1 (mTORC1). In this study, we identify TSC1 as a component of the transforming growth factor ß (TGF-ß)-Smad2/3 pathway. Here, TSC1 functions independently of TSC2. TSC1 interacts with the TGF-ß receptor complex and Smad2/3 and is required for their association with one another. TSC1 regulates TGF-ß-induced Smad2/3 phosphorylation and target gene expression and controls TGF-ß-induced growth arrest and epithelial-to-mesenchymal transition (EMT). Hyperactive Akt specifically activates TSC1-dependent cytostatic Smad signaling to induce growth arrest. Thus, TSC1 couples Akt activity to TGF-ß-Smad2/3 signaling. This has implications for cancer treatments targeting phosphoinositide 3-kinases and Akt because they may impair tumor-suppressive cytostatic TGF-ß signaling by inhibiting Akt- and TSC1-dependent Smad activation.