Accumulation of P-bodies in Candida albicans under different stress and filamentous growth conditions

Candida albicans is an opportunistic fungal pathogen that grows as budding yeast, pseudohyphal, and hyphal forms. In response to external signals, C. albicans switches rapidly among these forms. mRNA-containing cytoplasmic granules, termed processing bodies (P-bodies), have been reported to accumulate under various environmental stress conditions in diverse species from yeast to mammals. Here, we provide the first microscopic and genetic characterization of P-bodies in C. albicans. The core components of P-bodies, including the decapping machinery (Dcp2 and Dhh1), 5′–3′ exoribonuclease (Kem1/Xrn1), and the P-body scaffolding protein (Edc3), were identified and their localizations with respect to P-bodies were demonstrated. Various growth conditions, including glucose deprivation, hyperosmotic stress, and heat stress, stimulated the accumulation of P-bodies. In addition, we observed P-body aggregation during hyphal development. The deletion mutant strain edc3/edc3 had a defect in filamentation and exhibited a dramatic reduction in the number of P-bodies. These results suggest that Edc3 plays an essential role in the assembly and maintenance of P-bodies in C. albicans, and that the switch to filamentous growth appears to accompany P-body accumulation.     

A Heat-Sensitive Arabidopsis thaliana Kinase Substitutes for Human p70s6k Function In Vivo

In mammalian cells, mitogen-induced phosphorylation of ribosomal protein S6 by p70^s6k has been implicated in the selective translational upregulation of 5′TOP mRNAs. We demonstrate here that the homologous Arabidopsis thaliana protein, AtS6k2, ectopically expressed in human 293 cells or isolated from plant cells, phosphorylates specifically mammalian and plant S6 at 25°C but not at 37°C. When Arabidopsis suspension culture cells are shifted from 25 to 37°C, the kinase becomes rapidly inactivated, consistent with the observation that heat shock abrogates S6 phosphorylation in plants. Treatment with potato acid phosphatase reduced the specific activity of immunoprecipitated AtS6k2 threefold, an effect which was blocked in the presence of 4-nitrophenyl phosphate. In quiescent mammalian cells, AtS6k2 is activated by serum stimulation, a response which is abolished by the fungal metabolite wortmannin but is resistant to rapamycin. Treatment of mammalian cells with rapamycin abolishes in vivo S6 phosphorylation by p70^s6k; however, ectopic expression of AtS6k2 rescues the rapamycin block. Collectively, the data demonstrate that AtS6k2 is the functional plant homolog of mammalian p70^s6k and identify a new signalling pathway in plants.     

Lead toxicity induces autophagy to protect against cell death through mTORC1 pathway in cardiofibroblasts

Heavy metals, such as lead (Pb²⁺), are usually accumulated in human bodies and impair human''s health. Lead is a metal with many recognized adverse health side effects and yet the molecular processes underlying lead toxicity are still poorly understood. In the present study, we proposed to investigate the effects of lead toxicity in cultured cardiofibroblasts. After lead treatment, cultured cardiofibroblasts showed severe endoplasmic reticulum (ER) stress. However, the lead-treated cardiofibroblasts were not dramatically apoptotic. Further, we found that these cells determined to undergo autophagy through inhibiting mammalian target of rapamycin complex 1 (mTORC1) pathway. Moreover, inhibition of autophagy by 3-methyladenine (3-MA) may dramatically enhance lead toxicity in cardiofibroblasts and cause cell death. Our data establish that lead toxicity induces cell stress in cardiofibroblasts and protective autophagy is activated by inhibition of mTORC1 pathway. These findings describe a mechanism by which lead toxicity may promote the autophagy of cardiofibroblasts cells, which protects cells from cell stress. Our findings provide evidence that autophagy may help cells to survive under ER stress conditions in cardiofibroblasts and may set up an effective therapeutic strategy for heavy metal toxicity.     

Metabolic characterization of a paused-like pluripotent state

Embryonic diapause is a conserved reproductive strategy in which development arrests at the blastocyst phase. Recently mammalian target of rapamycin (mTOR) inhibition was shown to induce diapause on mouse blastocysts and a paused-like state on mouse embryonic stem cells (mESCs). In this work, we aimed to further characterize this new paused-pluripotent state, focusing on its glycolytic and oxidative metabolic function. We therefore exposed mESCs, to the mTOR inhibitor INK-128 and evaluated proliferation, pluripotency status and energy-related metabolism, as well as the mTOR inhibition status and translational function. Unexpectedly, in our hands INK-128 did not inhibit the phosphorylation of mTOR or its downstream targets after 48 h. Accordingly, no alterations on protein translational function were observed. Nonetheless, INK-128 could still successfully induce a paused-like state in naïve mESCs regardless of their culturing conditions, by greatly slowing proliferation without affecting pluripotency status. This effect was more prevalent in 2i cultured cells. Interestingly, in this paused-like state, mESCs present a glucose-related hypometabolic profile, which is a hallmark of diapaused blastocysts, with decreased glycolytic and oxidative metabolism and decreased nutrient uptake. Despite the lack of mTOR inhibition and translational suppression, INK-128 still induced a paused-like pluripotent state through cell cycle and metabolic modulation, rather than by translational suppression, suggesting more than one avenue for this type of pluripotent phenotype.     

Withdrawal of Essential Amino Acids Increases Autophagy by a Pathway Involving Ca2+/Calmodulin-dependent Kinase Kinase-β (CaMKK-β)

Autophagy is the main lysosomal catabolic process that becomes activated under stress conditions, such as amino acid starvation and cytosolic Ca²⁺ upload. However, the molecular details on how both conditions control autophagy are still not fully understood. Here we link essential amino acid starvation and Ca²⁺ in a signaling pathway to activate autophagy. We show that withdrawal of essential amino acids leads to an increase in cytosolic Ca²⁺, arising from both extracellular medium and intracellular stores, which induces the activation of adenosine monophosphate-activated protein kinase (AMPK) via Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β). Furthermore, we show that autophagy induced by amino acid starvation requires AMPK, as this induction is attenuated in its absence. Subsequently, AMPK activates UNC-51-like kinase (ULK1), a mammalian autophagy-initiating kinase, through phosphorylation at Ser-555 in a process that requires CaMKK-β. Finally, the mammalian target of rapamycin complex C1 (mTORC1), a negative regulator of autophagy downstream of AMPK, is inhibited by amino acid starvation in a Ca²⁺-sensitive manner, and CaMKK-β appears to be important for mTORC1 inactivation, especially in the absence of extracellular Ca²⁺. All these results highlight that amino acid starvation regulates autophagy in part through an increase in cellular Ca²⁺ that activates a CaMKK-β-AMPK pathway and inhibits mTORC1, which results in ULK1 stimulation.     

Inflammatory Stress Increases Hepatic CD36 Translational Efficiency via Activation of the mTOR Signalling Pathway

Inflammatory stress is an independent risk factor for the development of non-alcoholic fatty liver disease (NAFLD). Although CD36 is known to facilitate long-chain fatty acid uptake and contributes to NAFLD progression, the mechanisms that link inflammatory stress to hepatic CD36 expression and steatosis remain unclear. As the mammalian target of rapamycin (mTOR) signalling pathway is involved in CD36 translational activation, this study was undertaken to investigate whether inflammatory stress enhances hepatic CD36 expression via mTOR signalling pathway and the underlying mechanisms. To induce inflammatory stress, we used tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) stimulation of the human hepatoblastoma HepG2 cells in vitro and casein injection in C57BL/6J mice in vivo. The data showed that inflammatory stress increased hepatic CD36 protein levels but had no effect on mRNA expression. A protein degradation assay revealed that CD36 protein stability was not different between HepG2 cells treated with or without TNF-α or IL-6. A polysomal analysis indicated that CD36 translational efficiency was significantly increased by inflammatory stress. Additionally, inflammatory stress enhanced the phosphorylation of mTOR and its downstream translational regulators including p70S6K, 4E-BP1 and eIF4E. Rapamycin, an mTOR-specific inhibitor, reduced the phosphorylation of mTOR signalling pathway and decreased the CD36 translational efficiency and protein level even under inflammatory stress resulting in the alleviation of inflammatory stress-induced hepatic lipid accumulation. This study demonstrates that the activation of the mTOR signalling pathway increases hepatic CD36 translational efficiency, resulting in increased CD36 protein expression under inflammatory stress.     

REDD1, an inhibitor of mTOR signalling,is regulated by the CUL4A–DDB1 ubiquitin ligase

The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxia‐inducible factor‐1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post‐translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin‐mediated degradation mediated by the CUL4A–DDB1–ROC1–β‐TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3β. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.     

Heat-Stress and Light-Stress Induce Different Cellular Pathologies in the Symbiotic Dinoflagellate during Coral Bleaching

Coral bleaching is a significant contributor to the worldwide degradation of coral reefs and is indicative of the termination of symbiosis between the coral host and its symbiotic algae (dinoflagellate; Symbiodinium sp. complex), usually by expulsion or xenophagy (symbiophagy) of its dinoflagellates. Herein, we provide evidence that during the earliest stages of environmentally induced bleaching, heat stress and light stress generate distinctly different pathomorphological changes in the chloroplasts, while a combined heat- and light-stress exposure induces both pathomorphologies; suggesting that these stressors act on the dinoflagellate by different mechanisms. Within the first 48 hours of a heat stress (32°C) under low-light conditions, heat stress induced decomposition of thylakoid structures before observation of extensive oxidative damage; thus it is the disorganization of the thylakoids that creates the conditions allowing photo-oxidative-stress. Conversely, during the first 48 hours of a light stress (2007 µmoles m⁻² s⁻¹ PAR) at 25°C, condensation or fusion of multiple thylakoid lamellae occurred coincidently with levels of oxidative damage products, implying that photo-oxidative stress causes the structural membrane damage within the chloroplasts. Exposure to combined heat- and light-stresses induced both pathomorphologies, confirming that these stressors acted on the dinoflagellate via different mechanisms. Within 72 hours of exposure to heat and/or light stresses, homeostatic processes (e.g., heat-shock protein and anti-oxidant enzyme response) were evident in the remaining intact dinoflagellates, regardless of the initiating stressor. Understanding the sequence of events during bleaching when triggered by different environmental stressors is important for predicting both severity and consequences of coral bleaching.     

Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces a cellular stress response characterized by rapid and sustained activation of the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway and selective apoptosis of cells lacking functional p53. Here we have investigated how mTOR regulates ASK1 signaling using p53-mutant rhabdomyosarcoma cells. In Rh30 cells, ASK1 was found to physically interact with protein phosphatase 5 (PP5), previously identified as a negative regulator of ASK1. Rapamycin did not affect either protein level of PP5 or association of PP5 with ASK1. Instead, rapamycin caused rapid dissociation of the PP2A-B" regulatory subunit (PR72) from the PP5-ASK1 complex, which was associated with reduced phosphatase activity of PP5. This effect was dependent on expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Down-regulation of PP5 activity by rapamycin coordinately activated ASK1, leading to elevated phosphorylation of c-Jun. Amino acid deprivation, which like rapamycin inhibits mTOR signaling, also inhibited PP5 activity, caused rapid dissociation of PR72, and activated ASK1 signaling. Overexpression of PP5, but not the PP2A catalytic subunit, blocked rapamycin-induced phosphorylation of c-Jun, and protected cells from rapamycin-induced apoptosis. The results suggest that PP5 is downstream of mTOR, and positively regulated by the mTOR pathway. The findings suggest that in the absence of serum factors, mTOR signaling suppresses apoptosis through positive regulation of PP5 activity and suppression of cellular stress.     

S6 Kinase- and β-TrCP2-Dependent Degradation of p19Arf Is Required for Cell Proliferation

The kinase mTOR (mammalian target of rapamycin) promotes translation as well as cell survival and proliferation under nutrient-rich conditions. Whereas mTOR activates translation through ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4E-BP), how it facilitates cell proliferation has remained unclear. We have now identified p19^Arf, an inhibitor of cell cycle progression, as a novel substrate of S6K that is targeted to promote cell proliferation. Serum stimulation induced activation of the mTOR-S6K axis and consequent phosphorylation of p19^Arf at Ser⁷⁵. Phosphorylated p19^Arf was then recognized by the F-box protein β-TrCP2 and degraded by the proteasome. Ablation of β-TrCP2 thus led to the arrest of cell proliferation as a result of the stabilization and accumulation of p19^Arf. The β-TrCP2 paralog β-TrCP1 had no effect on p19^Arf stability, suggesting that phosphorylated p19^Arf is a specific substrate of β-TrCP2. Mice deficient in β-TrCP2 manifested accumulation of p19^Arf in the yolk sac and died in utero. Our results suggest that the mTOR pathway promotes cell proliferation via β-TrCP2-dependent p19^Arf degradation under nutrient-rich conditions.