Explore the vast range of titles available.

A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through β-adrenergic receptor (βARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known βAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted βAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from βARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.

Growth signals, such as extracellular nutrients and growth factors, have substantial effects on genome integrity; however, the direct underlying link remains unclear. Here, we show that the mechanistic target of rapamycin (mTOR)–ribosomal S6 kinase (S6K) pathway, a central regulator of growth signalling, phosphorylates RNF168 at Ser60 to inhibit its E3 ligase activity, accelerate its proteolysis and impair its function in the DNA damage response, leading to accumulated unrepaired DNA and genome instability. Moreover, loss of the tumour suppressor liver kinase B1 ( LKB1 ; also known as STK11 ) hyperactivates mTOR complex 1 (mTORC1)–S6K signalling and decreases RNF168 expression, resulting in defects in the DNA damage response. Expression of a phospho-deficient RNF168-S60A mutant rescues the DNA damage repair defects and suppresses tumorigenesis caused by Lkb1 loss. These results reveal an important function of mTORC1–S6K signalling in the DNA damage response and suggest a general mechanism that connects cell growth signalling to genome stability control. Xie and colleagues find that activated mTORC1 growth signalling impairs DNA repair through S6K-mediated phosphorylation and inhibition of the RNF168 ligase.

During pregnancy, the appropriate allocation of nutrients between the mother and the fetus is dominated by maternal–fetal interactions, which is primarily governed by the placenta. The syncytiotrophoblast (STB) lining at the outer surface of the placental villi is directly bathed in maternal blood and controls feto–maternal exchange. The STB is the largest multinucleated cell type in the human body, and is formed through syncytialization of the mononucleated cytotrophoblast. However, the physiological advantage of forming such an extensively multinucleated cellular structure remains poorly understood. Here, we discover that the STB uniquely adapts to nutrient stress by inducing the macropinocytosis machinery through repression of mammalian target of rapamycin (mTOR) signaling. In primary human trophoblasts and in trophoblast cell lines, differentiation toward a syncytium triggers macropinocytosis, which is greatly enhanced during amino acid shortage, induced by inhibiting mTOR signaling. Moreover, inhibiting mTOR in pregnant mice markedly stimulates macropinocytosis in the syncytium. Blocking macropinocytosis worsens the phenotypes of fetal growth restriction caused by mTOR-inhibition. Consistently, placentas derived from fetal growth restriction patients display: 1) Repressed mTOR signaling, 2) increased syncytialization, and 3) enhanced macropinocytosis. Together, our findings suggest that the unique ability of STB to undergo macropinocytosis serves as an essential adaptation to the cellular nutrient status, and support fetal survival and growth under nutrient deprivation.

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to nutrients, energy levels, and growth factors. It contains the atypical kinase mTOR and the RAPTOR subunit that binds to the Tor signalling sequence (TOS) motif of substrates and regulators. mTORC1 is activated by the small GTPase RHEB (Ras homologue enriched in brain) and inhibited by PRAS40. Here we present the 3.0 ångström cryo-electron microscopy structure of mTORC1 and the 3.4 ångström structure of activated RHEB–mTORC1. RHEB binds to mTOR distally from the kinase active site, yet causes a global conformational change that allosterically realigns active-site residues, accelerating catalysis. Cancer-associated hyperactivating mutations map to structural elements that maintain the inactive state, and we provide biochemical evidence that they mimic RHEB relieving auto-inhibition. We also present crystal structures of RAPTOR–TOS motif complexes that define the determinants of TOS recognition, of an mTOR FKBP12–rapamycin-binding (FRB) domain–substrate complex that establishes a second substrate-recruitment mechanism, and of a truncated mTOR–PRAS40 complex that reveals PRAS40 inhibits both substrate-recruitment sites. These findings help explain how mTORC1 selects its substrates, how its kinase activity is controlled, and how it is activated by cancer-associated mutations. The cryo-electron microscopy and crystal structures of several mTORC1 complexes, and accompanying biochemical analyses, shed light on how mTORC1 is regulated and how cancer mutations lead to its hyperactivation. mTORC1 structures shed light on function Mechanistic target of rapamycin complex 1 (mTORC1) is a protein complex that is important for regulating cell growth and homeostasis and is aberrantly regulated in many diseases such as cancer, diabetes and neurodegeneration. Here, Nikola Pavletich and colleagues use cryo-electron microscopy and crystallography to determine the structures of several mTORC1 complexes. The structures and accompanying biochemical analysis provide mechanistic insights into how mTORC1 is allosterically activated by the GTPase RHEB, how it is inhibited by PRAS40, and how it recognizes substrates via the TOS motif. The findings also shed light on how cancer mutations lead to hyperactivation of mTORC1.

Chromatin and metabolic states both influence lifespan, but how they interact in lifespan regulation is largely unknown. The COMPASS chromatin complex, which trimethylates lysine 4 on histone H3 (H3K4me3), regulates lifespan in Caenorhabditis elegans . However, the mechanism by which H3K4me3 modifiers affect longevity, and whether this mechanism involves metabolic changes, remain unclear. Here we show that a deficiency in H3K4me3 methyltransferase, which extends lifespan, promotes fat accumulation in worms with a specific enrichment of mono-unsaturated fatty acids (MUFAs). This fat metabolism switch in H3K4me3 methyltransferase-deficient worms is mediated at least in part by the downregulation of germline targets, including S6 kinase, and by the activation of an intestinal transcriptional network that upregulates delta-9 fatty acid desaturases. Notably, the accumulation of MUFAs is necessary for the lifespan extension of H3K4me3 methyltransferase-deficient worms, and dietary MUFAs are sufficient to extend lifespan. Given the conservation of lipid metabolism, dietary or endogenous MUFAs could extend lifespan and healthspan in other species, including mammals. A deficiency in H3K4me3 methyltransferase causes accumulation of mono-unsaturated fatty acids, which is important for lifespan extension in C. elegans and could be relevant in mammals. Longevity fuelled by fat The lifespan of a worm is extended by H3K4me3 methyltransferase deficiency, but how and why remains unclear. Here it is shown that the loss of H3K4me3 in the germline affects fat metabolism in the worm intestine, resulting in the accumulation of mono-unsaturated fatty acids (MUFAs), but not poly-unsaturated fatty acids (PUFAs). The fat switch appears to be mediated in part by the downregulation of specific targets in the germline, including S6K, and the activation of a transcriptional network in the intestine leading to the upregulation of conserved delta-9 fatty acid desaturases. MUFA accumulation is necessary for the increased longevity caused by H3K4me3-methyltransferase deficiency, and the authors found that dietary MUFAs, but not PUFAs, were sufficient to extend worm lifespan. Whether dietary or endogenous MUFAs could extend lifespan and healthspan in other species remains to be seen.

Background Cardamonin, a chalcone isolated from Alpiniae katsumadai , has anti-inflammatory and anti-tumor activities. However, the molecular mechanism by which cardamonin inhibits breast cancer progression largely remains to be determined. Methods CCK-8 and Hoechst 33258 staining were used to detect cell growth and apoptosis, respectively. HIF-1α driven transcription was measured by luciferase reporter assay. Glucose uptake and lactate content were detected with 2-NBDG and L-Lactate Assay Kit. Cell metabolism assays were performed on Agilent’s Seahorse Bioscience XF96 Extracellular Flux Analyzer. Mitochondrial membrane potential was measured with JC-1 probe. DCFH-DA was used to measure ROS level. Protein expression was detected by western blotting assay. Immunohistochemistry was performed to measure the expression of HIF-1α, LDHA and CD31 in tumor tissues. Results Cardamonin inhibited growth of the triple negative breast cancer cell line MDA-MB-231 in vitro and in vivo by suppressing HIF-1α mediated cell metabolism. Cardamonin inhibited the expression of HIF-1α at mRNA and protein levels by repressing the mTOR/p70S6K pathway, and subsequently enhanced mitochondrial oxidative phosphorylation and induced reactive oxygen species (ROS) accumulation. We also found that cardamonin inhibited the Nrf2-dependent ROS scavenging system which further increased intracellular ROS levels. Eventually, accumulation of the intracellular ROS induced apoptosis in breast cancer cells. In addition, cardamonin treatment reduced glucose uptake as well as lactic acid production and efflux, suggesting its function in repressing the glycolysis process. Conclusions These results reveal novel function of cardamonin in modulating cancer cell metabolism and suppressing breast cancer progression, and suggest its potential for breast cancer treatment.

Functioning as a master kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1) plays a fundamental role in phosphorylating and activating protein kinases A, B and C (AGC) family kinases, including AKT. However, upstream regulation of PDK1 remains largely elusive. Here we report that ribosomal protein S6 kinase beta 1 (S6K1), a member of AGC kinases and downstream target of mechanistic target of rapamycin complex 1 (mTORC1), directly phosphorylates PDK1 at its pleckstrin homology (PH) domain, and impairs PDK1 interaction with and activation of AKT. Mechanistically, S6K1-mediated phosphorylation of PDK1 augments its interaction with 14-3-3 adaptor protein and homo-dimerization, subsequently dissociating PDK1 from phosphatidylinositol 3,4,5 triphosphate (PIP 3 ) and retarding its interaction with AKT. Pathologically, tumor patient-associated PDK1 mutations, either attenuating S6K1-mediated PDK1 phosphorylation or impairing PDK1 interaction with 14-3-3, result in elevated AKT kinase activity and oncogenic functions. Taken together, our findings not only unravel a delicate feedback regulation of AKT signaling via S6K1-mediated PDK1 phosphorylation, but also highlight the potential strategy to combat mutant PDK1 -driven cancers. The direct upstream regulation of PDK1 is not fully understood. Here the authors demonstrate that S6K1 directly phosphorylates PDK1 to inhibit AKT kinase activity and its ability to drive tumourigenesis.

Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.

The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase-related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a complex of truncated mTOR and mammalian lethal with SEC13 protein 8 (mLST8) with an ATP transition state mimic and with ATP-site inhibitors. The structures reveal an intrinsically active kinase conformation, with catalytic residues and a catalytic mechanism remarkably similar to canonical protein kinases. The active site is highly recessed owing to the FKBP12–rapamycin-binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. mTOR-activating mutations map to the structural framework that holds these elements in place, indicating that the kinase is controlled by restricted access. In vitro biochemistry shows that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. Rapamycin–FKBP12 inhibits the kinase by directly blocking substrate recruitment and by further restricting active-site access. The structures also reveal active-site residues and conformational changes that underlie inhibitor potency and specificity. Co-crystal structures of a number of complexes involving truncated mammalian target of rapamycin, a phosphoinositide 3-kinase-related protein kinase, reveal an intrinsically active kinase conformation and show how rapamycin–FKBP12 directly blocks substrate recruitment to the kinase domain. Structure of mTOR kinase The mTOR (mammalian target of rapamycin) pathway is a central regulator of cell growth in response to environmental signals such as energy, nutrients and growth factors, and is misregulated in cancer and metabolic diseases. Here the first crystal structures of the mTOR kinase are presented. The 3.2 Å crystal structures of the enzyme bound to a positive regulator and to small-molecule ATP-competitive inhibitors reveal an intrinsically active kinase, and explain how the rapamycin–FKBP12 complex blocks recruitment of substrates to the kinase domain.

Insulin activates sterol regulatory element-binding protein-1c (SREBP-1c) in liver, thereby increasing fatty acid and triglyceride synthesis. We created a line of transgenic rats that produce epitope-tagged human SREBP-1c in liver under control of the constitutive apolipoprotein E promoter/enhancer. This system allows us to dissect the pathway by which insulin stimulates SREBP-1c processing without interference by the insulin-mediated increase in SREBP-1c mRNA. Rats are used because freshly isolated rat hepatocytes respond much more robustly to insulin than do mouse hepatocytes. The data reveal that insulin-mediated stimulation of SREBP-1c processing requires the mechanistic target of rapamycin complex 1 (mTORC1), which also is required for insulin-mediated SREBP-1c mRNA induction. However, in contrast to mRNA induction, insulin stimulation of SREBP-1c processing is blocked by an inhibitor of p70 S6-kinase. The data indicate that the pathways for insulin enhancement of SREBP-1c mRNA and proteolytic processing diverge after mTORC1. Stimulation of processing requires the mTORC1 target p70 S6-kinase, whereas induction of mRNA bypasses this enzyme. Insulin stimulation of both processes is blocked by glucagon. The transgenic rat system will be useful in further defining the molecular mechanism for insulin stimulation of lipid synthesis in liver in normal and diabetic states.