The polyglutamine neurodegenerative protein ataxin 3 regulates aggresome formation

The polyglutamine-containing neurodegenerative protein ataxin 3 (AT3) has deubiquitylating activity and binds ubiquitin chains with a preference for chains of four or more ubiquitins. Here we characterize the deubiquitylating activity of AT3 in vitro and show it trims/edits K48-linked ubiquitin chains. AT3 also edits polyubiquitylated (125)I-lysozyme and decreases its degradation by proteasomes. Cellular studies show that endogenous AT3 colocalizes with aggresomes and preaggresome particles of the misfolded cystic fibrosis transmembrane regulator (CFTR) mutant CFTRDeltaF508 and associates with histone deacetylase 6 and dynein, proteins required for aggresome formation and transport of misfolded protein. Small interfering RNA knockdown of AT3 greatly reduces aggresomes formed by CFTRDeltaF508, demonstrating a critical role of AT3 in this process. Wild-type AT3 restores aggresome formation; however, AT3 with mutations in the active site or ubiquitin interacting motifs cannot restore aggresome formation in AT3 knockdown cells. These same mutations decrease the association of AT3 and dynein. These data indicate that the deubiquitylating activity of AT3 and its ubiquitin interacting motifs as well play essential roles in CFTRDeltaF508 aggresome formation.     

Phospholipase D1 is an effector of Rheb in the mTOR pathway

The mammalian target of rapamycin (mTOR) assembles a signaling network essential for the regulation of cell growth, which has emerged as a major target of anticancer therapies. The tuberous sclerosis complex 1 and 2 (TSC1/2) proteins and their target, the small GTPase Rheb, constitute a key regulatory pathway upstream of mTOR. Phospholipase D (PLD) and its product phosphatidic acid are also upstream regulators of the mitogenic mTOR signaling. However, how the TSC/Rheb and PLD pathways interact or integrate in the rapamycin-sensitive signaling network has not been examined before. Here, we find that PLD1, but not PLD2, is required for Rheb activation of the mTOR pathway, as demonstrated by the effects of RNAi. The overexpression of Rheb activates PLD1 in cells in the absence of mitogenic stimulation, and the knockdown of Rheb impairs serum stimulation of PLD activation. Furthermore, the overexpression of TSC2 suppresses PLD1 activation, whereas the knockdown or deletion of TSC2 leads to elevated basal activity of PLD. Consistent with a TSC-Rheb-PLD signaling cascade, AMPK and PI3K, both established regulators of TSC2, appear to lie upstream of PLD as revealed by the effects of pharmacological inhibitors, and serum activation of PLD is also dependent on amino acid sufficiency. Finally, Rheb binds and activates PLD1 in vitro in a GTP-dependent manner, strongly suggesting that PLD1 is a bona fide effector for Rheb. Hence, our findings reveal an unexpected interaction between two cascades in the mTOR signaling pathways and open up additional possibilities for targeting this important growth-regulating network for the development of anticancer drugs.     

Neurofibromatosis-1 regulates mTOR-mediated astrocyte growth and glioma formation in a TSC/Rheb-independent manner

Converging evidence from the analysis of human brain tumors and genetically engineered mice has revealed that the mammalian target of rapamycin (mTOR) pathway is a central regulator of glial and glioma cell growth. In this regard, mutational inactivation of neurofibromatosis-1 (NF1), tuberous sclerosis complex (TSC), and PTEN genes is associated with glioma formation, such that pharmacologic inhibition of mTOR signaling results in attenuated tumor growth. This shared dependence on mTOR suggests that PTEN and NF1 (neurofibromin) glial growth regulation requires TSC/Rheb (Ras homolog enriched in brain) control of mTOR function. In this report, we use a combination of genetic silencing in vitro and conditional mouse transgenesis approaches in vivo to demonstrate that neurofibromin regulates astrocyte cell growth and glioma formation in a TSC/Rheb-independent fashion. First, we show that Nf1 or Pten inactivation, but not Tsc1 loss or Rheb overexpression, increases astrocyte cell growth in vitro. Second, Nf1-deficient increased mTOR signaling and astrocyte hyperproliferation is unaffected by Rheb shRNA silencing. Third, conditional Tsc1 inactivation or Rheb overexpression in glial progenitors of Nf1(+/-) mice does not lead to glioma formation. Collectively, these findings establish TSC/Rheb-independent mechanisms for mTOR-dependent glial cell growth control and gliomagenesis relevant to the design of therapies for individuals with glioma.     

Regulation of inducible nitric oxide synthase by aggresome formation

Misfolding and aggregation of proteins play an important part in the pathogenesis of several genetic and degenerative diseases. Recent evidence suggests that cells have evolved a pathway that involves sequestration of aggregated proteins into specialized "holding stations" called aggresomes. Here we show that cells regulate inducible NO synthase (iNOS), an important host defense protein, through aggresome formation. iNOS aggresome formation depends on a functional dynein motor and the integrity of the microtubules. The iNOS aggresome represents a "physiologic aggresome" and thus defines a new paradigm for cellular regulation of protein processing. This study indicates that aggresome formation in response to misfolded proteins may merely represent an acceleration of an established physiologic regulatory process for specific proteins whose regulation by aggresome formation is deemed necessary by the cell.     

Ricolinostat (ACY‐1215) induced inhibition of aggresome formation accelerates carfilzomib‐induced multiple myeloma cell death

Proteasome inhibition induces the accumulation of aggregated misfolded/ubiquitinated proteins in the aggresome; conversely, histone deacetylase 6 (HDAC6) inhibition blocks aggresome formation. Although this rationale has been the basis of proteasome inhibitor (PI) and HDAC6 inhibitor combination studies, the role of disruption of aggresome formation by HDAC6 inhibition has not yet been studied in multiple myeloma (MM). The present study aimed to evaluate the impact of carfilzomib (CFZ) in combination with a selective HDAC6 inhibitor (ricolinostat) in MM cells with respect to the aggresome‐proteolysis pathway. We observed that combination treatment of CFZ with ricolinostat triggered synergistic anti‐MM effects, even in bortezomib‐resistant cells. Immunofluorescent staining showed that CFZ increased the accumulation of ubiquitinated proteins and protein aggregates in the cytoplasm, as well as the engulfment of aggregated ubiquitinated proteins by autophagosomes, which was blocked by ricolinostat. Electron microscopy imaging showed increased autophagy triggered by CFZ, which was inhibited by the addition of ACY‐1215. Finally, an in vivo mouse xenograft study confirmed a decrease in tumour volume, associated with apoptosis, following treatment with CFZ in combination with ricolinostat. Our results suggest that ricolinostat inhibits aggresome formation, caused by CFZ‐induced inhibition of the proteasome pathway, resulting in enhanced apoptosis in MM cells.     

The physiologic aggresome mediates cellular inactivation of iNOS

Nitric Oxide (NO), produced by inducible nitric oxide synthase (iNOS), has been implicated in the pathogenesis of various biological and inflammatory disorders. Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed “physiologic aggresome,” is exemplified by aggresome formation of iNOS, an important host defense protein. The functional significance of cellular formation of the iNOS aggresome is hitherto unknown. In this study, we used live cell imaging, fluorescence microscopy, and intracellular fluorescence NO probes to map the subcellular location of iNOS and NO under various conditions. We found that NO production colocalized with cytosolic iNOS but aggresomes containing iNOS were distinctly devoid of NO production. Further, cells expressing iNOS aggresomes produced significantly less NO as compared with cells not expressing aggresomes. Importantly, primary normal human bronchial epithelial cells, stimulated by cytokines to express iNOS, progressively sequestered iNOS to the aggresome, a process that correlated with marked reduction of NO production. These results suggest that bronchial epithelial cells used the physiologic aggresome mechanism for iNOS inactivation. Our studies reveal a novel cellular strategy to terminate NO production via formation of the iNOS aggresome.     

Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that occurs upon mutation of either the TSC1 or TSC2 genes, which encode the protein products hamartin and tuberin, respectively. Here, we show that hamartin and tuberin function together to inhibit mammalian target of rapamycin (mTOR)-mediated signaling to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). First, coexpression of hamartin and tuberin repressed phosphorylation of 4E-BP1, resulting in increased association of 4E-BP1 with eIF4E; importantly, a mutant of TSC2 derived from TSC patients was defective in repressing phosphorylation of 4E-BP1. Second, the activity of S6K1 was repressed by coexpression of hamartin and tuberin, but the activity of rapamycin-resistant mutants of S6K1 were not affected, implicating mTOR in the TSC-mediated inhibitory effect on S6K1. Third, hamartin and tuberin blocked the ability of amino acids to activate S6K1 within nutrient-deprived cells, a process that is dependent on mTOR. These findings strongly implicate the tuberin-hamartin tumor suppressor complex as an inhibitor of mTOR and suggest that the formation of tumors within TSC patients may result from aberrantly high levels of mTOR-mediated signaling to downstream targets.     

Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells

Rheb is a unique member of the Ras superfamily GTP-binding proteins. We as well as others previously have shown that Rheb is a critical component of the TSC/TOR signaling pathway. In fission yeast, Rheb is encoded by the rhb1 gene. Rhb1p is essential for growth and directly interacts with Tor2p. In this article, we report identification of 22 single amino acid changes in the Tor2 protein that enable growth in the absence of Rhb1p. These mutants also exhibit decreased mating efficiency. Interestingly, the mutations are located in the C-terminal half of the Tor2 protein, clustering mainly within the FAT and kinase domains. We noted some differences in the effect of a mutation in the FAT domain (L1310P) and in the kinase domain (E2221K) on growth and mating. Although the Tor2p mutations bypass Rhb1p's requirement for growth, they are incapable of suppressing Rhb1p's requirement for resistance to stress and toxic amino acids, pointing to multiple functions of Rhb1p. In mammalian systems, we find that mammalian target of rapamycin (mTOR) carrying analogous mutations (L1460P or E2419K), although sensitive to rapamycin, exhibits constitutive activation even when the cells are starved for nutrients. These mutations do not show significant difference in their ability to form complexes with Raptor, Rictor, or mLST8. Furthermore, we present evidence that mutant mTOR can complex with wild-type mTOR and that this heterodimer is active in nutrient-starved cells.     

PPIs抑制P13K/Akt／mTOR信号通路逆转胃癌细胞的化疗多药耐药

背景：前期实验显示质子泵抑制剂（PPIs）可抑制空泡型质子泵（V-H＋-ATPases）和多药耐药蛋白P—gP、MRP1表达,增强胃癌细胞的化疗敏感性。目的：探讨PPIs抑制空泡型质子泵逆转胃癌细胞化疗多药耐药与P13K／Akt／mTOR信号通路的关系。方法：应用不同浓度埃索美拉唑或泮托拉唑预处理人胃腺癌细胞敏感株SGC7901和多药耐药株SGC7901／MDR,或以V—H＋-ATPasessiRNA干扰SGC7901／MDR细胞内的V-H＋-ATPases表达,或以雷帕霉素阻断mTOR表达,以蛋白质印迹法检测经不同方式处理的细胞内V—H＋ATPases、P—SP、MRPl蛋白表达以及P13K／Akt/mT0R／HIF—1α信号通路及其信号旁路TSCl／2-Rheb中的相关蛋白表达;以免疫荧光法检测经埃索美拉唑预处理的SGC7901／MDR细胞内的V-H＋ATPases、P—gP蛋白表达和定位。结果：PPIs可呈浓度依赖性地抑制SGC7901／MDR细胞内的V—H＋ATPases、P13K、Akt、roTOR、HIF-1仅、TSCI、TSC2、Rheb、P—gP、MRP1表达以及Akt底物和TSC2磷酸化,改变V-H＋ATPases、P—gP的胞内定位,对SGC7901细胞则无上述影响。以V—H＋-ATPasessiRNA抑制SGC7901／MDR细胞内的V—H＋-ATPases表达,作用与PPIs预处理相似。以雷帕霉素阻断mTOR可呈浓度依赖性地抑制SGC7901／MDR细胞内的HIF-1α、P—gP表达。结论：PPIs抑制空泡型质子泵逆转胃癌细胞化疗多药耐药的机制与抑制P13K／Akt／mTOR信号通路有关。     

The coordinate regulation of the p53 and mTOR pathways in cells

Cell growth and proliferation requires an intricate coordination between the stimulatory signals arising from nutrients and growth factors and the inhibitory signals arising from intracellular and extracellular stresses. Alteration of the coordination often causes cancer. In mammals, the mTOR (mammalian target of rapamycin) protein kinase is the central node in nutrient and growth factor signaling, and p53 plays a critical role in sensing genotoxic and other stresses. The results presented here demonstrate that activation of p53 inhibits mTOR activity and regulates its downstream targets, including autophagy, a tumor suppression process. Moreover, the mechanisms by which p53 regulates mTOR involves AMP kinase activation and requires the tuberous sclerosis (TSC) 1/TSC2 complex, both of which respond to energy deprivation in cells. In addition, glucose starvation not only signals to shut down mTOR, but also results in the transient phosphorylation of the p53 protein. Thus, p53 and mTOR signaling machineries can cross-talk and coordinately regulate cell growth, proliferation, and death.     

Deletion of tuberous sclerosis 1 in somatic cells of the murine reproductive tract causes female infertility

Tumors develop with dysregulated activation of mammalian target of rapamycin (mTOR), the kinase activity of which is kept in an inactive state by a tumor suppressor dimer containing tuberous sclerosis 1 (TSC1) and TSC2. We examined whether conditional deletion of TSC1 by a knock-in allele of the anti-Müllerian hormone type 2 receptor (Amhr2) driving Cre expression and subsequent activation of mTOR in granulosa cells and in oviductal and uterine stromal cells affects fertility in female mice. Increased phosphorylation of ribosomal protein S6, a downstream target of activated mTOR, was observed in all AMHR2-expressing tissues examined, indicating loss of TSC1 activity. TSC1 deletion in granulosa cells led to the detection of significantly fewer primordial follicles in mutant mice at 12 wk, suggesting premature ovarian insufficiency, which might be related to the significantly increased time mutant mice spent in estrus. Although the number of good-quality ovulated oocytes was not significantly different compared with controls, there was a significantly higher number of degenerated oocytes after normal and superovulation, suggesting compromised oocyte quality, as well. Natural mating also showed severalfold higher numbers of degenerate bodies in the mutants that collected in bilateral swellings resembling hydrosalpinges that formed in all mice examined because of occlusion of the proximal oviduct. Attempts to transfer control embryos into mutant uteri also failed, indicating that implantation was compromised. Endometrial epithelial cells continued to proliferate, and quantitative RT-PCR showed that mucin 1 expression persisted during the window of implantation in mutant uteri, without any changes in progesterone receptor mRNA expression, suggesting a mechanism that does not involve disrupted estradiol-regulated progesterone receptor expression. Homozygous deletion of TSC1 in reproductive tract somatic tissues of mice rendered females completely infertile, which is likely due to these pleiotropic effects on follicle recruitment, oviductal development, and blastocyst implantation.     

Mutant surfactant A2 proteins associated with familial pulmonary fibrosis and lung cancer induce TGF-β1 secretion

Mutations in the genes encoding the lung surfactant proteins are found in patients with interstitial lung disease and lung cancer, but their pathologic mechanism is poorly understood. Here we show that bronchoalveolar lavage fluid from humans heterozygous for a missense mutation in the gene encoding surfactant protein (SP)-A2 (SFTPA2) contains more TGF-β1 than control samples. Expression of mutant SP-A2 in lung epithelial cells leads to secretion of latent TGF-β1, which is capable of autocrine and paracrine signaling. TGF-β1 secretion is not observed in lung epithelial cells expressing the common SP-A2 variants or other misfolded proteins capable of increasing cellular endoplasmic reticulum stress. Activation of the unfolded protein response is necessary for maximal TGF-β1 secretion because gene silencing of the unfolded protein response transducers leads to an ∼50% decrease in mutant SP-A2–mediated TGF-β1 secretion. Expression of the mutant SP-A2 proteins leads to the coordinated increase in gene expression of TGF-β1 and two TGF-β1–binding proteins, LTBP-1 and LTBP-4; expression of the latter is necessary for secretion of this cytokine. Inhibition of the TGF-β autocrine positive feedback loop by a pan–TGF-β–neutralizing antibody, a TGF-β receptor antagonist, or LTBP gene silencing results in the reversal of TGF-β–mediated epithelial-to-mesenchymal transition and cell death. Because secretion of latent TGF-β1 is induced specifically by mutant SP-A2 proteins, therapeutics targeted to block this pathway may be especially beneficial for this molecularly defined subgroup of patients.     

A small-molecule scaffold induces autophagy in primary neurons and protects against toxicity in a Huntington disease model

Autophagy is an intracellular turnover pathway. It has special relevance for neurodegenerative proteinopathies, such as Alzheimer disease, Parkinson disease, and Huntington disease (HD), which are characterized by the accumulation of misfolded proteins. Although induction of autophagy enhances clearance of misfolded protein and has therefore been suggested as a therapy for proteinopathies, neurons appear to be less responsive to classic autophagy inducers than nonneuronal cells. Searching for improved inducers of neuronal autophagy, we discovered an N¹⁰-substituted phenoxazine that, at proper doses, potently and safely up-regulated autophagy in neurons in an Akt- and mTOR-independent fashion. In a neuron model of HD, this compound was neuroprotective and decreased the accumulation of diffuse and aggregated misfolded protein. A structure/activity analysis with structurally similar compounds approved by the US Food and Drug Administration revealed a defined pharmacophore for inducing neuronal autophagy. This pharmacophore should prove useful in studying autophagy in neurons and in developing therapies for neurodegenerative proteinopathies.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy

Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min after mTOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, et al. (2014) Nature 513(7518):440–443]. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth.The balance between overall rates of protein synthesis and degradation determines whether a cell grows or atrophies. It has long been proposed that overall rates of protein synthesis and degradation can be coordinately regulated (1). For example, when hormones (e.g., insulin and insulin-like growth factor-1) and nutrients are plentiful, rates of protein synthesis are high and protein degradation is suppressed, whereas in starving cells, synthesis falls and overall degradation rises. One critical factor coordinating overall synthesis and degradation is the Ser/Thr protein kinase mammalian target of rapamycin (mTOR), which promotes protein translation (2) while suppressing autophagy (lysosomal proteolysis) (3).mTOR’s pleiotropic functions are catalyzed by two distinct kinase complexes: mTORC1, the key component of which is Raptor, and mTORC2, which instead contains Rictor (4, 5). Growth factors act through class I PI3K and Akt kinases to inhibit the tumor suppressor TSC2, and thereby activate mTORC1 (6). Adequate supply of amino acids, especially leucine, can also activate mTORC1, but through a distinct mechanism involving Rag GTPase and lysosomal recruitment of mTORC1 (4). Unlike mTORC1, mTORC2 is insensitive to amino acid supply but is activated by growth factors via mechanisms that remain unclear.Rapamycin is a natural product that selectively inhibits mTORC1 but not mTORC2. Torin1 is a synthetic mTOR inhibitor that blocks ATP-binding to mTOR and thus inactivates both mTORC1 and mTORC2 (7). Both Torin1 and rapamycin inhibit overall protein synthesis, induce autophagosome formation, and thus mimic the effects of starvation. However, Torin1 is much more effective than rapamycin in affecting these two processes because rapamycin inhibits mTORC1 incompletely (7, 8). Because many types of cancer are associated with overactivation of mTOR, rapamycin and other novel mTOR inhibitors are useful in the treatment of certain cancers (9). Rapamycin is widely used in the clinic as an immune suppressor (10). Nevertheless, rapamycin extends lifespan in aged mice, perhaps by triggering similar changes as occur with dietary caloric restriction (11). Additionally, stimulating autophagy by rapamycin can help clear intracellular protein aggregates as they accumulate in many neurodegenerative diseases and reduce their toxicity (12).Eukaryotic cells degrade proteins by both the autophagy-lysosome system and the ubiquitin proteasome system (UPS). Macroautophagy delivers cytoplasmic proteins or organelles into autophagic vacuoles for degradation, and autophagosome formation is rapidly activated by starvation or mTOR inhibition (3). The UPS is responsible for the degradation of most cytosolic and nuclear proteins in mammalian cells, including the short-lived regulatory and misfolded proteins as well as the bulk of cell constituents, which are long-lived components (13, 14). Degradation by the UPS is highly selective, involving the attachment of a ubiquitin (Ub) chain to the substrate through the sequential actions of a Ub-activating enzyme (E1), Ub-conjugating enzymes (E2s), and one of the cell’s many Ub ligases (E3s) (15). Proteins conjugated to Ub chains are rapidly degraded by the 26S proteasome, whereas the Ub molecules are recycled by proteasome-associated deubiquitinating enzymes (DUBs) (16). Although the degradation of specific short-lived proteins has been studied extensively, our understanding of the global mechanisms controlling proteasomal degradation of the bulk of cell constituents is very limited.The best-characterized function of mTOR is to enhance protein translation through mTORC1-mediated phosphorylation of 4E-BPs and S6Ks (2). The simultaneous suppression of protein degradation is generally attributed to the ability of mTORC1 to inhibit autophagy, which seems to occur by the phosphorylation and inactivation of Atg1/Ulks (3). The goal of this study was to test whether the increased proteolysis upon mTOR inhibition may also occur through activation of the UPS. We demonstrate herein that mTOR inhibition not only enhances lysosomal proteolysis, but also rapidly stimulates the ubiquitination and proteasomal degradation of many proteins. We also investigated the mechanisms by which the UPS is activated, the role of mTORC1 or mTORC2, and the nature of the proteins whose stability is affected by mTOR.Together with the enhancement of autophagy, this activation of proteolysis by the UPS upon mTOR inhibition appears to represent an important adaptation to starvation that helps slow growth and provide essential amino acids. A very different role of mTORC1 in regulating protein degradation was recently proposed by Zhang et al. (17), who surprisingly reported that rapamycin did not rapidly increase proteolysis, but after 16 h actually reduced overall proteolysis by decreasing proteasome expression (17). Such a suppression is inconsistent with the well-established increase in proteolysis and autophagy in starving cells, and various observations about the regulation of the UPS (see below). Zhang et al.’s conclusions probably result from their potentially misleading pulse-chase methods to measure protein degradation and cell culture conditions (18). In this study, we have used well-validated pulse-chase approaches (14) to measure proteolysis by proteasomal and lysosomal systems after mTOR inhibition and have also monitored overall Ub conjugation and proteasome content.     

Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2–mediated suppression of mTORC1

Reactive intermediates such as reactive nitrogen species play essential roles in the cell as signaling molecules but, in excess, constitute a major source of cellular damage. We found that nitrosative stress induced by steady-state nitric oxide (NO) caused rapid activation of an ATM damage-response pathway leading to downstream signaling by this stress kinase to LKB1 and AMPK kinases, and activation of the TSC tumor suppressor. As a result, in an ATM-, LKB1-, TSC-dependent fashion, mTORC1 was repressed, as evidenced by decreased phosphorylation of S6K, 4E-BP1, and ULK1, direct targets of the mTORC1 kinase. Decreased ULK1 phosphorylation by mTORC1 at S757 and activation of AMPK to phosphorylate ULK1 at S317 in response to nitrosative stress resulted in increased autophagy: the LC3-II/LC3-I ratio increased as did GFP-LC3 puncta and acidic vesicles; p62 levels decreased in a lysosome-dependent manner, confirming an NO-induced increase in autophagic flux. Induction of autophagy by NO correlated with loss of cell viability, suggesting that, in this setting, autophagy was functioning primarily as a cytotoxic response to excess nitrosative stress. These data identify a nitrosative-stress signaling pathway that engages ATM and the LKB1 and TSC2 tumor suppressors to repress mTORC1 and regulate autophagy. As cancer cells are particularly sensitive to nitrosative stress, these data open another path for therapies capitalizing on the ability of reactive nitrogen species to induce autophagy-mediated cell death.Autophagy is a self-digestion process by which a eukaryotic cell degrades and recycles aggregate-prone proteins, macromolecules, and organelles. During autophagy, cytoplasmic contents are sequestered in double-membrane bound vesicles called autophagosomes and delivered to lysosomes for degradation, thereby allowing cells to eliminate and recycle the contents (1–3). Autophagy participates in both prosurvival (recycling of cellular building blocks) and prodeath (excess catalysis) pathways. A comprehensive understanding of signaling pathways that regulate autophagy holds great promise for new therapeutic opportunities by opening the possibility to compromise prosurvival autophagic pathways that enable tumor cells to evade therapy, or by promoting prodeath autophagic pathways to kill cancer cells.The classical pathway regulating autophagy in mammalian cells involves the serine/threonine kinase, mammalian target of rapamycin (mTOR). Active mTOR kinase in the mTORC1 complex phosphorylates and inhibits ULK1, a key proautophagy adapter involved in nucleation of the autophagophore membrane. Inactivation of mTORC1, either pharmacologically with rapamycin or via activation of the tuberous sclerosis complex (TSC) tumor suppressor, leads to downstream dephosphorylation events, including loss of ULK1 phosphorylation at S757. The TSC1/2 heterodimer is itself regulated by upstream kinases, including the AMP-activated protein kinase (AMPK), which regulates several metabolic processes and activates the TSC to repress mTORC1 under conditions of energy stress (4–6). AMPK also directly regulates autophagy by phosphorylating and activating ULK1 at S317 (7).Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes extending from its role as a chemical messenger and antibacterial agent to an integral component of the cardiovascular system and immune response to pathogens (8). NO has also been documented to play both promotional and inhibitory roles in cancer etiology. NO is produced endogenously by tumor cells of various histogenetic origins and has been associated with tumor proliferation and resistance to anticancer drugs. Although cancer cells may maintain endogenous NO at elevated levels that facilitate tumor growth, this strategy carries a survival liability, as high levels of NO can cause cytostasis or cytotoxicity (9). Thus, cancer cells may be more sensitive to NO-induced nitrosative stress, which could potentially provide a therapeutic avenue for modulating nitrosative stress to induce cell death. However, understanding at the molecular level regarding how NO participates in regulation of either prosurvival or prodeath autophagic pathways is limited.Recently, a novel signaling pathway involving activation of ATM to suppress mTORC1 in response to oxidative damage was identified (10). In the study reported here, we demonstrate that NO engages this pathway to suppress mTORC1 and promote autophagy. In response to nitrosative stress induced by steady-state exposure to NO, ATM is activated, signaling to AMPK via LKB1 to activate the TSC tumor suppressor and suppress mTORC1. Concomitant with suppression of mTORC1, autophagy is induced, accompanied by loss of cell viability. Our data provide strong evidence that NO regulates autophagy, with implications both for understanding the physiological role of NO-induced signaling and the development of therapies that can modulate nitrosative stress to kill cancer cells.     

TSC1/TSC2 and Rheb have different effects on TORC1 and TORC2 activity

Target of rapamycin (TOR) plays a central role in cell growth regulation by integrating signals from growth factors, nutrients, and cellular energy levels. TOR forms two distinct physical and functional complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). TORC1, which is sensitive to rapamycin, regulates translation and cell growth, whereas TORC2, which is insensitive to rapamycin, regulates cell morphology and cell growth. The Ras homology enriched in brain (Rheb) small GTPase is known to be a key upstream activator of TORC1, although the mechanism of Rheb in TORC1 activation remains to be determined. However, the function of Rheb in the TORC2 regulation has not been elucidated. By measuring Akt and S6K phosphorylation as a functional assay for TORC1 and -2, here, we report that dRheb has an inhibitory effect on dTORC2 activity in Drosophila S2 cells. This negative effect of dRheb on dTORC2 is possibly due to a feedback mechanism involving dTORC1 and dS6K. We also observed that Rheb does not activate TORC2 in human embryonic kidney 293 cells, although it potently stimulates TORC1. Furthermore, tuberous sclerosis complex 1 (TSC1) and TSC2, which are negative regulators of Rheb, have negative and positive effects on TORC1 and -2, respectively. Our observations suggest that TSC1/2 and Rheb have different effects on the activity of TORC1 and -2, further supporting the complexity of TOR regulation.     

Deficiencies in tRNA synthetase editing activity cause cardioproteinopathy

Misfolded proteins are an emerging hallmark of cardiac diseases. Although some misfolded proteins, such as desmin, are associated with mutations in the genes encoding these disease-associated proteins, little is known regarding more general mechanisms that contribute to the generation of misfolded proteins in the heart. Reduced translational fidelity, caused by a hypomorphic mutation in the editing domain of alanyl-tRNA synthetase (AlaRS), resulted in accumulation of misfolded proteins in specific mouse neurons. By further genetic modulation of the editing activity of AlaRS, we generated mouse models with broader phenotypes, the severity of which was directly related to the degree of compromised editing. Severe disruption of the editing activity of AlaRS caused embryonic lethality, whereas an intermediate reduction in AlaRS editing efficacy resulted in ubiquitinated protein aggregates and mitochondrial defects in cardiomyocytes that were accompanied by progressive cardiac fibrosis and dysfunction. In addition, autophagic vacuoles accumulated in mutant cardiomyocytes, suggesting that autophagy is insufficient to eliminate misfolded proteins. These findings demonstrate that the pathological consequences of diminished tRNA synthetase editing activity, and thus translational infidelity, are dependent on the cell type and the extent of editing disruption, and provide a previously unidentified mechanism underlying cardiac proteinopathy.Proteins are the building blocks and major signaling molecules of cells, and as such, their synthesis and degradation are tightly regulated. Disruption of protein homeostasis (proteostasis) can result in the accumulation of abnormal proteins that lead to cellular pathogenesis. Like the neuroproteopathies, misfolding of specific proteins in cardiomyocytes can be caused by genetic mutations that alter the primary structure of aggregated proteins. For example, mutations in the desmin gene, which encodes a muscle-specific intermediate filament protein, or in the gene encoding αB-crystallin, a molecular chaperone for desmin, result in aggregation of these proteins and are a primary cause of hereditary cardiomyopathy (1, 2). Systemic amyloidosis, the extracellular accumulation of abnormal proteinaceous fibrils, can also occur in the heart with detrimental effects on cardiac function (3, 4). In addition, misfolded proteins and changes in the ubiquitin proteasome system or autophagy have been widely reported in failing hearts (4, 5), demonstrating the importance of protein quality control systems for cardiomyocyte homeostasis. However, whether such proteostatic changes induce pathological changes, or are consequences of the diseased state, is not clear.In addition to genetic mutations, defective proteins can be generated by inaccurate translation. Translational fidelity is largely controlled by the precise aminoacylation of tRNAs with their cognate amino acids, a function carried out by the aminoacyl-tRNA synthetases (aaRSs). To improve substrate specificity, an editing site, which hydrolyses misactivated noncognate amino acids or mischarged tRNAs and is distinct from the aminoacylation domain, is found in approximately half of the aaRSs (6, 7). Failure of proofreading by these aaRSs may result in incorporation of the wrong amino acid into the nascent peptide, which could be detrimental to protein folding and function.Although errors in aminoacylation are relatively low (8, 9), even small decreases in aaRS proofreading have dramatic effects on cell survival (10–12). Bacterial alanyl-tRNA synthetase (AlaRS) can misactivate glycine or serine, and Escherichia coli expressing a severe editing-deficient form (C666A) of AlaRS had increased death when grown in elevated concentrations of these amino acids (11). In mice, a point mutation (A734E) in the AlaRS editing domain in the mutant strain “sticky” caused a twofold increase in Ser-mischarged tRNA^Ala that resulted in formation of ubiquitinated protein aggregates in cerebellar Purkinje cells and degeneration of these neurons (10). However, because cell loss in other regions of the brain or in other tissues was not observed in the sti mutant mouse, the importance of AlaRS editing activity in other mammalian cell types remains unknown.Here we hypothesized that further decreases in AlaRS editing function could lead to misfolded protein accumulation in additional cell types. Indeed mouse embryos homozygous for a point mutation at C723 (which corresponds to the C666 amino acid in the editing domain of bacterial AlaRS) died by midgestation. Furthermore, decreasing the amount of the sti-associated AlaRS^A734E enzyme or placing the sti mutation and the more severe AlaRS (Aars^C723A) mutation in trans caused extensive aggregation of misfolded proteins in mouse cardiomyocytes, eventually leading to cardiomyopathy. Using this proteome-wide mistranslation system, we demonstrate that different cell types are distinctly sensitive to errors in protein synthesis and that mistranslation can induce cardiac proteinopathy, which leads to cardiac fibrosis and decrements in heart function.     

Tsc1 regulates tight junction independent of mTORC1

Tuberous sclerosis complex 1 (Tsc1) is a tumor suppressor that functions together with Tsc2 to negatively regulate the mechanistic target of rapamycin complex 1 (mTORC1) activity. Here, we show that Tsc1 has a critical role in the tight junction (TJ) formation of epithelium, independent of its role in Tsc2 and mTORC1 regulation. When an epithelial cell establishes contact with neighboring cells, Tsc1, but not Tsc2, migrates from the cytoplasm to junctional membranes, in which it binds myosin 6 to anchor the perijunctional actin cytoskeleton to β-catenin and ZO-1. In its absence, perijunctional actin cytoskeleton fails to form. In mice, intestine-specific or inducible, whole-body Tsc1 ablation disrupts adherens junction/TJ structures in intestine or skin epithelia, respectively, causing Crohn’s disease–like symptoms in the intestine or psoriasis-like phenotypes on the skin. In patients with Crohn’s disease or psoriasis, junctional Tsc1 levels in epithelial tissues are markedly reduced, concomitant with the TJ structure impairment, suggesting that Tsc1 deficiency may underlie TJ-related diseases. These findings establish an essential role of Tsc1 in the formation of cell junctions and underpin its association with TJ-related human diseases.

Epithelium is a thin tissue covering the body surface, lining alimentary spaces, and other structures inside the body. It is composed of a layer of attached epithelial cells, such that it blocks the paracellular diffusion of solutes and water, as well as preventing infectious microorganisms entering the body (1). This paracellular blockage is achieved by a tripartite apical junctional complex, which constitutes tight junctions (TJs), adherens junctions (AJs), and desmosomes arranged in sequential order, from the apical end to the basal end of the junction. In this arrangement, TJs establish barrier functions. Consequently, TJ dysfunction is associated with a myriad of human diseases, including Crohn’s disease, ulcerative colitis, celiac disease (leak-flux diarrhea), cystic fibrosis, atopic dermatitis (AD), and psoriasis (1–3).TJs are composed of networks of strands formed by transmembrane proteins. The extracellular domains of these membrane proteins are tethered together, and their cytoplasmic domains are anchored to the actin cytoskeleton via cytoplasmic scaffolding proteins. More than 40 different proteins have been found in TJs, including transmembrane proteins, claudins, junctional adhesion molecules, coxsackie adenovirus receptors, and TJ-associated marvel proteins, such as occludin, tricellulin, marvelD3 proteins, and cytoplasmic scaffolding proteins of the ZO family (4–7). While AJ and TJ structural components and organization are well studied, the mechanisms controlling their assembly and stability of established adhesive contacts remain unclear. Several studies have shown that AJ formation precedes TJ and is essential for TJ formation (8, 9). The attachment of the cadherin α-catenin–β-catenin adhesion complex to perijunctional cortex actin filaments establishes AJs (10, 11). The subsequent recruitment of ZO-1 to the α-catenin–β-catenin complex is believed to initiate TJ formation from the existing AJs (12).Tsc1 (hamartin) is a tumor suppressor protein encoded by TSC1, a causative gene for tuberous sclerosis complex (TSC) syndrome (13–16). Tsc1 functions with Tsc2, a GTPase-activating protein (GAP), to restrict Rheb activation, a Ras-like small GTPase and activator of mechanistic targets of rapamycin complex 1 (mTORC1). Tsc1 binds Tsc2 directly to stabilize the latter, preventing it from proteasomal degradation (17, 18). The GAP activity of Tsc2 leads to Rheb inactivation and subsequent mTORC1 inhibition (19, 20). mTORC1 is a central signaling hub controlling cell growth, metabolism, survival, and autophagy in response to nutrient availability and growth factors (21–25). Its abnormal activation in Tsc1- and Tsc2-deficient cells is believed to be the main pathogenic cause behind TSC syndrome (20, 26, 27).While studying the function of Tsc1 in intestinal epithelial cells, we serendipitously observed that intestinal, epithelial-specific Tsc1 ablation caused symptoms and histopathological alterations in mice, commonly associated with TJ defects (28, 29). This observation led us to investigate a previously unknown role of Tsc1 in TJs. Here, we show that Tsc1 is a key regulator of cell–cell adhesion that controls TJ formation independent of its role in mTORC1 regulation. Reduced Tsc1 levels at the junctional membrane are associated with TJ-related diseases in humans.