The mTORC1 pathway stimulates glutamine metabolism and cell proliferation by repressing SIRT4.

Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mammalian target of rapamycin complex 1 (mTORC1) activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP-responsive element binding 2 (CREB2). Thus, a relationship between mTORC1, SIRT4, and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation, and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach.

Metabolic stress controls mTORC1 lysosomal localization and dimerization by regulating the TTT-RUVBL1/2 complex.

The metabolism of glucose and glutamine, primary carbon sources utilized by mitochondria to generate energy and macromolecules for cell growth, is directly regulated by mTORC1. We show that glucose and glutamine, by supplying carbons to the TCA cycle to produce ATP, positively feed back to mTORC1 through an AMPK-, TSC1/2-, and Rag-independent mechanism by regulating mTORC1 assembly and its lysosomal localization. We discovered that the ATP-dependent TTT-RUVBL1/2 complex was disassembled and repressed by energy depletion, resulting in its decreased interaction with mTOR. The TTT-RUVBL complex was necessary for the interaction between mTORC1 and Rag and formation of mTORC1 obligate dimers. In cancer tissues, TTT-RUVBL complex mRNAs were elevated and positively correlated with transcripts encoding proteins of anabolic metabolism and mitochondrial function-all mTORC1-regulated processes. Thus, the TTT-RUVBL1/2 complex responds to the cell's metabolic state, directly regulating the functional assembly of mTORC1 and indirectly controlling the nutrient signal from Rags to mTORC1.

HIV-mediated phosphatidylinositol 3-kinase/serine-threonine kinase activation in APCs leads to programmed death-1 ligand upregulation and suppression of HIV-specific CD8 T cells.

Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.

ATM: Promoter of metabolic "cost" reduction and "savings" usage during hypoxia through mTORC1 regulation.

Cells must sense environmental conditions and adjust to maintain metabolic homeostasis and survive stress conditions; in this issue, Cam et al. (2010) show that the tumor suppressor kinase ATM is activated by hypoxia, phosphorylates and stabilizes HIF-1α, and inhibits mTORC1.

DNA-based influenza vaccines: evaluating their potential to provide universal protection.

The recent outbreaks of the H5N1 and H1N1 pandemic influenza have highlighted the importance of developing fast, effective therapeutic strategies to prevent and/or limit the spread of future influenza outbreaks. Although current vaccines against influenza are generally effective, several limitations, including those associated with the amount of available vaccine, the time to vaccine production and vaccine efficacy, may encumber a mass vaccination strategy and effective targeting against future outbreaks. This feature review discusses the prospects of SynCon-derived DNA vaccines against influenza; such vaccines are expected to be effective at targeting many currently circulating influenza virus strains, as well as potentially targeting strains that may be associated with future outbreaks. Because of advantages associated with safety, time to production and ease of production, as well as the generation of more effective immune responses, influenza DNA vaccines provide a promising potential solution to a global medical concern.

Glucose addiction of TSC null cells is caused by failed mTORC1-dependent balancing of metabolic demand with supply.

The mTORC1-signaling pathway integrates environmental conditions into distinct signals for cell growth by balancing anabolic and catabolic processes. Accordingly, energetic stress inhibits mTORC1 signaling predominantly through AMPK-dependent activation of TSC1/2. Thus, TSC1/2-/- cells are hypersensitive to glucose deprivation, and this has been linked to increased p53 translation and activation of apoptosis. Herein, we show that mTORC1 inhibition during glucose deprivation prevented not only the execution of death, but also induction of energetic stress. mTORC1 inhibition during glucose deprivation decreased AMPK activation and allowed ATP to remain high, which was both necessary and sufficient for protection. This effect was not due to increased catabolic activities such as autophagy, but rather exclusively due to decreased anabolic processes, reducing energy consumption. Specifically, TSC1/2-/- cells become highly dependent on glutamate dehydrogenase-dependent glutamine metabolism via the TCA cycle for survival. Therefore, mTORC1 inhibition during energetic stress is primarily to balance metabolic demand with supply.

HIV-1 Vpr: regulator of viral survival.

The HIV-1 Vpr protein is a viral accessory protein that plays a number of important roles during HIV infection. The activities of Vpr are numerous and include the induction of apoptosis, the modulation of cell cycle arrest, as well as control of viral transcription. Study of HIV clones lacking Vpr in vitro and analysis of HIV variants isolated from long-term nonprogressors in vivo highlight the importance of Vpr for viral replication as well as immune suppression and cell death. Vpr may therefore be considered among the most important accessory proteins encoded by HIV.

Not all substrates are treated equally: implications for mTOR, rapamycin-resistance and cancer therapy.

The mTORC1 signaling pathway is a critical regulator of cell growth and is hyper activated in many different cancers. Rapamycin, an allosteric inhibitor of mTORC1, has been approved for treatment against renal cell carcinomas and is being evaluated for other cancers. Mechanistically, mTORC1 controls cell growth in part through its two well-characterized substrates S6K1 and 4E-BP1. In this review, we discuss the implications of a recent finding that showed differential inhibition of S6K1 and 4E-BP1 by rapamycin, leading to cell-type-specific repression of cap-dependent translation. We discuss potential mechanisms for this effect, and propose that mTOR-specific kinase inhibitors, instead of rapamycin, should be considered for mTOR-targeted cancer therapy.

Anti-tumor activity mediated by protein and peptide transduction of HIV viral protein R (Vpr).

Peptides that are capable of traversing the cell membrane, via protein transduction domains (PTDs), are attractive either directly as drugs or indirectly as carriers for the delivery of therapeutic molecules. For example, an HIV-1 Tat derived peptide has successfully delivered a large variety of "cargoes" including proteins, peptides and nucleic acids into cells when conjugate to the PTD. There also exists other naturally occurring membrane permeable peptides which have potential as PTDs. Specifically, one of the accessory proteins of HIV (viral protein R; i.e., Vpr), which is important in controlling viral pathogenesis, possesses cell transduction domain characteristics. Related to these characteristics, Vpr has also been demonstrated to induce cell cycle arrest and host/target cell apoptosis, suggesting a potential anti-cancer activity for this protein. In this report we assessed the ability of Vpr protein or peptides, with or without conjugation to a PTD, to mediate anti-cancer activity against several tumor cell lines. Specifically, several Vpr peptides spanning carboxy amino acids 65-83 induced significant (i.e., greater than 50%) in vitro growth inhibition/toxicity of murine B16.F10 melanoma cells. Likewise, in in vitro experiments with other tumor cell lines, conjugation of Vpr to the Tat derived PTD and transfection of this construct into cells enhanced the induction of in vitro apoptosis by this protein when compared to the effects of transfection of cells with unconjugated Vpr. These results underscore the potential for Vpr based reagents as well as PTDs to enhance anti-tumor activity, and warrants further examination of Vpr protein and derived peptides as potential therapeutic agents against progressive cell proliferative diseases such as cancer.

Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation.

The mammalian translational initiation machinery is a tightly controlled system that is composed of eukaryotic initiation factors, and which controls the recruitment of ribosomes to mediate cap-dependent translation. Accordingly, the mTORC1 complex functionally controls this cap-dependent translation machinery through the phosphorylation of its downstream substrates 4E-BPs and S6Ks. It is generally accepted that rapamycin, a specific inhibitor of mTORC1, is a potent translational repressor. Here we report the unexpected discovery that rapamycin's ability to regulate cap-dependent translation varies significantly among cell types. We show that this effect is mechanistically caused by rapamycin's differential effect on 4E-BP1 versus S6Ks. While rapamycin potently inhibits S6K activity throughout the duration of treatment, 4E-BP1 recovers in phosphorylation within 6 h despite initial inhibition (1-3 h). This reemerged 4E-BP1 phosphorylation is rapamycin-resistant but still requires mTOR, Raptor, and mTORC1's activity. Therefore, these results explain how cap-dependent translation can be maintained in the presence of rapamycin. In addition, we have also defined the condition by which rapamycin can control cap-dependent translation in various cell types. Finally, we show that mTOR catalytic inhibitors are effective inhibitors of the rapamycin-resistant phenotype.

Human immunodeficiency virus type 1 Nef induces programmed death 1 expression through a p38 mitogen-activated protein kinase-dependent mechanism.

Chronic viral infection is characterized by the functional impairment of virus-specific T-cell responses. Recent evidence has suggested that the inhibitory receptor programmed death 1 (PD-1) is specifically upregulated on antigen-specific T cells during various chronic viral infections. Indeed, it has been reported that human immunodeficiency virus (HIV)-specific T cells express elevated levels of PD-1 and that this expression correlates with the viral load and inversely with CD4(+) T-cell counts. More importantly, antibody blockade of the PD-1/PD-L1 pathway was sufficient to both increase and stimulate virus-specific T-cell proliferation and cytokine production. However, the mechanisms that mediate HIV-induced PD-1 upregulation are not known. Here, we provide evidence that the HIV type 1 (HIV-1) accessory protein Nef can transcriptionally induce the expression of PD-1 during infection in vitro. Nef-induced PD-1 upregulation requires its proline-rich motif and the activation of the downstream kinase p38. Further, inhibition of Nef activity by p38 MAPK inhibitor effectively blocked PD-1 upregulation, suggesting that p38 MAPK activation is an important initiating event in Nef-mediated PD-1 expression in HIV-1-infected cells. These data demonstrate an important signaling event of Nef in HIV-1 pathogenesis.

Mind the GAP: Wnt steps onto the mTORC1 train.

The TSC1/2 tumor-suppressor complex controls protein synthesis through the regulation of mTOR. In this issue of Cell, Inoki et al. (2006) report that the kinases GSK3 and AMPK cooperate in the activation of TSC2 to inhibit mTOR activity. Surprisingly, the phosphorylation of TSC2 by GSK3 is markedly suppressed by Wnt signaling. This suggests that components of the mTOR pathway may be therapeutic targets for diseases linked to hyperactive Wnt signaling.

TORgeting oncogene addiction for cancer therapy.

The PI3K-Akt-mTOR growth-regulating pathway is conserved from mammals to flies and hyperactivated in many cancers. Accordingly, rapamycin analogs, which are inhibitors of mTOR-Raptor signaling, have recently garnered much interest as potential therapeutic agents against cancer. However, due to the heterogeneity of tumors, prior knowledge of the genetic and biochemical background of cancer cells will be required for effective targeted therapy. Thus, the identification of biological markers against activated oncogenic pathways is needed. In the January issue of Nature Medicine, Thomas et al. identify the loss of VHL tumor suppressor gene as a potential determining factor in tumor sensitivity to rapamycin.

The HIV-1 Vpr and glucocorticoid receptor complex is a gain-of-function interaction that prevents the nuclear localization of PARP-1.

The Vpr protein of HIV-1 functions as a vital accessory gene by regulating various cellular functions, including cell differentiation, apoptosis, nuclear factor of kappaB (NF-kappaB) suppression and cell-cycle arrest of the host cell. Several reports have indicated that Vpr complexes with the glucocorticoid receptor (GR), but it remains unclear whether the GR pathway is required for Vpr to function. Here, we report that Vpr uses the GR pathway as a recruitment vehicle for the NF-kappaB co-activating protein, poly(ADP-ribose) polymerase-1 (PARP-1). The GR interaction with Vpr is both necessary and sufficient to facilitate this interaction by potentiating the formation of a Vpr-GR-PARP-1 complex. The recruitment of PARP-1 by the Vpr-GR complex prevents its nuclear localization, which is necessary for Vpr to suppress NF-kappaB. The association of GR with PARP-1 is not observed with steroid (glucocorticoid) treatment, indicating that the GR association with PARP-1 is a gain of function that is solely attributed to HIV-1 Vpr. These data provide important insights into Vpr biology and its role in HIV pathogenesis.

Mapping of immune responses following wild-type and mutant ABeta42 plasmid or peptide vaccination in different mouse haplotypes and HLA Class II transgenic mice.

Although the recent clinical trial of the ABeta42 peptide vaccine against Alzheimer's Disease (AD) has been halted due to adverse events, the apparent clinical utility of this approach underscores the need to further improve the safety of the vaccine, as well as to understand the potential immunological basis for complications. In this study, we examine both humoral and cellular immune responses elicited by immunization with peptide or DNA encoding wild-type and the Flemish and Dutch mutations of ABeta42 (i.e. the beta amyloid peptide spanning amino acids 1-42) in mice of different immune haplotypes as well as HLA Class II transgenic mice. The Flemish and Dutch mutations have been associated with cerebrovascular hemorrhages in affected individuals. These data allow determination of potential immunological responses that could mediate pathology observed with mutant forms of amyloid beta, as well as lead to the generation of safer vaccine preparations. Following peptide or plasmid immunization, antibody responses were measured against the different ABeta42 peptides in an ELISA assay, while T cell epitopes were analyzed through interferon gamma ELISPOT and lymphocyte proliferation assays. B cell mapping studies indicated that sera from all of the haplotype mice vaccinated with any of the ABeta42 peptides reacted specifically to the first 10 amino acids of ABeta42 with the ABeta42 mutants eliciting higher immune responses. ELISPOT analysis, which accessed cellular immune responses indicated that mice expressed differences in Class I epitopes dependent on the different immune haplotypes. These results may have implications for the design of future ABeta42 based vaccines against Alzheimer's Disease.

HIV-1 Viral protein-r (Vpr) protects against lethal superantigen challenge while maintaining homeostatic T cell levels in vivo.

The HIV-1 accessory protein Vpr exhibits many interesting features related to macrophage and T cell biology. As a viral protein or as a soluble molecule it can suppress immune cell activation and cytokine production in vitro in part by targeted inhibition of NF-kappaB. In this regard we sought to test its effects in vivo on an NF-kappaB-dependent immune pathway. We examined the activity of Vpr in a lethal toxin-mediated challenge model in mice. Intravenous delivery of Vpr was sufficient to protect mice from lethal challenge with staphylococcal endotoxin B (SEB). Furthermore, Vpr protected host CD4+ T cells from in vivo depletion likely by preventing induction of AICD of SEB-exposed cells in a post-toxin-binding fashion. Understanding the biology of Vpr's activities in this model may allow for new insight into potential mechanisms of hyperinflammatory disease and into Vpr pathobiology in the context of HIV infection.