Increased Protein Translation Research Articles

Exposure of Pancreatic β-Cells to Excess Glucose Results in Bimodal Activation of mTORC1 and mTOR-Dependent Metabolic Acceleration.

SummaryChronic exposure of pancreatic β-cells to excess glucose can lead to metabolic acceleration and loss of stimulus-secretion coupling. Here, we examined how exposure to excess glucose (defined here as concentrations above 5 mM) affects mTORC1 signaling and the metabolism of β-cells. Acute exposure to excess glucose stimulated glycolysis-dependent mTORC1 signaling, without changes in the PI3K or AMPK pathways. Prolonged exposure to excess glucose led to hyperactivation of mTORC1 and metabolic acceleration, characterized by higher basal respiration and maximal respiratory capacity, increased energy demand, and enhanced flux through mitochondrial pyruvate metabolism. Inhibition of pyruvate transport to the mitochondria decelerated the metabolism of β-cells chronically exposed to excess glucose and re-established glucose-dependent mTORC1 signaling, disrupting a positive feedback loop for mTORC1 hyperactivation. mTOR inhibition had positive and negative impacts on various metabolic pathways and insulin secretion, demonstrating a role for mTOR signaling in the long-term metabolic adaptation of β-cells to excess glucose.

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway.

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its nonstructural protein NS3 (BTV-NS3), is able to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eukaryotic translation initiation factor 4E (eIF4E). By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease in the MAPK/ERK activation by BTV, supporting a model wherein BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the alpha/beta interferon response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel a new essential function of NS3 during BTV infection.IMPORTANCE Bluetongue virus (BTV) is responsible of the arthropod-borne disease bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its nonstructural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway, and this activation is BRAF dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.

Maintenance of Nucleolar Homeostasis by CBX4 Alleviates Senescence and Osteoarthritis

CBX4, a component of polycomb repressive complex 1 (PRC1), plays important roles in the maintenance of cell identity and organ development through gene silencing. However, whether CBX4 regulates human stem cell homeostasis remains unclear. Here, we demonstrate that CBX4 counteracts human mesenchymal stem cell (hMSC) aging via the maintenance of nucleolar homeostasis. CBX4 protein is downregulated in aged hMSCs, whereas CBX4 knockout in hMSCs results in destabilized nucleolar heterochromatin, enhanced ribosome biogenesis, increased protein translation, and accelerated cellular senescence. CBX4 maintains nucleolar homeostasis by recruiting nucleolar protein fibrillarin (FBL) and heterochromatin protein KRAB-associated protein 1 (KAP1) at nucleolar rDNA, limiting the excessive expression of rRNAs. Overexpression of CBX4 alleviates physiological hMSC aging and attenuates the development of osteoarthritis in mice. Altogether, our findings reveal a critical roleof CBX4 in counteracting cellular senescence by maintaining nucleolar homeostasis, providing a potential therapeutic target for aging-associated disorders.

Abstract 449: Targeting PKR-like endoplasmic reticulum kinase modulates metabolism to promote T-cell effector function and PD1 immunotherapy responsiveness

Abstract The development of immunotherapy is a major recent advance in clinical oncology. However the majority of patients do not respond and those who do, experience resistance and relapse. Therefore, understanding the molecular mechanisms of immune checkpoint inhibitor resistance is critical to develop combinatorial drug strategies to potentiate therapeutic responsiveness to reduce mortality. Highly secretory cells, such as T-cells, have a larger endoplasmic reticulum (ER) to handle the increased protein translation capacity required by the cell. Therefore, T-cells may be highly sensitive to ER stress. The high nutrient needs of the tumor deplete resources in the microenvironment subjugating infiltrating T-cells to reduced nutrient availability resulting in stress. Our data indicate that co-culturing melanoma cells with T-cells increase T-cell specific unfolded protein response (UPR) signaling. Stimulating ER stress decreased cytolytic T-cell function in TALL-104 T-cells, enabling Mun2b melanoma survival. Knockdown of PERK restored T-cell killing capacity in ER stress induced cells. In an ex-vivo model of antigen-specific mediated T-cell death, Pmel-1 T-cells stimulated with gp100 displayed increased B16 melanoma killing when treated with GSK2606414 (a small molecule PERK inhibitor). PERK inhibition also elevated glycolysis proteins and mitochondrial bioenergetics in T-cells, suggesting PERK inhibition increases T-cell metabolism. As proof-of-concept, we determined the effect of PERK inhibition in a syngeneic B16 melanoma model. Male C57/Bl6 mice were inoculated with B16 melanoma cells. At 5 days post injection, mice were treated with a control IgG, antisense morpholino targeting PERK, PD1 antibody, or a combination of PERK morpholino and PD1 antibody. PERK morpholino treatment alone was sufficient to reduce tumor volume by 46.5%. PD1 antibody therapy alone reduced tumor volume by 47.4%. The combination of PD1 antibody and PERK targeting therapy significantly reduced B16 melanoma tumor volume by 64.5% demonstrating that targeting PERK in vivo enhanced immunocheckpoint therapy efficacy. Treated B16 tumors were homogenized and infiltrating T-cells isolated by flow cytometry. Gated CD3+ infiltrating leukocytes were then counted for CD8 and PD1 expression. Tumor infiltrating CD8+ T-cells doubled with PERK inhibition alone, giving further proof indicating that PERK may be a novel immune checkpoint target. Small molecule PERK inhibitors were previously shown to cause β-islet damage and insulin resistance; therefore we tested the effect of our PERK morpholino on glucose tolerance in the B16 melanoma model. PERK morpholino treatment had no overall impact on glucose tolerance and therefore may be a novel therapeutic to induce T-cell metabolism and potentiate immune checkpoint therapy efficacy with reduced off-target toxicities. Citation Format: David R. Soto-Pantoja, Yismeilin R. Feliz-Mosquea, Kenysha YJ Clear, Adam S. Wilson, Katherine L. Cook. Targeting PKR-like endoplasmic reticulum kinase modulates metabolism to promote T-cell effector function and PD1 immunotherapy responsiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 449.

Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells.

Increased protein translation in cells and various factors in the tumor microenvironment can induce endoplasmic reticulum (ER) stress, which initiates the unfolded protein response (UPR). We have previously reported that factors released from cancer cells mounting a UPR induce a de novo UPR in bone marrow-derived myeloid cells, macrophages, and dendritic cells that facilitates protumorigenic characteristics in culture and tumor growth in vivo. We investigated whether this intercellular signaling, which we have termed transmissible ER stress (TERS), also operates between cancer cells and what its functional consequences were within the tumor. We found that TERS signaling induced a UPR in recipient human prostate cancer cells that included the cell surface expression of the chaperone GRP78. TERS also activated Wnt signaling in recipient cancer cells and enhanced resistance to nutrient starvation and common chemotherapies such as the proteasome inhibitor bortezomib and the microtubule inhibitor paclitaxel. TERS-induced activation of Wnt signaling required the UPR kinase and endonuclease IRE1. However, TERS-induced enhancement of cell survival was predominantly mediated by the UPR kinase PERK and a reduction in the abundance of the transcription factor ATF4, which prevented the activation of the transcription factor CHOP and, consequently, the induction of apoptosis. When implanted in mice, TERS-primed cancer cells gave rise to faster growing tumors than did vehicle-primed cancer cells. Collectively, our data demonstrate that TERS is a mechanism of intercellular communication through which tumor cells can adapt to stressful environments.

Abstract B43: Global upregulation of ribosomal protein gene pathway in normal cerebellar progenitor development and in pathogenesis of sonic hedgehog subtype medulloblastomas

Abstract Co-regulated transcription of ribosomal protein genes (RPGs) is at the heart of efficient ribosome biogenesis; however, mechanisms that regulate RPG pathways in relation to normal vertebrate development and disease are not well understood. Granule progenitors in the external granule layer (EGL) of the developing mouse cerebellum show a sharp proliferative spike during the first two weeks after birth in a sonic hedgehog (Shh)-dependent manner. Medulloblastomas (MBs) constitute the most common malignant pediatric brain tumor originating in the cerebellum. In particular, Shh subtype MBs result from unregulated proliferation of granule progenitors, and understanding mechanisms underlying their proliferative capacity can provide insights into MB pathogenesis and therapeutics. Interestingly, increased ribosomal protein (RP) levels in MBs have been associated with poor patient survival. Here, we describe an unexpected role of global upregulation of the RPG pathway in normal cerebellar granule progenitor development and in pathogenesis of Shh subtype MBs. Using unbiased RNA-seq analysis, we detected global transcriptional upregulation of RPG pathway in mouse cerebellum during postnatal development compared to adults. In situ hybridization and immunohistochemistry of individual RPs demonstrated increased transcript and protein levels of individual RPs specifically in the EGL, respectively. Increased RP levels in EGL progenitors resulted predominantly by transcriptional mechanisms, as opposed to post-mitotic purkinje neurons showing selective upregulation of RPG protein but not transcript levels. Translational analysis of nascent polypeptide chains by incorporation of O-propargyl-puromycin by flow cytometry and immunofluorescence revealed increased protein translation in EGL with respect to rest of the neonatal cerebellum, suggesting functional ribosomal biogenesis in addition to global upregulation of RPG pathway. To examine if reducing RP levels affect normal tissue homeostasis, we analyzed brain and cerebellum development in a hypomorphic allele of an individual RP (Rpl24 Bst/+). Although RPG pathway was transcriptionally downegulated in Rpl24 Bst/+ neonatal cerebellum with respect to wild type, gross cerebellar morphology and granule neuron numbers in adult animals were not affected. In addition, neurogenesis in the subventricular zone of the lateral ventricle and subgranular zone of the dentate gyrus of Rpl24 Bst/+ mice were not affected. Thus, partial reduction of RPs does not impact upon normal brain development. Interestingly, Shh subtype MBs in mice demonstrated global upregulation of RPG transcripts and increased individual protein levels in comparison to postnatal cerebellum, which already has higher levels of RPG pathway compared to adults. Human Shh-MB samples also demonstrated higher RP levels with respect to normal cerebellar tissue. Analysis of RNA-seq datasets in TCGA suggested that global transcriptional upregulation of the RPG pathway is not a common feature of all human tumors, in particular only ~50% of tumor subtypes showed global upregulation. However, global upregulation of RPG pathway in Shh-MBs is remarkably significant both in the extent and severity of the increase. Interestingly, we observed reduced tumor size in mice with Rpl24 hypomorphism compared with control mice expressing only constitutively active Smoothened (SmoM2) in neural stem/progenitors, suggesting that the RPG pathway determines tumorigenic burden in Shh-MBs. Thus, unlike cerebellar progenitor development, Shh-MBs show dependency on global RPG transcription. Characterizing the RPG pathway in human MB subtypes with respect to patient prognosis and tumor recurrence, and/or therapeutic strategies targeting ribosomal biogenesis might be transformative in developing effective Shh-MB therapies. Citation Format: Issei Shimada, Somatilaka Bandarigoda, John Shelton, Zhenyu Xuan, Veena Rajaram, Saikat Mukhopadhyay. Global upregulation of ribosomal protein gene pathway in normal cerebellar progenitor development and in pathogenesis of sonic hedgehog subtype medulloblastomas. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B43.

IL-3 up-regulates and activates human eosinophil CD32 and αMβ2 integrin causing degranulation.

Eosinophils contribute to the pathogenesis of multiple diseases, including asthma. Treatment with antibodies targeting IL-5 or IL-5 receptor α reduces the frequency of asthma exacerbations. Eosinophil receptors for IL-5 share a common ß-chain with IL-3 and GM-CSF receptors. We recently reported that IL-3 is more potent than IL-5 or GM-CSF in maintaining the ERK/p90S6K/RPS6 ribosome-directed signaling pathway, leading to increased protein translation. We aimed to determine disease-relevant consequences of prolonged eosinophil stimulation with IL-3. Human blood eosinophils were used to establish the impact of activation with IL-3 on IgG-driven eosinophil degranulation. When compared to IL-5, continuing exposure to IL-3 further induced degranulation of eosinophils on aggregated IgG via increased production and activation of both CD32 (low affinity IgG receptor) and αMß2 integrin. In addition, unlike IL-5 or GM-CSF, IL-3 induced expression of CD32B/C (FCGRIIB/C) subtype proteins, without changing CD32A (FCGRIIA) protein and CD32B/C mRNA expression levels. Importantly, these in vitro IL-3-induced modifications were recapitulated in vivo on airway eosinophils. We observed for the first time upregulation of CD32B/C on eosinophils, and identified IL-3 as a potent inducer of CD32- and αMß2-mediated eosinophil degranulation.

ONC212 Is a Potent Member of the Imipridone Class of Anti-Cancer Compounds That Induces p53-Independent Apoptosis in Hematological Malignancies

The functional or genetic inactivation of p53 hampers human tumor treatment. Therefore, novel therapeutic strategies are needed. ONC201 is a p53-independent inducer of apoptosis that is the founding member of the imipridone class of novel anti-cancer compounds, which possess a unique pharmacophore. We discovered that ONC201 exerts anti-tumor effects via ATF4 induction through activation of an atypical integrated stress response (ISR) (Ishizawa et al. and Kline et al, Sci Signal, 2016). Several clinical trials of ONC201 are ongoing in advanced cancers, showing a promising safety profiling and signs of clinical activity in both solid tumors and hematopoietic malignancies. In this study, we investigated the effects of ONC212, which has emerged as a highly potent member of the imipridone family, in preclinical models of hematological malignancies.ONC212 exerted potent and prominent apoptogenic effects on acute myeloid leukemia (AML) and mantle cell lymphoma (MCL) cell lines (e.g., ED50s of 141.0 nM in p53 wild-type OCI-AML3 cells, 105.7 nM in MOLM13 cells, and 265.2 nM in p53-null JeKo-1 cell lines). Time course analysis of apoptosis in OCI-AML3 cells showed that ONC212 takes more than 36 hours to start to induce apoptosis, which is similar to observations with ONC201. Next, we further examined similarities between ONC212 and ONC201 by evaluating the in vitro efficacy of ONC212 in ONC201-resistant (ONC201-R) cell lines that we have generated by chronic exposure of MCL and AML cell lines to ONC201, of which ED50s for ONC201 treatment at 72 hrs were all > 5 μM. Interestingly, the ONC201-R cell lines were more resistant to ONC212 than the isogenic ONC201-naïve cells (Figure 1), indicating that these cell lines are cross-resistant to ONC212.We previously proved that increased protein translation of the transcription factor ATF4 is one of the major molecular events involved in ONC201-induced apoptosis (Ishizawa et al., Sci Signal, 2016). Similarly, ATF4 protein abundance was increased by 24-hour treatment with ONC212. DDIT3 (CHOP) gene, a target of ATF4, was transcriptionally upregulated in parallel with its target genes GADD34, DR5 and TRIB3 in ONC212-treated JeKo-1 and OCI-AML3 cells by 24 hrs after treatment (Figure 2). Of note, ONC201 was reported to transcriptionally induce TRAIL in a p53-independent manner in solid tumors (Allen et al., Sci Transl Med, 2013), but it was not operational in hematological cell lines (Ishizawa et al., Sci Sig 2016). Consistently, we also confirmed that ONC212 does not increase TRAIL mRNA in MCL (JeKo-1) and AML (OCI-AML3) cells.BCL-2 is a protective factor for cells under endoplasmic reticulum stress, which is one way to activate ISR. Therefore, we investigated whether the BCL-2 inhibitor ABT-199 sensitizes hematopoietic malignant cells to ONC212. Apoptosis was significantly higher in the combination than either drug alone in MCL and AML cell lines even in THP-1 and OCI-AML3 cells that are relatively resistant to ONC201/212 and/or ABT-199 (Figure 3), suggesting that this combination could overcome the resistance to either of agents. Indeed, the combination was also synergistic in the OCI-AML3 ONC201-R cell line (Figure 3).Taken together, our preclinical studies suggest that ONC212 is a promising and potent new member of the impridone class of anti-cancer compounds that warrants further development in hematological malignancies. The combination of ONC212 with ABT-199 is attractive, considering that acquired resistance after a short-term response remains a clinical challenge with ABT-199. [Display omitted] [Display omitted] DisclosuresKonopleva:AbbVie: Research Funding; Genentech: Research Funding. Allen:Oncoceutics Inc.: Employment. Stogniew:Oncoceutics Inc.: Employment, Equity Ownership. Andreeff:Oncoceutics Inc.: Membership on an entity's Board of Directors or advisory committees.

Transforming Growth Factor-β1 Increases DNA Methyltransferase 1 and 3a Expression through Distinct Post-transcriptional Mechanisms in Lung Fibroblasts

DNA methylation is a fundamental epigenetic mark that plays a critical role in differentiation and is mediated by the actions of DNA methyltransferases (DNMTs). TGF-β1 is one of the most potent inducers of fibroblast differentiation, and although many of its actions on fibroblasts are well described, the ability of TGF-β1 to modulate DNA methylation in mesenchymal cells is less clear. Here, we examine the ability of TGF-β1 to modulate the expression of various DNMTs in primary lung fibroblasts (CCL210). TGF-β1 increased the protein expression, but not RNA levels, of both DNMT1 and DNMT3a. The increases in DNMT1 and DNMT3a were dependent on TGF-β1 activation of focal adhesion kinase and PI3K/Akt. Activation of mammalian target of rapamycin complex 1 by Akt resulted in increased protein translation of DNMT3a. In contrast, the increase in DNMT1 by TGF-β1 was not dependent on new protein synthesis and instead was due to decreased protein degradation. TGF-β1 treatment led to the phosphorylation and inactivation of glycogen synthase kinase-3β, which resulted in inhibition of DNMT1 ubiquitination and proteosomal degradation. The phosphorylation and inactivation of glycogen synthase kinase-3β was dependent on mammalian target of rapamycin complex 1. These results demonstrate that TGF-β1 increases expression of DNMT1 and DNMT3a through different post-transcriptional mechanisms. Because DNA methylation is critical to many processes including development and differentiation, for which TGF-β1 is known to be crucial, the ability of TGF-β1 to increase expression of both DNMT1 and DNMT3a demonstrates a novel means by which TGF-β1 may regulate DNA methylation in these cells.

Instant and Persistent Antidepressant Response of Gardenia Yellow Pigment Is Associated with Acute Protein Synthesis and Delayed Upregulation of BDNF Expression in the Hippocampus.

Gardenia yellow pigment (GYP) is a collection of compounds with shared structure of crocin, which confers antidepressant activity. GYP is remarkably enriched in Gardenia jasminoides Ellis, implicated in rapid antidepressant effects that are exerted through enhanced neuroplasticity. This study aims to investigate the rapid antidepressant-like activity of GYP and its underlying mechanism. After the optimal dose was determined, antidepressant responses in tail suspension test or forced swim test were monitored at 30 min, 1 day, 3 days, and 7 days post a single GYP administration. Rapid antidepressant potential was tested using learned helplessness paradigm. The expression of proteins involved in hippocampal neuroplasticity was determined. The effect of blockade of protein synthesis on GYP's antidepressant response was examined. Antidepressant response was detected at 30 min, and lasted for at least 3 days post a single administration of GYP. A single administration of GYP also reversed the deficits in learned helplessness test. Thirty minutes post GYP administration, ERK signaling was activated, and its downstream effector phosphorylated eukaryotic elongation factor 2 was inhibited, contributing to increased protein translation. Expression of synaptic proteins GluR1 and synapsin 1 was upregulated. Blockade of protein synthesis with anisomycin blunted the immediate antidepressant response of GYP. CREB signaling and BDNF expression were upregulated at 24 h, but not at 30 min. In conclusion, GYP-induced immediate antidepressant response was dependent on synthesis of proteins, including synaptic proteins. This was followed by enhanced expression of CREB and BDNF, which likely mediated the persistent antidepressant responses.

Abstract B33: Assessing the role of DGAT activity on lipid homeostasis and cancer cell survival

Abstract Studies on the effects of modulating the lipid composition of cells have found that increases in saturated fatty acid levels can lead to ER stress, activation of the unfolded protein response (UPR) and cell death. These effects may occur through saturation of the phospholipid pool and subsequent deterioration of ER membrane function. Changes in the composition of the ER membrane can be sensed by the two UPR stress sensors IRE1 and PERK and lead to activation of UPR targets. Rapidly proliferating cancer cells frequently exhibit elevated ER stress due to their increased protein translation rate. In addition, the tumor microenvironment is frequently hypoxic and a lack of oxygen can inhibit the activity of the fatty acid desaturase SCD1. This leads to saturation of the fatty acid pool and renders the ER less able to cope with high levels of protein synthesis, making the maintenance of adequate lipid homeostasis of paramount importance to cancer cells within hypoxic tumor domains. Tumor cells adapt to these conditions through a variety of mechanisms, including the uptake of exogenous unsaturated lipid. Both hypoxia and the presence of certain oncogenic drivers have been found to increase lipid uptake, which was found to protect cancer cells against inhibition of SCD1. Since fatty acids can be converted to triglycerides and stored within lipid droplets, we asked whether cancer cells also cope with saturation of their fatty acid pool by storing excess saturated fatty acids as triglycerides. Clear cell renal cell carcinoma (ccRCC) tumors are very fatty and lipidomic analyses have found high levels of triglycerides in ccRCC compared to normal kidney tissue. We inhibited triglyceride synthesis in A498 ccRCC cells by shRNA –mediated knockdown of DGAT enzymes, which catalyze the final step in the synthesis of triglycerides from fatty acids. We tested whether loss of DGAT activity causes sensitivity to tumor-like stresses and investigated its effect on xenograft tumor growth. Remarkably, while loss of DGAT activity has little effect on cancer cell proliferation under oxygen- and nutrient-rich conditions, loss of DGAT activity has large effects on xenograft tumor growth. We also find that loss of DGAT activity is often accompanied by increases in markers of ER stress. We are currently assessing the lipid composition of these tumors and determining what aspects of the tumor microenvironment sensitize cells to loss of DGAT activity. Citation Format: Daniel Ackerman, Bo Qiu, Hong Xie, Jurre Kamphorst, M. Celeste Simon. Assessing the role of DGAT activity on lipid homeostasis and cancer cell survival. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B33.

The RiboPuromycylation Method Reveals Rapid IL12-Dependent Antigen-Independent Activation of Bone Marrow Virtual Memory T cells (INC1P.403)

Abstract Protein translation is an essential and intricately regulated cellular process that can potentially be used to monitor immune cell activation. We extended the RiboPuromycylation Method (RPM) to measure protein translation in vivo. Unexpectedly, 24 h following either vaccinia virus (VACV) or vesicular stomatitis virus (VSV) infection, 15-30% of bone marrow (BM) CD4+ and CD8+ T cells, all CD44+, increase protein translation. This is days before protein translation is increased in T cells in the draining lymph node or spleen. Bystander BM but not splenic OT-1 T cells demonstrate increased protein translation after VACV infection. Early virus-induced BM T cell translation is absent in IL-12 -/- mice. In BM cultures from naïve mice, addition of IL-12, and especially IL-12 with IL-18 increase T cell translation within hours, with IL1-2 and IL-18 eventually leading to T cell division. Our findings indicate that the BM is a unique environment where bystander T cells are rapidly activated by innate immune response to viruses. We hypothesize that this may allow memory and memory-like T cells to be activated in the absence of cognate antigen to facilitate their functionality when they encounter the cognate pathogen.

Intracellular Sensing and Transport of Amino Acids and the Downstream Effects on mTORC1 Activation: Implications for Muscle Protein Synthesis

It is widely established that the rate of muscle protein synthesis is elevated with amino acid availability. However, when there is a lack in amino acid availability homeostatic processes are implemented in order to prioritize the energy demands necessitating amino acid scarcity. To this respect, it is understood that amino acid availability is a rate-limiting factor underlying the mediation of muscle protein synthesis (MPS). The mammalian target of rapamycin complex 1 (mTORC1) is a master regulatory complex that plays a significant role in meticulously coordinating the myriad of signaling cascades in order to up-regulate MPS activity through increased protein translation initiation. The presence of adequate amino acids such as leucine is evidenced to be necessary for the activation of mTORC1. Moreover, amino acids have been known as subsequent mediators that trigger mTORC1 activation independent of the traditional upstream signaling cascades associated with growth factors. It has recently been observed that the activation of mTORC1 is implemented at the surface of the lysosome membrane. As a result, considerable advances have occurred which demonstrate various protein components which relay on amino acids to relay signals to mTORC1. This review will focalize upon describing the present understanding of the multiple signaling components that mediate signals in conditions of adequate amino acid levels to mTORC1. Furthermore, this review will also present recent advancements behind amino acid sensing mechanisms through the discussion of upstream components of mTORC1, amino acid transporters, as well as proposed underlying mechanisms of intracellular signaling relative to amino acid sensing.

Enzymatic Characterization of ER Stress-Dependent Kinase, PERK, and Development of a High-Throughput Assay for Identification of PERK Inhibitors

PERK is serine/threonine kinase localized to the endoplasmic reticulum (ER) membrane. PERK is activated and contributes to cell survival in response to a variety of physiological stresses that affect protein quality control in the ER, such as hypoxia, glucose depravation, increased lipid biosynthesis, and increased protein translation. Pro-survival functions of PERK are triggered by such stresses, suggesting that development of small-molecule inhibitors of PERK may be efficacious in a variety of disease scenarios. Hence, we have conducted a detailed enzymatic characterization of the PERK kinase to develop a high-throughput-screening assay (HTS) that will permit the identification of small-molecule PERK inhibitors. In addition to establishing the Km of PERK for both its primary substrate, eIF2α, and for adenosine triphosphate, further mechanistic studies revealed that PERK targets its substrate via either a random/steady-state ordered mechanism. For HTS, we developed a time-resolved fluorescence resonance energy transfer–based assay that yielded a robust Z′ factor and percent coefficient of variation value, enabling the successful screening of 79,552 compounds. This approach yielded one compound that exhibited good in vitro and cellular activity. These results demonstrate the validity of this screen and represent starting points for drug discovery efforts.

Monoterpene Glycoside ESK246 from Pittosporum Targets LAT3 Amino Acid Transport and Prostate Cancer Cell Growth

The l-type amino acid transporter (LAT) family consists of four members (LAT1–4) that mediate uptake of neutral amino acids including leucine. Leucine is not only important as a building block for proteins, but plays a critical role in mTORC1 signaling leading to protein translation. As such, LAT family members are commonly upregulated in cancer in order to fuel increased protein translation and cell growth. To identify potential LAT-specific inhibitors, we established a function-based high-throughput screen using a prefractionated natural product library. We identified and purified two novel monoterpene glycosides, ESK242 and ESK246, sourced from a Queensland collection of the plant Pittosporum venulosum. Using Xenopus laevis oocytes expressing individual LAT family members, we demonstrated that ESK246 preferentially inhibits leucine transport via LAT3, while ESK242 inhibits both LAT1 and LAT3. We further show in LNCaP prostate cancer cells that ESK246 is a potent (IC50 = 8.12 μM) inhibitor of leucine uptake, leading to reduced mTORC1 signaling, cell cycle protein expression and cell proliferation. Our study suggests that ESK246 is a LAT3 inhibitor that can be used to study LAT3 function and upon which new antiprostate cancer therapies may be based.

HSF1 in Translation

The master regulator of the classical cytoprotective "heat shock" response, heat shock factor 1 (HSF1), is increasingly implicated in cancer pathogenesis, but the mechanisms remain poorly understood. A recent study connects increased protein translation to activation of HSF1 in malignant cells and demonstrates the therapeutic benefit of targeting this link.

The RiboPuroMycylation method reveals surprising protein synthesis in bone marrow T cells within a day of vaccinia virus infection (P3385)

Abstract Protein translation is an essential and intricately regulated cellular process. We recently described the RiboPuroMycylation Method (RPM) to measure protein translation in cultured cells. RPM is based on ribosome-catalyzed addition of puromycin to nascent chains followed by detection with a monoclonal antibody specific for puromycin in fixed and permeabilized cells. To apply RPM in vivo, we administered puromycin intravenously and measured puromycin staining via flow cytometry. We were able to detect a robust signal that was blocked by ribosome targeting antibiotics that prevent nascent chain puromycylation. After infection with either vaccinia virus (VACV) or influenza virus, we find profound increases in protein translation in activated T and B cells. In mice with adoptively transferred OT-1 T cells we find that translation of individual cells peaks 3 days after a foot pad infection, i.e. several days before T cell numbers peak. One day after an intra-peritoneal VACV infection, almost 40% of the CD4+ and CD8+ T cells in the bone marrow (BM) have increased protein translation as compared to only 3% of the T cells in the spleen. Using transgenic OT-1 T cells and VACV with or without SIINFEKL, we find that the increased protein translation in the spleen and BM is antigen-dependent. Taken together, these findings illustrate the ability of in vivo RPM to provide novel information regarding the participation of translational regulation in workings of the immune system.

Intra-Axonal Translation of RhoA Promotes Axon Growth Inhibition by CSPG

Chondroitin sulfate proteoglycans (CSPGs) are a major component of the glial scar that contributes to the limited regeneration of the CNS after axonal injury. However, the intracellular mechanisms that mediate the effects of CSPGs are not fully understood. Here we show that axonal growth inhibition mediated by CSPGs requires intra-axonal protein synthesis. Application of CSPGs to postnatal rat dorsal root ganglia axons results in an increase in the axonal levels of phosphorylated 4E-BP1, a marker of increased protein translation. Axons grown in media containing CSPGs exhibit markedly reduced growth rates, which can be restored by the selective application of protein synthesis inhibitors to distal axons. We show that these axons contain transcripts encoding RhoA, a regulator of the cytoskeleton that is commonly used by the signaling pathways activated by many inhibitors of axon growth. We also show that selective application of CSPGs to axons results in increased intra-axonal synthesis of RhoA and that depletion of RhoA transcripts from axons results in enhanced growth of axons in the presence of CSPGs. These data identify local translation as an effector pathway of CSPGs and demonstrate that local translation of RhoA contributes to the axon growth inhibitory effect of CSPGs.

Anti-tumor effects of an engineered “killer” transfer RNA

A hallmark of cancer cells is their ability to continuously divide; and rapid proliferation requires increased protein translation. Elevating levels of misfolded proteins can elicit growth arrest due to ER stress and decreased global translation. Failure to correct prolonged ER stress eventually results in cell death via apoptosis. tRNASer(AAU) is an engineered human tRNASer with an anticodon coding for isoleucine. Here we test the possibility that tRNASer(AAU) can be an effective killing agent of breast cancer cells and can effectively inhibit tumor-formation in mice. We found that tRNASer(AAU) exert strong effects on breast cancer translation activity, cell viability, and tumor formation. Translation is strongly inhibited by tRNASer(AAU) in both tumorigenic and non-tumorigenic cells. tRNASer(AAU) significantly decreased the number of viable cells over time. A short time treatment with tRNASer(AAU) was sufficient to eliminate breast tumor formation in a xenograft mouse model. Our results indicate that tRNASer(AAU) can inhibit breast cancer metabolism, growth and tumor formation. This RNA has strong anti-cancer effects and presents an opportunity for its development into an anti-tumor agent. Because tRNASer(AAU) corrupts the protein synthesis mechanism that is an integral component of the cell, it would be extremely difficult for tumor cells to evolve and develop resistance against this anti-tumor agent.

Drosophila TRPML Is Required for TORC1 Activation

Loss-of-function mutations in TRPML1 (transient receptor potential mucolipin 1) cause the lysosomal storage disorder, mucolipidosis type IV (MLIV). Here, we report that flies lacking the TRPML1 homolog displayed incomplete autophagy and reduced viability during the pupal period--a phase when animals rely on autophagy for nutrients. We show that TRPML was required for fusion of amphisomes with lysosomes, and its absence led to accumulation of vesicles of significantly larger volume and higher luminal Ca(2+). We also found that trpml(1) mutant cells showed decreased TORC1 (target of rapamycin complex 1) signaling and a concomitant upregulation of autophagy induction. Both of these defects in the mutants were reversed by genetically activating TORC1 or by feeding the larvae a high-protein diet. The high-protein diet also reduced the pupal lethality and the increased volume of acidic vesicles. Conversely, further inhibition of TORC1 activity by rapamycin exacerbated the mutant phenotypes. Finally, TORC1 exerted reciprocal control on TRPML function. A high-protein diet caused cortical localization of TRPML, and this effect was blocked by rapamycin. Our findings delineate the interrelationship between the TRPML and TORC1 pathways and raise the intriguing possibility that a high-protein diet might reduce the severity of MLIV.