Inhibition Of Global Protein Synthesis Research Articles

Energy stress promotes P-bodies formation via lysine-63-linked polyubiquitination of HAX1

Energy stress, characterized by the reduction of intracellular ATP, has been implicated in various diseases, including cancer. Here, we show that energy stress promotes the formation of P-bodies in a ubiquitin-dependent manner. Upon ATP depletion, the E3 ubiquitin ligase TRIM23 catalyzes lysine-63 (K63)-linked polyubiquitination of HCLS1-associated protein X-1 (HAX1). HAX1 ubiquitination triggers its liquid‒liquid phase separation (LLPS) and contributes to P-bodies assembly induced by energy stress. Ubiquitinated HAX1 also interacts with the essential P-body proteins, DDX6 and LSM14A, promoting their condensation. Moreover, we find that this TRIM23/HAX1 pathway is critical for the inhibition of global protein synthesis under energy stress conditions. Furthermore, high HAX1 ubiquitination, and increased cytoplasmic localization of TRIM23 along with elevated HAX1 levels, promotes colorectal cancer (CRC)-cell proliferation and correlates with poor prognosis in CRC patients. Our data not only elucidate a ubiquitination-dependent LLPS mechanism in RNP granules induced by energy stress but also propose a promising target for CRC therapy.

The E3 ubiquitin-protein ligase synoviolin (Syvn1/Hrd1) promotes adaptive decreases in cardiac myocyte protein synthesis via eIF2α/ATF4 pathway activation

As the heart undergoes pathologic hypertrophy in response to many disease conditions, an imbalance between protein synthesis and degradation in cardiac myocytes, due to the increases in protein synthesis relative to protein degradation, happens, which challenges protein folding and endoplasmic reticulum (ER) proteostasis. This leads to ER stress and the activation of the ER stress response. Synoviolin (Syvn1), also known as HMG-CoA reductase degradation protein 1 (Hrd1), is an ER-localized protein that is upregulated in cardiac myocytes during ER stress. We previously found that Syvn1 mediates the ubiquitylation and consequent degradation of toxic misfolded proteins and preserved cardiac function in a model of pressure overload-induced cardiac pathology. The objective of this study was to examine whether Syvn1 is involved in regulating protein synthesis, and if so, what its function is during hypertrophic growth of cardiac myocytes. Given that cardiac hypertrophy is caused by increases in protein synthesis, we hypothesized that Syvn1 provides an adaptive response in part through regulation of protein synthesis and consequent decrease in cardiac hypertrophic response. We examined the effects of Syvn1 on ventricular myocyte growth in vitro and found that overexpression of Syvn1 decreased cardiac myocyte surface area as well as protein synthesis when treated with phenylephrine (PE), a treatment which normally leads to increased protein synthesis and increased cardiac myocyte size, mimicking pathological cardiac hypertrophy. We next focused on identifying the mechanism of Syvn1-mediated growth inhibition and found that increases in Syvn1 lead to activation of the PERK (protein kinase RNA (PKR)-like ER kinase) branch of the ER stress response, which leads to increased phosphorylation of the α-subunit of translation initiation factor 2 (eIF2α) at Ser51, thereby inhibiting global translational initiation. PERK activation also results in selective translation of a subset of mRNAs, including activating transcription factor 4 (ATF4), which activates the transcription of a wide range of genes involved in adaptation to stress conditions. We found increased ATF4 levels and increased ATF4 target gene expression with Syvn1 overexpression during PE treatment, compared to control PE treatment. Inhibition of the PERK pathway blocked Syvn1-mediated decrease in cardiac myocyte size and protein synthesis as well as PERK activation and ATF4 target gene induction. Additionally, we found that an adeno-associated virus encoding Syvn1, administered in a therapeutically-relevant manner after the onset of pathological cardiac hypertrophy induced by transverse-aortic constriction in mice, blunted pathological remodeling and preserved cardiac function. We posit that the Syvn1/eIF2α/ATF4 pathway constitutes a newly-discovered mechanism for balancing the proteome in cardiac myocytes. Here we found that Syvn1 upregulation blunts maladaptive increase in cardiac myocyte size during pathologic hypertrophy. In conclusion, these findings suggest that Syvn1 is a critical regulator of cardiac myocyte protein synthesis and subsequent growth and that increases in Syvn1 lead to the activation of the PERK branch of the ER stress response, which results in inhibition of global protein synthesis, and activation of ATF4. This work was supported by the National Institutes of Health (R01HL157027) and the University of Arizona Health Sciences Career Development Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cell competition: emerging signaling and unsolved questions.

Multicellular communities have an intrinsic mechanism that optimizes their structure and function via cell-cell communication. One of the driving forces for such self-organization of the multicellular system is cell competition, the elimination of viable unfit or deleterious cells via cell-cell interaction. Studies in Drosophila and mammals have identified multiple mechanisms of cell competition caused by different types of mutations or cellular changes. Intriguingly, recent studies have found that different types of "losers" of cell competition commonly show reduced protein synthesis. In Drosophila, the reduction in protein synthesis levels in loser cells is caused by phosphorylation of the translation initiation factor eIF2α via a bZip transcription factor Xrp1. Given that a variety of cellular stresses converge on eIF2α phosphorylation and thus global inhibition of protein synthesis, cell competition may be a machinery that optimizes multicellular fitness by removing stressed cells. In this review, we summarize and discuss emerging signaling mechanisms and critical unsolved questions, as well as the role of protein synthesis in cell competition.

Differential requirements for P stalk components in activating yeast protein kinase Gcn2 by stalled ribosomes during stress

The General Amino Acid Control is a conserved response to amino acid starvation involving activation of protein kinase Gcn2, which phosphorylates eukaryotic initiation factor 2 (eIF2α) with attendant inhibition of global protein synthesis and increased translation of yeast transcriptional activator GCN4. Gcn2 can be activated by either amino acid starvation or conditions that stall elongating ribosomes without reducing aminoacylation of tRNA, but it is unclear whether distinct molecular mechanisms operate in these two circumstances. We identified three regimes that activate Gcn2 in yeast cells by starvation-independent (SI) ribosome-stalling: treatment with tigecycline, eliminating the sole gene encoding tRNAArgUCC, and depletion of translation termination factor eRF1. We further demonstrated requirements for the tRNA- and ribosome-binding domains of Gcn2, the positive effector proteins Gcn1/Gcn20, and the tethering of at least one of two distinct P1/P2 heterodimers to the uL10 subunit of the ribosomal P stalk, for detectable activation by SI-ribosome stalling. Remarkably, no tethered P1/P2 proteins were required for strong Gcn2 activation elicited by starvation for histidine or branched-chain amino acids isoleucine/valine. These results indicate that Gcn2 activation has different requirements for the P stalk depending on how ribosomes are stalled. We propose that accumulation of deacylated tRNAs in amino acid-starved cells can functionally substitute for the P stalk in binding to the histidyl-tRNA synthetase-like domain of Gcn2 for eIF2α kinase activation by ribosomes stalled with A sites devoid of the eEF1A∙GTP∙aminoacyl-tRNA ternary complex.

Velcrin-induced selective cleavage of tRNALeu(TAA) by SLFN12 causes cancer cell death.

Velcrin compounds kill cancer cells expressing high levels of phosphodiesterase 3A (PDE3A) and Schlafen family member 12 (SLFN12) by inducing complex formation between these two proteins, but the mechanism of cancer cell killing by the PDE3A-SLFN12 complex is not fully understood. Here, we report that the physiological substrate of SLFN12 RNase is tRNALeu(TAA). SLFN12 selectively digests tRNALeu(TAA), and velcrin treatment promotes the cleavage of tRNALeu(TAA) by inducing PDE3A-SLFN12 complex formation in vitro. We found that distinct sequences in the variable loop and acceptor stem of tRNALeu(TAA) are required for substrate digestion. Velcrin treatment of sensitive cells results in downregulation of tRNALeu(TAA), ribosome pausing at Leu-TTA codons and global inhibition of protein synthesis. Velcrin-induced cleavage of tRNALeu(TAA) by SLFN12 and the concomitant global inhibition of protein synthesis thus define a new mechanism of apoptosis initiation.

Asparagine bioavailability regulates the translation of MYC oncogene.

Amino acid restriction has recently emerged as a compelling strategy to inhibit tumor growth. Recent work suggests that amino acids can regulate cellular signaling in addition to their role as biosynthetic substrates. Using lymphoid cancer cells as a model, we found that asparagine depletion acutely reduces the expression of c-MYC protein without changing its mRNA expression. Furthermore, asparagine depletion inhibits the translation of MYC mRNA without altering the rate of MYC protein degradation. Of interest, the inhibitory effect on MYC mRNA translation during asparagine depletion is not due to the activation of the general controlled nonderepressible 2 (GCN2) pathway and is not a consequence of the inhibition of global protein synthesis. In addition, both the 5' and 3' untranslated regions (UTRs) of MYC mRNA are not required for this inhibitory effect. Finally, using a MYC-driven mouse B cell lymphoma model, we found that shRNA inhibition of asparagine synthetase (ASNS) or pharmacological inhibition of asparagine production can significantly reduce the MYC protein expression and tumor growth when environmental asparagine becomes limiting. Since MYC is a critical oncogene, our results uncover a molecular connection between MYC mRNA translation and asparagine bioavailability and shed light on a potential to target MYC oncogene post-transcriptionally through asparagine restriction.

Abstract 1839: Nelfinavir induced-cytotoxicity towards high-grade serous ovarian cancer cells involves induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition

Abstract High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, the rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction of HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction of clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. Western blot analysis of underlying molecular mechanisms revealed activation by NFV of the three signaling arms of the unfolded protein response (UPR): PERK, IRE1alpha and ATF6 - in a similar manner to the classical endoplasmic reticulum (ER) stressor tunicamycin. NFV-mediated modulation of the UPR was accompanied by the inhibition of global protein synthesis as analyzed through a non-radioactive method by labelling the nascent polypeptides with puromycin. NFV maintained a proapoptotic environment through the UPR, evidenced by a dose- and time-dependent enhanced expression of ATF4, CHOP, increased Bax/Bcl2 ratio, and cleavage of executioner caspase-7. Enhanced gamma-H2AX suggested NFV-mediated induction of DNA damage, which was associated with decreased proliferation signals driven by the AKT and ERK pathways. NFV increased the level of autophagosome-associated protein LC3II in HGSOC cells of varying platinum sensitivities; however, the autophagic flux was not increased during co-treatment with NFV and the lysosomal inhibitor bafilomycin A1, suggesting a likely impairment of the lysosomes by NFV impeding autophagosome clearance. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile. Citation Format: Mahbuba R. Subeha, Alicia A. Goyeneche, Prisca Bustamante, Michael A. Lisio, Julia V. Burnier, Carlos M. Telleria. Nelfinavir induced-cytotoxicity towards high-grade serous ovarian cancer cells involves induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1839.

Abstract 879: SLFN12-mediated depletion of tRNA-Leu-TAA by velcrin treatment induces ribosome pausing at Leu-TTA codons and represses global translation initiation

Abstract Velcrin compounds kill cancer cells expressing high levels of PDE3A and SLFN12 by inducing complex formation between these two proteins, but the mechanism of cancer cell killing by the PDE3A-SLFN12 complex is not fully understood. We previously determined that SLFN12 is an RNase, that PDE3A binding upregulates SLFN12 RNase activity, and that SLFN12 RNase activity is required for velcrin response. Here, we report that the depletion of tRNA-Leu-TAA by velcrin treatment induces ribosome pausing at Leu-TTA codons and global inhibition of protein synthesis. We performed ribosome profiling in HeLa cells treated with DMSO or DNMDP and found that ribosome footprints containing Leu-TTA codons at the A-site of the ribosome were significantly more abundant in DNMDP-treated cells than in cells treated with DMSO, indicating a pausing or stalling of ribosomes at Leu-TTA upon velcrin treatment. Although the overall number of ribosome pausing sites across the transcriptome was doubled by DNMDP treatment, ribosome pausing at Leu-TTA was increased &gt 1000-fold, also resulting in an increased number of genes with TTA pause sites. Even though Leu-TTA is a rare codon, over 70% of all transcripts contain at least one TTA codon, implying that ribosome pausing at TTA codons could directly impact many genes. Genes with TTA pause sites most enriched upon velcrin treatment prominently include those involved in control of mRNA translation and protein homeostasis. These results imply that, in addition to direct effects on mRNA translation due to ribosome pausing, indirect effects could occur due to decreased availability of particular components of the translation machinery, resulting in a widespread impact on protein synthesis. To test our hypothesis, we measured the effects of velcrin treatment on nascent protein abundance. Treatment of HeLa cells with DNMDP resulted in downregulation of newly synthesized proteins, whereas no significant changes in total protein content were observed. Downregulation of protein translation was observed only in velcrin-sensitive cell lines, indicating that inhibition of protein translation could be the mechanism of action of cancer cell killing by PDE3A-SLFN12 complex formation. Citation Format: Stephanie Hoyt, Sooncheol Lee, Joseph McGaunn, Andrew Cherniack, Matthew Meyerson, Heidi Greulich. SLFN12-mediated depletion of tRNA-Leu-TAA by velcrin treatment induces ribosome pausing at Leu-TTA codons and represses global translation initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 879.

Mitochondrial Inhibition by Sodium Azide Induces Assembly of eIF2α Phosphorylation-Independent Stress Granules in Mammalian Cells.

Mitochondrial stress is involved in many pathological conditions and triggers the integrated stress response (ISR). The ISR is initiated by phosphorylation of the eukaryotic translation initiation factor (eIF) 2α and results in global inhibition of protein synthesis, while the production of specific proteins important for the stress response and recovery is favored. The stalled translation preinitiation complexes phase-separate together with local RNA binding proteins into cytoplasmic stress granules (SG), which are important for regulation of cell signaling and survival under stress conditions. Here we found that mitochondrial inhibition by sodium azide (NaN3) in mammalian cells leads to translational inhibition and formation of SGs, as previously shown in yeast. Although mammalian NaN3-induced SGs are very small, they still contain the canonical SG proteins Caprin 1, eIF4A, eIF4E, eIF4G and eIF3B. Similar to FCCP and oligomycine, other mitochodrial stressors that cause SG formation, NaN3-induced SGs are formed by an eIF2α phosphorylation-independent mechanisms. Finally, we discovered that as shown for arsenite (ASN), but unlike FCCP or heatshock stress, Thioredoxin 1 (Trx1) is required for formation of NaN3-induced SGs.

Oxidative Stress Suppresses Nrf2 Protein Synthesis through Global Protein Synthesis Inhibition

Oxidative and electrophilic stresses are common aggressors in chronic diseases ranging from arthritis to cancer to neurodegenerative disease. The transcription factor Nrf2 is an essential mediator of the cell’s cytoprotective response to these stressors. Nrf2 is constantly synthesized, ubiquitinated, and degraded with a short half‐life of 20 minutes that maintains low basal Nrf2 protein levels. Electrophiles and oxidative stress cause Nrf2 to accumulate and subsequently to activate cytoprotective genes by modifying cysteines of the Keap1 protein, the repressor that targets Nrf2 for ubiquitination. We previously found that paradoxically, reactive oxygen species (ROS) stimulate the production of cytoprotective proteins, but inhibit Nrf2 protein synthesis, revealing a “Dr. Jekyll and Mr. Hyde” effect of ROS on Nrf2 accumulation. We tested the hypothesis that oxidative conditions that activate the Nrf2 pathway simultaneously inhibit global protein synthesis, thereby suppressing Nrf2 protein synthesis. We investigated both ROS generated by redox cycling of the oxidizable phenol, di‐tert‐butylhydroquinone (dtBHQ), and direct addition of H2O2 to HaCaT keratinocyte cells. We found that 100 μM to 400 μM H2O2 and 50 μM dtBHQ inhibit both Nrf2 protein synthesis and global protein synthesis. Addition of antioxidants, such as the manganese porphyrin MnTMPyP, rescued both Nrf2‐specific and global protein synthesis. Therefore, antioxidants such as MnTMPyP that mitigate the suppressive effects of ROS on global protein synthesis during Nrf2 activation could enhance the Nrf2/cytoprotective response under conditions of electrophilic and oxidative stress.

Oxidative Stress Suppresses Nrf2 Protein Synthesis through Global Protein Synthesis Inhibition

Oxidative Stress Suppresses Nrf2 Protein Synthesis through Global Protein Synthesis Inhibition

Abstract 3153: Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment

Abstract Recent advances in cancer metabolism suggest that targeting amino acid metabolism represents a promising strategy for the development of novel therapeutic agents. Tumor, stromal and myeloid-derived suppressor cells (MDSC) within the tumor microenvironment (TME) create a nutrient-poor environment that inhibit immune function and support tumor growth. GCN2 (general control nonderepressible 2), a stress response kinase, plays a key role in maintaining cellular homeostasis under a wide range of stressors. Phosphorylation of GCN2 (pGCN2) in response to stress leads to inhibition of global protein synthesis and subsequently leads to 1) T cell anergy and apoptosis, 2) enhanced MDSC-dependent immune suppression and 3) tumor cell survival. Collectively, these roles suggest that GCN2 inhibition could have both a direct anticancer and an immune-activating effect. Treating nutrient-deprived T cells in vitro with a RAPT GCN2 inhibitor (RPT-GCN2i) rescued CD4+ and CD8+ T cell proliferation and effector functions. The RPT-GCN2i also reversed T cell suppression mediated by MDSCs derived from healthy donors or cancer patients. Using syngeneic mouse tumor models, we demonstrated that translational induction of activating transcription factor 4 (ATF4) is a strong marker of GCN2 pathway activation in vivo. Oral administration of an RPT-GCN2i exhibited notable drug-target occupancy and potently inhibited GCN2 kinase and ATF4 in the TME. RPT-GCN2i as a single agent and in combination with checkpoint blockade or angiogenesis inhibitor (anti-VEGFR) led to delays in tumor growth rate in various syngeneic tumor models. In addition, GCN2 inhibition redirected MDSC within the TME from a suppressive to inflammatory phenotype through downregulation of Arg1 and iNOS. Our results show that inhibition of GCN2 is an attractive approach for enhancing antitumor immune response and therefore GCN2 is a promising therapeutic target for the treatment of cancer. Citation Format: Lisa Marshall, Buvana Ravishankar, Urvi Kolhatkar, Mengshu Xu, Lavanya Adusumilli, Deepa Pookot, Thant Zaw, Chandru Ramana, Raashi Sreenivasan, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Omar Robles, Anqi Ma, Andrew Ng, Anton Shakhmin, Justy Guagua, Scott Jacobson, Steve Wong, Delia Bradford, Tracy L. McGaha, M Teresa Ciudad, James E. Talmadge, Holly C. Britton, George Katibah, Gene Cutler, David Wustrow, Paul D. Kassner, Dirk G. Brockstedt. Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3153.

Impact of Hydrogen Peroxide on Protein Synthesis in Yeast.

Cells must be able to respond and adapt to different stress conditions to maintain normal function. A common response to stress is the global inhibition of protein synthesis. Protein synthesis is an expensive process consuming much of the cell’s energy. Consequently, it must be tightly regulated to conserve resources. One of these stress conditions is oxidative stress, resulting from the accumulation of reactive oxygen species (ROS) mainly produced by the mitochondria but also by other intracellular sources. Cells utilize a variety of antioxidant systems to protect against ROS, directing signaling and adaptation responses at lower levels and/or detoxification as levels increase to preclude the accumulation of damage. In this review, we focus on the role of hydrogen peroxide, H2O2, as a signaling molecule regulating protein synthesis at different levels, including transcription and various parts of the translation process, e.g., initiation, elongation, termination and ribosome recycling.

Poly(U)-specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element-mediated inhibition.

Upstream open reading frames (uORFs) are known to negatively affect translation of the downstream ORF. The regulatory proteins involved in relieving this inhibition are however poorly characterized. In response to cellular stress, eIF2α phosphorylation leads to an inhibition of global protein synthesis, while translation of specific factors such as CHOP is induced. We analyzed a 105‐nt inhibitory uORF in the transcript of human CHOP (huORFchop) and found that overexpression of the zebrafish or human ENDOU poly(U)‐endoribonuclease (Endouc or ENDOU‐1, respectively) increases CHOP mRNA translation also in the absence of stress. We also found that Endouc/ENDOU‐1 binds and cleaves the huORFchop transcript at position 80G‐81U, which induces CHOP translation independently of phosphorylated eIF2α. However, both ENDOU and phospho‐eIF2α are nonetheless required for maximal translation of CHOP mRNA. Increased levels of ENDOU shift a huORFchop reporter as well as endogenous CHOP transcripts from the monosome to polysome fraction, indicating an increase in translation. Furthermore, we found that the uncapped truncated huORFchop‐69‐105‐nt transcript contains an internal ribosome entry site (IRES), facilitating translation of the cleaved transcript. Therefore, we propose a model where ENDOU‐mediated transcript cleavage positively regulates CHOP translation resulting in increased CHOP protein levels upon stress. Specifically, CHOP transcript cleavage changes the configuration of huORFchop thereby releasing its inhibition and allowing the stalled ribosomes to resume translation of the downstream ORF.

Salubrinal Mediated Fetal Hemoglobin Induction through the eIF2α-ATF4 Signaling Pathway

Sickle cell disease (SCD) is a genetic disorder caused by a mutation in the adult β-globin gene, affecting ~100,000 people in the United States and millions worldwide. Clinical symptoms of SCD include anemia, pain, and progressive organ damage creating a great burden to annual healthcare costs. An effective therapeutic intervention for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents to ameliorate clinical symptoms. Hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD, however, it is not effective in all people. Therefore, the goal of this study is to determine the ability of Salubrinal (SAL), to induce HbF. Salubrinal is a selective inhibitor of protein phosphatase 1 leading to increased levels of p-eIF2α (phosphorylated eukaryotic initiation factor 2α) and inhibition of global protein synthesis. Activating transcription factor 4 (ATF4) is a downstream target of p-eIF2α activated during oxidative stress. The main function of these signaling events is to attenuate stress to the endoplasmic reticulum. Previously we identified a Gγ-globin cAMP response element (G-CRE) that binds ATF2, a binding partner of ATF4 involved in HbF induction (Sangerman J et al. Blood 2006). Furthermore, ENCODE analysis showed ATF4 sites at -822Gγ and β-globin gene second intron. Thus, studies were performed to determine if p-eIF2α-ATF4 signaling is involved in mechanisms of HbF induction by SAL. Initial experiments involved the use of day 8 erythroid progenitors generated from human CD34+ stem cells; treatments included SAL 12, 18 and 24 µM, and 0.5% DMSO (vehicle control) for 48 h; cell viability remained >90% in all drug conditions. The level of γ-globin mRNA increased 1.2-fold and 1.3-fold at SAL 18 and 24 µM respectively (p<0.05). Comparable, HbF was induced by SAL 24 µM alone and combined SAL/HU treatments to 1.8-fold. To gain insights into mechanisms of HbF induction by SAL, we next quantified the level of p-eIF2α. We observed a 1.7-fold increase in p-eIF2α with SAL 12 and 24 µM and parallel increase in ATF4 (4.8-fold). Flow cytometry revealed SAL increased F-cells (HbF positive cells) from 30.9% (DMSO treated) to 90.6%. Similarly, studies were performed using sickle erythroid progenitors generated from peripheral blood mononuclear cells. On day 8, SAL (9, 18, 24 µM) dissolved in water was added for 48 h; cell viability remained >90% for all drug conditions. SAL (18 μM) increased γ-globin mRNA 3.2-fold and F-cells 2.5-fold (p<0.05) compared to the untreated control. We used mean fluorescence intensity (MFI) to quantify HbF protein per cells, which showed a dose-dependent increase with SAL treatment. Since sickle red blood cells are under oxidative stress, we measured the levels of reactive oxygen species (ROS) by flow cytometry. SAL 12, 18, 24 µM decreased ROS levels in a dose-dependent manner by 7.6%, 8.7% and 10% respectively. Interestingly, SAL/HU treatment decreased ROS levels by 10.2% compared to a 4.3% mediated by the nitric oxide donor HU. Western blot analysis showed a dose-dependent increase in HbF and a 3.3-fold increase in p-eIF2α (p<0.05) and ATF4, without changing HbS expression. To generate data for clinical development, we utilized the Townes SCD mouse model. SCD mice (n=5 per group) were treated with SAL (3 and 5mg/kg), HU (100mg/kg; positive control) or water control (vehicle), 5 days a week for 4 weeks. Blood was drawn at week 0 (baseline), 2 and 4 at treatment completion. All data were normalized for each group and treatment response at week 2 and 4 compared to week 0 using a paired t-test and ANOVA to compare across treatment groups; statistical significance set at p<0.05. All groups showed normal weight gain and no significant changes in complete blood counts, differential or reticulocyte counts. Flow cytometry of peripheral blood showed that SAL (3mg/kg) produced a 2-fold increase in F-cells by 2 and 4 weeks while SAL (5mg/kg) produced a further 3.1-fold increase in F-cells by week 4 (p<0.05) without toxicity. Our initial in vitro findings, supports HbF induction by SAL involving p-eIF2α-ATF4 signaling. The interaction of ATF4 in the G-CRE and/or other predicted binding sites will be investigated. To support clinical trials, studies in the SCD preclinical model support the ability of SAL to induce HbF in vivo; additional studies are underway. Defining the mechanism of HbF induction by SAL has the potential to impact treatment for SCD. Disclosures No relevant conflicts of interest to declare.

Direct Antiviral Activity of IFN-Stimulated Genes Is Responsible for Resistance to Paramyxoviruses in ISG15-Deficient Cells.

IFNs, produced during viral infections, induce the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, whereas others regulate the cellular response. Besides functioning as an antiviral effector, ISG15 is a negative regulator of IFN signaling, and inherited ISG15 deficiency leads to autoinflammatory IFNopathies, in which individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15-/- cells and (using A549-UBA7-/- cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type I IFN response. ISG15-deficient cells pretreated with IFN-α were resistant to paramyxovirus infection. We also showed that IFN-α treatment of ISG15-deficient cells led to significant inhibition of global protein synthesis, leading us to ask whether resistance was due to the direct antiviral activity of ISGs or whether cells were nonpermissive because of translation defects. We took advantage of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal antiviral ISG for parainfluenza virus 5. Knockdown of IFIT1 restored parainfluenza virus 5 infection in IFN-α-pretreated, ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of the IFN response. However, resistance could be induced if cells were pretreated with IFN-α for longer times, presumably because of inhibition of protein synthesis. These data show that the cause of virus resistance is 2-fold; ISG15 deficiency leads to the early overexpression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15-dependent loss of translational control.

PERK (Protein Kinase RNA-Like ER Kinase) Branch of the Unfolded Protein Response Confers Neuroprotection in Ischemic Stroke by Suppressing Protein Synthesis.

Background and Purpose- Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods- Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with Perkf/f mice. Transient middle cerebral artery occlusion was used to induce stroke. Short- and long-term stroke outcomes were evaluated. Protein synthesis in the brain was assessed using a surface-sensing-of-translation approach. Results- After tamoxifen-induced deletion of Perk in forebrain neurons was confirmed in PERK-cKO mice, PERK-cKO and control mice were subjected to transient middle cerebral artery occlusion and 3 days or 3 weeks recovery. PERK-cKO mice had larger infarcts and worse neurological outcomes compared with control mice, suggesting that PERK-induced eIF2α phosphorylation and subsequent suppression of translation protects neurons from ischemic stress. Indeed, better stroke outcomes were observed in PERK-cKO mice that received postischemic treatment with salubrinal, which can restore the ischemia-induced increase in phosphorylated eIF2α in these mice. Finally, our data showed that post-treatment with salubrinal improved functional recovery after stroke. Conclusions- Here, we presented the first evidence that postischemic suppression of translation induced by PERK activation promotes recovery of neurological function after stroke. This confirms and further extends our previous observations that recovery of ER function impaired by ischemic stress critically contributes to stroke outcome. Therefore, future research should include strategies to improve stroke outcome by targeting unfolded protein response branches to restore protein homeostasis in neurons.

(1–4)-Thiodisaccharides as anticancer agents. Part 5. Evaluation of anticancer activity and investigation of mechanism of action

(1–4)-Thiodisaccharides, thiosugars with the 1–4-thio bridge, were recently shown to induce oxidative stress, as well as, apoptosis in cancer cells in the low micromolar range; however, the detailed mechanism of their anticancer action still remains unknown. In order to clarify the mechanism of (1–4)- thiodisaccharides action, we performed a series of tests including cytotoxic, clonogenic and apoptosis assays using an in vitro glioma cancer model with one ATCC cell line U87 and two novel glioma cell lines derived from cancer patients – H6PX and H7PX. We also evaluated the ability of (1–4)-thiodisaccharides to interfere with protein folding and synthesis processes, as well as, the thioredoxin system. (1–4)-thiodisaccharides induced glioma cell death, which were found to be accompanied with endoplasmic reticulum stress, inhibition of global protein synthesis, reduced overall cellular thiol level and thioredoxin reductase activity. We also performed a RT-PCR and Elisa analysis of (1–4)-thiodisaccharides-treated glioma cells to identify any changes within the pathway affected by (1–4)-thiodisaccharides. We observed a significant increase of expression in key markers of endoplasmic reticulum stress and pro-apoptotic protein, FASLG. We proposed that (1–4)-thiodisaccharides react with cellular thiols and disturb any cellular thiol-depended processes like thioredoxin system or protein folding.

Cross Talk between eIF2α and eEF2 Phosphorylation Pathways Optimizes Translational Arrest in Response to Oxidative Stress.

SummaryThe cellular stress response triggers a cascade of events leading to transcriptional reprogramming and a transient inhibition of global protein synthesis, which is thought to be mediated by phosphorylation of eukaryotic initiation factor-2α (eIF2α). Using mouse embryonic fibroblasts (MEFs) and the fission yeast S. pombe, we report that rapid translational arrest and cell survival in response to hydrogen peroxide-induced oxidative stress do not rely on eIF2α kinases and eIF2α phosphorylation. Rather, H2O2 induces a block in elongation through phosphorylation of eukaryotic elongation factor 2 (eEF2). Kinetic and dose-response analyses uncovered cross talk between the eIF2α and eEF2 phosphorylation pathways, indicating that, in MEFs, eEF2 phosphorylation initiates the acute shutdown in translation, which is maintained by eIF2α phosphorylation. Our results challenge the common conception that eIF2α phosphorylation is the primary trigger of translational arrest in response to oxidative stress and point to integrated control that may facilitate the survival of cancer cells.

24(S)-Hydroxycholesterol induces ER dysfunction-mediated unconventional cell death

Endoplasmic reticulum (ER) stress induced by disruption of protein folding activates the unfolded protein response (UPR), which while generally pro-survival in effect can also induce cell death under severe ER stress. 24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the ER of neurons, plays an important role in maintaining brain cholesterol homeostasis but also shows neurotoxicity when subjected to esterification by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in the ER. In this study, we demonstrated that the accumulation of 24S-OHC esters in human neuroblastoma SH-SY5Y cells evoked the UPR with substantially no pro-survival adaptive response but with significant activation of pro-death UPR signaling via regulated IRE1-dependent decay (RIDD). We further found that accumulation of 24S-OHC esters caused disruption of ER membrane integrity and release of ER luminal proteins into cytosol. We also found that de novo synthesis of global proteins was robustly suppressed in 24S-OHC-treated cells. Collectively, these results show that ER dysfunction and the accompanying RIDD-mediated pro-death UPR signaling and global protein synthesis inhibition are responsible for 24S-OHC ester-induced unconventional cell death.