IU Indianapolis ScholarWorks :: Browsing by Subject "Translation"

Browsing by Subject "Translation"

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Activation of Gcn2 by Pharmacological Agents Designed to be Inhibitors
(2023-01) Carlson, Kenneth Reed; Wek, Ronald C.; Georgiadis, Millie M.; Liu, Yunlong; Staschke, Kirk A.; Turchi, John J.
The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stressadaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. This thesis reports that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, probes the mechanism by which this activation occurs, and compares the mechanism of Gcn2 activation by Gcn2iB to that of uncharged tRNA. In this study, Gcn2 activation was measured in cultured human cells by immunoblot and luciferase reporter assays making use of a genetic complementation assay to assess the contribution of various Gcn2 residues to its activation. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived fromGcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. However, compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.
Adaptation of the Client Diagnostic Questionnaire for East Africa
(Public Library of Science, 2024-03-19) Kwobah, Edith Kamaru; Goodrich, Suzanne; Kulzer, Jayne Lewis; Kanyesigye, Michael; Obatsa, Sarah; Cheruiyot, Julius; Kiprono, Lorna; Kibet, Colma; Ochieng, Felix; Bukusi, Elizabeth A.; Ofner, Susan; Brown, Steven A.; Yiannoutsos, Constantin T.; Atwoli, Lukoye; Wools-Kaloustian, Kara; Medicine, School of Medicine
Research increasingly involves cross-cultural work with non-English-speaking populations, necessitating translation and cultural validation of research tools. This paper describes the process of translating and criterion validation of the Client Diagnostic Questionnaire (CDQ) for use in a multisite study in Kenya and Uganda. The English CDQ was translated into Swahili, Dholuo (Kenya) and Runyankole/Rukiga (Uganda) by expert translators. The translated documents underwent face validation by a bilingual committee, who resolved unclear statements, agreed on final translations and reviewed back translations to English. A diagnostic interview by a mental health specialist was used for criterion validation, and Kappa statistics assessed the strength of agreement between non-specialist scores and mental health professionals' diagnoses. Achieving semantic equivalence between translations was a challenge. Validation analysis was done with 30 participants at each site (median age 32.3 years (IQR = (26.5, 36.3)); 58 (64.4%) female). The sensitivity was 86.7%, specificity 64.4%, positive predictive value 70.9% and negative predictive value 82.9%. Diagnostic accuracy by the non-specialist was 75.6%. Agreement was substantial for major depressive episode and positive alcohol (past 6 months) and alcohol abuse (past 30 days). Agreement was moderate for other depressive disorders, panic disorder and psychosis screen; fair for generalized anxiety, drug abuse (past 6 months) and Post Traumatic Stress Disorder (PTSD); and poor for drug abuse (past 30 days). Variability of agreement between sites was seen for drug use (past 6 months) and PTSD. Our study successfully adapted the CDQ for use among people living with HIV in East Africa. We established that trained non-specialists can use the CDQ to screen for common mental health and substance use disorders with reasonable accuracy. Its use has the potential to increase case identification, improve linkage to mental healthcare, and improve outcomes. We recommend further studies to establish the psychometric properties of the translated tool.
As Extracellular Glutamine Levels Decline, Asparagine Becomes an Essential Amino Acid
(Elsevier, 2018-02-06) Pavlova, Natalya N.; Hui, Sheng; Ghergurovich, Jonathan M.; Fan, Jing; Intlekofer, Andrew M.; White, Richard M.; Rabinowitz, Joshua D.; Thompson, Craig B.; Zhang, Ji; Pediatrics, School of Medicine
When mammalian cells are deprived of glutamine, exogenous asparagine rescues cell survival and growth. Here we report that this rescue results from use of asparagine in protein synthesis. All mammalian cell lines tested lacked cytosolic asparaginase activity and could not utilize asparagine to produce other amino acids or biosynthetic intermediates. Instead, most glutamine-deprived cell lines are capable of sufficient glutamine synthesis to maintain essential amino acid uptake and production of glutamine-dependent biosynthetic precursors, with the exception of asparagine. While experimental introduction of cytosolic asparaginase could enhance the synthesis of glutamine and increase tricarboxylic acid cycle anaplerosis and the synthesis of nucleotide precursors, cytosolic asparaginase suppressed the growth and survival of cells in glutamine-depleted medium in vitro and severely compromised the in vivo growth of tumor xenografts. These results suggest that the lack of asparaginase activity represents an evolutionary adaptation to allow mammalian cells to survive pathophysiologic variations in extracellular glutamine.
Autophagy-related 7 (ATG7) regulates food intake and liver health during asparaginase exposure
(Elsevier, 2025) Zalma, Brian A.; Ibrahim, Maria; Rodriguez-Polanco, Flavio C.; Bhavsar, Chintan T.; Rodriguez, Esther M.; Cararo-Lopes, Eduardo; Farooq, Saad A.; Levy, Jordan L.; Wek, Ronald C.; White, Eileen; Anthony, Tracy G.; Biochemistry and Molecular Biology, School of Medicine
Amino acid starvation by the chemotherapy agent asparaginase is a potent activator of the integrated stress response (ISR) in the liver and can upregulate autophagy in some cell types. We hypothesized that autophagy-related 7 (ATG7), a protein that is essential for autophagy and an ISR target gene, was necessary during exposure to asparaginase to maintain liver health. We knocked down Atg7 systemically (Atg7Δ/Δ) or in hepatocytes only (ls-Atg7KO) in mice before exposure to pegylated asparaginase for 5 days. Intact mice injected with asparaginase lost body weight due to reduced food intake and increased energy expenditure. Systemic Atg7 ablation reduced liver protein synthesis and increased liver injury in vehicle-injected mice but did not further reduce liver protein synthesis, exacerbate steatosis or liver injury, or alter energy expenditure following 5 days of asparaginase exposure. Atg7Δ/Δ mice were unexpectantly protected from asparaginase-induced anorexia and weight loss. This protection corresponded with reduced phosphorylation of hepatic GCN2 and blunted increases in ISR gene targets including growth differentiation factor 15 (GDF15), a negative regulator of food intake. Interestingly, asparaginase elevated serum GDF15 and reduced food intake in ls-Atg7KO mice, similar to intact mice. Liver triglycerides and production of the hepatokine fibroblast growth factor 21, another ISR gene target, were suppressed in asparaginase-exposed Atg7Δ/Δ and ls-Atg7KO mice. This work identifies a bidirectional relationship between autophagy and the ISR in the liver during asparaginase, affecting food intake and liver health.
Bacterial sepsis triggers an antiviral response that causes translation shutdown
(American Society for Clinical Investigation, 2019-01-02) Hato, Takashi; Maier, Bernhard; Syed, Farooq; Myslinski, Jered; Zollman, Amy; Plotkin, Zoya; Eadon, Michael T.; Dagher, Pierre C.; Medicine, School of Medicine
In response to viral pathogens, the host upregulates antiviral genes that suppress translation of viral mRNAs. However, induction of such antiviral responses may not be exclusive to viruses, as the pathways lie at the intersection of broad inflammatory networks that can also be induced by bacterial pathogens. Using a model of Gram-negative sepsis, we show that propagation of kidney damage initiated by a bacterial origin ultimately involves antiviral responses that result in host translation shutdown. We determined that activation of the eukaryotic translation initiation factor 2-α kinase 2/eukaryotic translation initiation factor 2α (Eif2ak2/Eif2α) axis is the key mediator of translation initiation block in late-phase sepsis. Reversal of this axis mitigated kidney injury. Furthermore, temporal profiling of the kidney translatome revealed that multiple genes involved in formation of the initiation complex were translationally altered during bacterial sepsis. Collectively, our findings imply that translation shutdown is indifferent to the specific initiating pathogen and is an important determinant of tissue injury in sepsis.
Cap-independent translation directs stress-induced differentiation of the protozoan parasite Toxoplasma gondii
(Elsevier, 2024) Dey, Vishakha; Holmes, Michael J.; Bastos, Matheus S.; Wek, Ronald C.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
Translational control mechanisms modulate the microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5'-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5'-cap is impaired by insertion of a 5'-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we determined that a trans-acting RNA-binding protein called BFD2/ROCY1 is necessary for the cap-independent translation of BFD1 through its binding to the 5'-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.
Chronic high fat feeding restricts islet mRNA translation initiation independently of ER stress via DNA damage and p53 activation
(Springer Nature, 2017-06-19) Hatanaka, Masayuki; Anderson-Baucum, Emily; Lakhter, Alexander; Kono, Tatsuyoshi; Maier, Bernhard; Tersey, Sarah A.; Tanizawa, Yukio; Evans-Molina, Carmella; Mirmira, Raghavendra G.; Sims, Emily K.; Pediatrics, School of Medicine
Under conditions of high fat diet (HFD) consumption, glucose dyshomeostasis develops when β-cells are unable to adapt to peripheral insulin demands. Few studies have interrogated the molecular mechanisms of β-cell dysfunction at the level of mRNA translation under such conditions. We sought to address this issue through polyribosome profile analysis of islets from mice fed 16-weeks of 42% HFD. HFD-islet analysis revealed clear trends toward global reductions in mRNA translation with a significant reduction in the polyribosome/monoribosome ratio for Pdx1 mRNA. Transcriptional and translational analyses revealed endoplasmic reticulum stress was not the etiology of our findings. HFD-islets demonstrated evidence of oxidative stress and DNA damage, as well as activation of p53. Experiments in MIN-6 β-cells revealed that treatment with doxorubicin to directly induce DNA damage mimicked our observed effects in islets. Islets from animals treated with pioglitazone concurrently with HFD demonstrated a reversal of effects observed from HFD alone. Finally, HFD-islets demonstrated reduced expression of multiple ribosome biogenesis genes and the key translation initiation factor eIF4E. We propose a heretofore unappreciated effect of chronic HFD on β-cells, wherein continued DNA damage owing to persistent oxidative stress results in p53 activation and a resultant inhibition of mRNA translation.
Developing A Technical Translation Program: A Needs Assessment Study
(Midwest Research-to-Practice Conference in Adult, Continuing, and Community Education, 2003) Sukhtankar, Prajakta; Cseh, Maria
This paper presents a needs assessment study designed to determine the skills needed by technical translators to become successful in their work and content areas to be covered to develop these skills in a new technical translation program at the Department of Modern Languages and Literatures at Oakland University. A mixed qualitative and quantitative research design was used to collect data from professional technical translators. Content analysis and descriptive statistics of interview and survey data revealed several insights and suggestions regarding prerequisites for the students entering into the program, program content, and instructional strategies. Based on the findings, the paper concludes with twelve recommendations for the design of the program.
Effects of PERK eIF2α Kinase Inhibitor against Toxoplasma gondii
(American Society for Microbiology, 2018-10-24) Augusto, Leonardo; Martynowicz, Jennifer; Staschke, Kirk A.; Wek, Ronald C.; Sullivan, William J., Jr.; Biochemistry and Molecular Biology, School of Medicine
Toxoplasma gondii is an obligate intracellular parasite that has infected one-third of the population. Upon infection of warm-blooded vertebrates, the replicating form of the parasite (tachyzoite) converts into a latent form (bradyzoite) present in tissue cysts. During immune deficiency, bradyzoites can reconvert into tachyzoites and cause life-threatening toxoplasmosis. We previously reported that translational control through phosphorylation of the α subunit of T. gondii eukaryotic initiation factor 2 (eIF2α) (TgIF2α) is a critical component of the parasite stress response. Diverse stresses can induce the conversion of tachyzoites to bradyzoites, including those disrupting the parasite's endoplasmic reticulum (ER) (ER stress). Toxoplasma possesses four eIF2α kinases, one of which (TgIF2K-A) localizes to the parasite ER analogously to protein kinase R-like endoplasmic reticulum kinase (PERK), the eIF2α kinase that responds to ER stress in mammalian cells. Here, we investigated the effects of a PERK inhibitor (PERKi) on Toxoplasma Our results show that the PERKi GSK2606414 blocks the enzymatic activity of TgIF2K-A and reduces TgIF2α phosphorylation specifically in response to ER stress. PERKi also significantly impeded multiple steps of the tachyzoite lytic cycle and sharply lowered the frequency of bradyzoite differentiation in vitro Pretreatment of host cells with PERKi prior to infection did not affect parasite infectivity, and PERKi still impaired parasite replication in host cells lacking PERK. In mice, PERKi conferred modest protection from a lethal dose of Toxoplasma Our findings represent the first pharmacological evidence supporting TgIF2K-A as an attractive new target for the treatment of toxoplasmosis.
The Essential Role of the Non-Essential Amino Acid Asparagine in Lymphoid Malignancies
(2023-05) Srivastava, Sankalp; Zhang, Ji; Dong, X. Charlie; Mosley, Amber L.; Wek, Ronald C.
Cancer cells display increased metabolic demands to support their proliferation and biosynthetic needs. It has been extensively shown in cancers, that amino acids have functions beyond the role of mRNA translation. The breadth of functions makes amino acid restriction an effective strategy for cancer therapy; hence an important line of research involves targeting amino acid acquisition and metabolism therapeutically. Currently, asparagine depletion via L-Asparaginase in acute lymphoblastic leukemia (ALL) remains the only clinically approved therapy to date. In the first project, we showed that ALL cells are auxotrophic for asparagine and rely on exogenous sources for this non-essential amino acid. However, sensitivity to L-Asparaginase therapy is mitigated by the expression of the enzyme asparagine synthetase (ASNS), involved in de novo asparagine biosynthesis. We showed that this adaptive response requires two essential steps; demethylation of the ASNS promoter and recruitment of activating transcription factor 4 (ATF4) to the promoter to drive ASNS transcription. Our follow-up study in ALL cells showed that asparagine bioavailability (through de novo biosynthesis or exogenous sources) is essential to maintain the expression of the critical oncogene c-MYC. c-MYC is a potent transcription factor and is dysregulated in over 60% of cancers, including hematopoietic malignancies. We showed that this regulation by asparagine is primarily at the translation level and c-MYC expression is rescued only when exogenous asparagine is available or when cells can undertake de novo biosynthesis. At the biochemical level, asparagine depletion also causes an induction of ATF4 mediated stress response and suppression of global translation mediated by decreased mammalian target of rapamycin complex 1 (mTORC1) activity. However, we found that neither inhibition of the stress response or rescuing global translation rescued c-MYC protein expression. We also extended this observation to c-MYC-driven lymphomas using cell lines and orthotopic in vivo models. We showed that genetic inhibition of ASNS or pharmacological inhibition of asparagine production can significantly limit c-MYC protein and tumor growth when environmental asparagine is limiting. Overall, our work shows an essential role for asparagine in lymphoid cancers and has expanded on the usage of L-Asparaginase to resistant leukemias and lymphomas.

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