56 results on '"Bo-Jhih Guan"'
Search Results
2. Translational control of breast cancer plasticity
- Author
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Michael Jewer, Laura Lee, Matthew Leibovitch, Guihua Zhang, Jiahui Liu, Scott D. Findlay, Krista M. Vincent, Kristofferson Tandoc, Dylan Dieters-Castator, Daniela F. Quail, Indrani Dutta, Mackenzie Coatham, Zhihua Xu, Aakshi Puri, Bo-Jhih Guan, Maria Hatzoglou, Andrea Brumwell, James Uniacke, Christos Patsis, Antonis Koromilas, Julia Schueler, Gabrielle M. Siegers, Ivan Topisirovic, and Lynne-Marie Postovit
- Subjects
Science - Abstract
Protein synthesis suppression protects breast cancer cells from clinically relevant stresses like hypoxia. Here, the authors show that unique mRNA isoforms that govern stem cell-like phenotypes escape translational repression to drive tumor progression and chemoresistance.
- Published
- 2020
- Full Text
- View/download PDF
3. Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response.
- Author
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Mohammed R Alzahrani, Bo-Jhih Guan, Leah L Zagore, Jing Wu, Chien-Wen Chen, Donny D Licatalosi, Kristian E Baker, and Maria Hatzoglou
- Subjects
Medicine ,Science - Abstract
Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation in the acute phase of the UPR is not well understood. Here we analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase. We have previously shown that global translational repression induced by the acute UPR was characterized by decreased translation and increased stability of XBP1 mRNA. We demonstrate here that this stabilization is independent of new transcription. In contrast, we show XBP1 mRNA newly synthesized during the acute phase accumulates with long poly(A) tails and escapes translational repression. Inhibition of newly synthesized RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. Furthermore, during the chronic phase of the UPR, levels of XBP1 mRNA with long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced mRNAs show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. We conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation.
- Published
- 2022
- Full Text
- View/download PDF
4. PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation
- Author
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Kenneth T Farabaugh, Dawid Krokowski, Bo-Jhih Guan, Zhaofeng Gao, Xing-Huang Gao, Jing Wu, Raul Jobava, Greeshma Ray, Tristan J de Jesus, Massimiliano G Bianchi, Evelyn Chukwurah, Ovidio Bussolati, Michael Kilberg, David A Buchner, Ganes C Sen, Calvin Cotton, Christine McDonald, Michelle Longworth, Parameswaran Ramakrishnan, and Maria Hatzoglou
- Subjects
Hyperosmotic stress ,TonEBP ,NF-κB p65 ,NF-κB c-Rel ,PACT ,PKR ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The inability of cells to adapt to increased environmental tonicity can lead to inflammatory gene expression and pathogenesis. The Rel family of transcription factors TonEBP and NF-κB p65 play critical roles in the switch from osmoadaptive homeostasis to inflammation, respectively. Here we identified PACT-mediated PKR kinase activation as a marker of the termination of adaptation and initiation of inflammation in Mus musculus embryonic fibroblasts. We found that high stress-induced PACT-PKR activation inhibits the interaction between NF-κB c-Rel and TonEBP essential for the increased expression of TonEBP-dependent osmoprotective genes. This resulted in enhanced formation of TonEBP/NF-κB p65 complexes and enhanced proinflammatory gene expression. These data demonstrate a novel role of c-Rel in the adaptive response to hyperosmotic stress, which is inhibited via a PACT/PKR-dependent dimer redistribution of the Rel family transcription factors. Our results suggest that inhibiting PACT-PKR signaling may prove a novel target for alleviating stress-induced inflammatory diseases.
- Published
- 2020
- Full Text
- View/download PDF
5. GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells
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Dawid Krokowski, Bo-Jhih Guan, Jing Wu, Yuke Zheng, Padmanabhan P. Pattabiraman, Raul Jobava, Xing-Huang Gao, Xiao-Jing Di, Martin D. Snider, Ting-Wei Mu, Shijie Liu, Brian Storrie, Eric Pearlman, Anna Blumental-Perry, and Maria Hatzoglou
- Subjects
Biology (General) ,QH301-705.5 - Abstract
Summary: GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR) pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER)-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome. : Here, Krokowski et al. show that GADD34/PP1 protects the microtubule network, prevents Golgi fragmentation, and preserves protein trafficking independent of its action in the integrated stress response (ISR). In osmoadaptation, GADD34 facilitates trans-Golgi-mediated processing of the endoplasmic reticulum (ER)-synthesized amino acid transporter SNAT2, which in turn increases amino acid uptake. Keywords: SNAT2, GADD34, hyperosmotic stress, amino acid transport, Golgi fragmentation, ISR
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- 2017
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- View/download PDF
6. Oncogenic PIK3CA mutations reprogram glutamine metabolism in colorectal cancer
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Yujun Hao, Yardena Samuels, Qingling Li, Dawid Krokowski, Bo-Jhih Guan, Chao Wang, Zhicheng Jin, Bohan Dong, Bo Cao, Xiujing Feng, Min Xiang, Claire Xu, Stephen Fink, Neal J. Meropol, Yan Xu, Ronald A. Conlon, Sanford Markowitz, Kenneth W. Kinzler, Victor E. Velculescu, Henri Brunengraber, Joseph E. Willis, Thomas LaFramboise, Maria Hatzoglou, Guo-Fang Zhang, Bert Vogelstein, and Zhenghe Wang
- Subjects
Science - Abstract
Cancer cells rely on glutamine to replenish the TCA cycle. Here, the authors show that oncogenic PIK3CAmutations drive this metabolic rewiring in colorectal cancer cells by up-regulating glutamate pyruvate transaminase expression, thus increasing sensitivity to glutamine starvation.
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- 2016
- Full Text
- View/download PDF
7. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response
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Xing-Huang Gao, Dawid Krokowski, Bo-Jhih Guan, Ilya Bederman, Mithu Majumder, Marc Parisien, Luda Diatchenko, Omer Kabil, Belinda Willard, Ruma Banerjee, Benlian Wang, Gurkan Bebek, Charles R. Evans, Paul L. Fox, Stanton L. Gerson, Charles L. Hoppel, Ming Liu, Peter Arvan, and Maria Hatzoglou
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protein sulfhydration ,hydrogen sulfide ,Integrated Stress Response ,quantitative proteomics ,Cystathionine gamma-lyase (CSE) ,metabolic reprogramming ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.
- Published
- 2015
- Full Text
- View/download PDF
8. Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response
- Author
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Mohammed R. Alzahrani, Bo-Jhih Guan, Leah L. Zagore, Jing Wu, Donny D. Licatalosi, Kristian E. Baker, and Maria Hatzoglou
- Abstract
Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation in the acute phase of the UPR is not well understood. Here we analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase. We have previously shown that global translational repression induced by the acute UPR was characterized by decreased translation and increased stability of XBP1 mRNA. We demonstrate here that this stabilization is independent of new transcription. In contrast, we show XBP1 mRNA newly synthesized during the acute phase accumulates with long poly(A) tails and escapes translational repression. Inhibition of nascent RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. Furthermore, during the chronic phase of the UPR, levels of XBP1 mRNA with long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced mRNAs show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. We conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation.
- Published
- 2022
9. Translational control of breast cancer plasticity
- Author
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Christos Patsis, Aakshi Puri, Julia Schueler, Michael Jewer, Daniela F. Quail, Antonis E. Koromilas, Scott D. Findlay, Krista Vincent, Guihua Zhang, Andrea Brumwell, Laura Lee, Bo-Jhih Guan, James Uniacke, Dylan Dieters-Castator, Indrani Dutta, Maria Hatzoglou, Jiahui Liu, Ivan Topisirovic, Zhihua Xu, Matthew Leibovitch, Lynne-Marie Postovit, Kristofferson Tandoc, Gabrielle M. Siegers, and Mackenzie Coatham
- Subjects
0301 basic medicine ,Homeobox protein NANOG ,Nodal Protein ,Science ,Eukaryotic Initiation Factor-2 ,General Physics and Astronomy ,Antineoplastic Agents ,Breast Neoplasms ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Metastasis ,Stress signalling ,03 medical and health sciences ,Breast cancer ,0302 clinical medicine ,Cell Line, Tumor ,RNA Isoforms ,medicine ,Humans ,Integrated stress response ,Phosphorylation ,lcsh:Science ,Multidisciplinary ,Nanog Homeobox Protein ,General Chemistry ,medicine.disease ,ISRIB ,Cell Hypoxia ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Protein Biosynthesis ,030220 oncology & carcinogenesis ,Cancer cell ,MCF-7 Cells ,Cancer research ,Translational Activation ,Female ,lcsh:Q ,Snail Family Transcription Factors ,5' Untranslated Regions ,Reprogramming - Abstract
Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5’ Untranslated Regions (5’UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and “fate-switching” toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-cell-like phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis., Protein synthesis suppression protects breast cancer cells from clinically relevant stresses like hypoxia. Here, the authors show that unique mRNA isoforms that govern stem cell-like phenotypes escape translational repression to drive tumor progression and chemoresistance.
- Published
- 2020
10. Stress-induced perturbations in intracellular amino acids reprogram mRNA translation in osmoadaptation independently of the ISR
- Author
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Dawid Krokowski, Raul Jobava, Krzysztof J. Szkop, Chien-Wen Chen, Xu Fu, Sarah Venus, Bo-Jhih Guan, Jing Wu, Zhaofeng Gao, Wioleta Banaszuk, Marek Tchorzewski, Tingwei Mu, Phil Ropelewski, William C. Merrick, Yuanhui Mao, Aksoylu Inci Sevval, Helen Miranda, Shu-Bing Qian, Maria Manifava, Nicholas T. Ktistakis, Anastasios Vourekas, Eckhard Jankowsky, Ivan Topisirovic, Ola Larsson, and Maria Hatzoglou
- Subjects
Amino Acid Transport System A ,Amino Acid Transport Systems ,Protein Biosynthesis ,TOR Serine-Threonine Kinases ,Amino Acids ,General Biochemistry, Genetics and Molecular Biology - Abstract
The integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.
- Published
- 2021
11. eIF2A-knockout mice reveal decreased life span and metabolic syndrome
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Anton A. Komar, Anchal Agarwal, Bo-Jhih Guan, Roman V. Kondratov, David A. Buchner, Arnab Ghosh, Maria Hatzoglou, William C. Merrick, Barsanjit Mazumder, William M. Baldwin, Nina Dvorina, Taras Y. Nazarko, Jackson Casteel, and Richard Anderson
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Endoplasmic reticulum ,Translation (biology) ,Biology ,Biochemistry ,Article ,Cell biology ,Internal ribosome entry site ,Eukaryotic initiation factor ,Knockout mouse ,Genetics ,Unfolded protein response ,Protein biosynthesis ,Integrated stress response ,Molecular Biology ,Biotechnology - Abstract
Eukaryotic initiation factor 2A (eIF2A) is a 65 kDa protein that functions in minor initiation pathways, which affect the translation of only a subset of messenger ribonucleic acid (mRNAs), such as internal ribosome entry site (IRES)-containing mRNAs and/or mRNAs harboring upstream near cognate/non-AUG start codons. These non-canonical initiation events are important for regulation of protein synthesis during cellular development and/or the integrated stress response. Selective eIF2A knockdown in cellular systems significantly inhibits translation of such mRNAs, which rely on alternative initiation mechanisms for their translation. However, there exists a gap in our understanding of how eIF2A functions in mammalian systems in vivo (on the organismal level) and ex vivo (in cells). Here, using an eIF2A-knockout (KO) mouse model, we present evidence implicating eIF2A in the biology of aging, metabolic syndrome and central tolerance. We discovered that eIF2A-KO mice have reduced life span and that eIF2A plays an important role in maintenance of lipid homeostasis, the control of glucose tolerance, insulin resistance and also reduces the abundance of B lymphocytes and dendritic cells in the thymic medulla of mice. We also show the eIF2A KO affects male and female mice differently, suggesting that eIF2A may affect sex-specific pathways. Interestingly, our experiments involving pharmacological induction of endoplasmic reticulum (ER) stress with tunicamycin did not reveal any substantial difference between the response to ER stress in eIF2A-KO and wild-type mice. The identification of eIF2A function in the development of metabolic syndrome bears promise for the further identification of specific eIF2A targets responsible for these changes.
- Published
- 2021
12. Adaptation to chronic ER stress enforces pancreatic β-cell plasticity
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Maria Hatzoglou, Clayton E. Mathews, Mohammed R. Alzahrani, Ivan C. Gerling, Chien-Wen Chen, Syrena Bracey, Hugo Lee, Leena Haataja, Bo-Jhih Guan, Zhaofeng Gao, Thomas LaFramboise, Boaz Tirosh, Ashleigh E. Schaffer, Peter Arvan, Feyza Engin, Klaus H. Kaestner, Ilya Bederman, and Long Gao
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Pathogenesis ,medicine.anatomical_structure ,Chemistry ,Endoplasmic reticulum ,Cell Plasticity ,Cell ,Unfolded protein response ,medicine ,Beta (finance) ,Reprogramming ,Proinsulin ,Cell biology - Abstract
Pancreatic β-cells undergo high levels of endoplasmic reticulum (ER) stress due to their role in insulin secretion. Hence, they require sustainable and efficient adaptive stress responses to cope with the stress. Whether duration and episodes of chronic ER stress directly compromises β-cell identity is largely unknown. We show that under reversible, chronic ER stress, β-cells undergo a distinct transcriptional and translational reprogramming. During reprogramming, expression of master regulators of β-cell function and identity and proinsulin processing is impaired. Upon recovery from stress, β-cells regain their identity, highlighting a high-degree of adaptive β-cell plasticity. Remarkably, when stress episodes exceed a certain threshold, β-cell identity is gradually lost. Single cell RNA-seq analysis of islets from type 1 diabetes (T1D) patients, identifies the severe deregulation of the chronic stress-adaptation program, and reveals novel biomarkers for progression of T1D. Our results suggest β-cell adaptive exhaustion (βEAR) is a significant component of the pathogenesis of T1D.
- Published
- 2021
13. Adaptation to chronic ER stress enforces pancreatic β-cell plasticity
- Author
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Chien-Wen Chen, Bo-Jhih Guan, Mohammed R. Alzahrani, Zhaofeng Gao, Long Gao, Syrena Bracey, Jing Wu, Cheikh A. Mbow, Raul Jobava, Leena Haataja, Ajay H. Zalavadia, Ashleigh E. Schaffer, Hugo Lee, Thomas LaFramboise, Ilya Bederman, Peter Arvan, Clayton E. Mathews, Ivan C. Gerling, Klaus H. Kaestner, Boaz Tirosh, Feyza Engin, and Maria Hatzoglou
- Subjects
Multidisciplinary ,Insulin-Secreting Cells ,Cell Plasticity ,General Physics and Astronomy ,Humans ,Insulin ,General Chemistry ,Endoplasmic Reticulum ,Endoplasmic Reticulum Stress ,Adaptation, Physiological ,General Biochemistry, Genetics and Molecular Biology - Abstract
Pancreatic β-cells are prone to endoplasmic reticulum (ER) stress due to their role in insulin secretion. They require sustainable and efficient adaptive stress responses to cope with this stress. Whether episodes of chronic stress directly compromise β-cell identity is unknown. We show here under reversible, chronic stress conditions β-cells undergo transcriptional and translational reprogramming associated with impaired expression of regulators of β-cell function and identity. Upon recovery from stress, β-cells regain their identity and function, indicating a high degree of adaptive plasticity. Remarkably, while β-cells show resilience to episodic ER stress, when episodes exceed a threshold, β-cell identity is gradually lost. Single cell RNA-sequencing analysis of islets from type 1 diabetes patients indicates severe deregulation of the chronic stress-adaptation program and reveals novel biomarkers of diabetes progression. Our results suggest β-cell adaptive exhaustion contributes to diabetes pathogenesis.
- Published
- 2021
14. Author response for 'A tale of two proteins: PACT and PKR and their roles in inflammation'
- Author
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Parameswaran Ramakrishnan, Evelyn Chukwurah, Maria Hatzoglou, Kenneth T. Farabaugh, and Bo-Jhih Guan
- Subjects
business.industry ,Immunology ,Medicine ,Inflammation ,medicine.symptom ,business ,Pact ,Protein kinase R - Published
- 2020
15. A tale of two proteins: PACT and PKR and their roles in inflammation
- Author
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Kenneth T. Farabaugh, Parameswaran Ramakrishnan, Maria Hatzoglou, Evelyn Chukwurah, and Bo-Jhih Guan
- Subjects
0301 basic medicine ,viruses ,Inflammation ,Biology ,Pact ,Biochemistry ,Article ,Proinflammatory cytokine ,03 medical and health sciences ,eIF-2 Kinase ,0302 clinical medicine ,Interferon ,medicine ,Humans ,Molecular Biology ,Activator (genetics) ,RNA-Binding Proteins ,Inflammasome ,Cell Biology ,Protein kinase R ,Cell biology ,030104 developmental biology ,030220 oncology & carcinogenesis ,medicine.symptom ,Signal transduction ,medicine.drug ,Signal Transduction - Abstract
Inflammation is a pathological hallmark associated with bacterial and viral infections, autoimmune diseases, genetic disorders, obesity and diabetes, as well as environmental stresses including physical and chemical trauma. Among numerous proteins regulating proinflammatory signaling, very few such as Protein kinase R (PKR), have been shown to play an all-pervading role in inflammation induced by varied stimuli. PKR was initially characterized as an interferon-inducible gene activated by viral double-stranded RNA with a role in protein translation inhibition. However, it has become increasingly clear that PKR is involved in multiple pathways that promote inflammation in response to stress activation, both dependent on and independent of its cellular protein activator of PKR (PACT). In this review, we discuss the signaling pathways that contribute to the initiation of inflammation, including Toll-like receptor, interferon, and RIG-I-like receptor signaling, as well as inflammasome activation. We go on to discuss the specific roles that PKR and PACT play in such proinflammatory signaling, as well as in metabolic syndrome- and environmental stress-induced inflammation.
- Published
- 2020
16. Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress
- Author
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Yuanhui Mao, Dawid Krokowski, Erica Shu, Di Hu, Raul Jobava, Eckhard Jankowsky, Evelyn Chukwurah, Leah L. Zagore, Xin Qi, Youwei Zhang, Shu-Bing Qian, Ivan Topisirovic, Zhaofeng Gao, Donny D. Licatalosi, Bo-Jhih Guan, William C. Merrick, Maria Hatzoglou, and Jing Wu
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Eukaryotic translation ,Mechanism (biology) ,Integrated stress response ,Translation (biology) ,Fragmentation (cell biology) ,Mitochondrion ,Biology ,Ribosome ,Function (biology) ,Cell biology - Abstract
SummaryMammalian cells have to adapt to environmental challenges that range from mild to severe stress. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe stress conditions, cells induce a transient hibernation-like mechanism that anticipates recovery. We demonstrate that this Adaptive Pausing Response (APR) is a coordinated cellular response that limits ATP supply and consumption though mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery from severe stress. We further show that recovery from severe stress involves adaptive ISR (Integrated Stress Response) signaling that in turn permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress through the APR, a mechanism that preserves vital elements of cellular function under harsh environmental conditions.
- Published
- 2020
17. Adipocyte-specific deletion of zinc finger protein 407 results in lipodystrophy and insulin resistance in mice
- Author
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Justine Ngo, Maria Hatzoglou, Xuan Xu, Alyssa Charrier, David A. Buchner, Bo-Jhih Guan, and Anthony Wynshaw-Boris
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0301 basic medicine ,Male ,medicine.medical_specialty ,Lipodystrophy ,Glucose uptake ,Adipose Tissue, White ,Adipose tissue ,Embryonic Development ,030209 endocrinology & metabolism ,White adipose tissue ,Biology ,Diet, High-Fat ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Gene Knockout Techniques ,Mice ,0302 clinical medicine ,Endocrinology ,Insulin resistance ,Adipose Tissue, Brown ,Internal medicine ,Adipocyte ,Brown adipose tissue ,medicine ,Adipocytes ,Animals ,Insulin ,RNA, Small Interfering ,Molecular Biology ,Adipogenesis ,Gene Expression Regulation, Developmental ,3T3 Cells ,medicine.disease ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,Glucose ,chemistry ,Gene Expression Regulation ,Female ,Insulin Resistance - Abstract
PPARγ deficiency in humans and model organisms impairs the transcriptional control of adipogenesis and mature adipocyte function resulting in lipodystrophy and insulin resistance. Zinc finger protein 407 (ZFP407) positively regulates PPARγ target gene expression and insulin-stimulated glucose uptake in cultured adipocytes. The in vivo physiological role of ZFP407 in mature adipocytes, however, remains to be elucidated. Here we generated adipocyte-specific ZFP407 knockout (AZKO) mice and discovered a partial lipodystrophic phenotype with reduced fat mass, hypertrophic adipocytes in inguinal and brown adipose tissue, and reduced adipogenic gene expression. The lipodystrophy was further exacerbated in AZKO mice fed a high-fat diet. Glucose and insulin tolerance tests revealed decreased insulin sensitivity in AZKO mice compared to control littermates. Cell-based assays demonstrated that ZFP407 is also required for adipogenesis, which may also contribute to the lipodystrophic phenotype. These results demonstrate an essential in vivo role of ZFP407 in brown and white adipose tissue formation and organismal insulin sensitivity.
- Published
- 2020
18. Retrograde signaling by a mtDNA-encoded non-coding RNA preserves mitochondrial bioenergetics
- Author
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Danyelle M. Townsend, C. E. Nelson, N. D. Molyneaux, H. Kenche, A. Blumental-Perry, Naftali Kaminski, Farida Ahangari, Maria Hatzoglou, E. Prendergas, N. A. Perry, Zhi-Wei Ye, Bo-Jhih Guan, Raul Jobava, Dawid Krokowski, K. Pandit, A. Miron, Min-Jong Kang, Susan H. Guttentag, Jie Zhang, Jing Wu, Y. Perry, A. J. Degar, P. A. Linden, and Ilya Bederman
- Subjects
0301 basic medicine ,Mitochondrial DNA ,RNA, Untranslated ,Bioenergetics ,Medicine (miscellaneous) ,Pathogenesis ,Biology ,Mitochondrion ,DNA, Mitochondrial ,General Biochemistry, Genetics and Molecular Biology ,Article ,Non-coding RNAs ,Cell Line ,Cigarette Smoking ,Electron Transport ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,Animals ,Gene ,lcsh:QH301-705.5 ,Cell Nucleus ,RNA ,Non-coding RNA ,Cell biology ,Mitochondria ,Mice, Inbred C57BL ,MicroRNAs ,030104 developmental biology ,lcsh:Biology (General) ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,Alveolar Epithelial Cells ,Retrograde signaling ,Female ,General Agricultural and Biological Sciences ,Energy Metabolism ,Signal Transduction - Abstract
Alveolar epithelial type II (AETII) cells are important for lung epithelium maintenance and function. We demonstrate that AETII cells from mouse lungs exposed to cigarette smoke (CS) increase the levels of the mitochondria-encoded non-coding RNA, mito-RNA-805, generated by the control region of the mitochondrial genome. The protective effects of mito-ncR-805 are associated with positive regulation of mitochondrial energy metabolism, and respiration. Levels of mito-ncR-805 do not relate to steady-state transcription or replication of the mitochondrial genome. Instead, CS-exposure causes the redistribution of mito-ncR-805 from mitochondria to the nucleus, which correlated with the increased expression of nuclear-encoded genes involved in mitochondrial function. These studies reveal an unrecognized mitochondria stress associated retrograde signaling, and put forward the idea that mito-ncRNA-805 represents a subtype of small non coding RNAs that are regulated in a tissue- or cell-type specific manner to protect cells under physiological stress., Blumental-Perry et al. find that exposure to cigarette smoke causes the redistribution of the mitochondria-encoded non-codling RNA, mito-ncR-805, from mitochondria to the nucleus in alveolar epithelial cells. This relocation event correlates with increased expression of nuclear genes involved in mitochondrial function, suggesting that this response has a protective role.
- Published
- 2020
19. Retrograde Signaling by Mitochondria-Encoded Mito-ncR-805 Preserves Mitochondrial Function of Alveolar Epithelial Type 2 Cells During Exposure to Cigarette Smoke
- Author
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Danyelle M. Townsend, Ilya Bederman, Zhi-Wei Ye, Gerald S. Shadel, Y. Perry, N. D. Molyneaux, Raul Jobava, Min-Jong Kang, Jing Wu, Maria Hatzoglou, Naftali Kaminski, A. Miron, S.H. Guttentag, N. A. Perry, Bo-Jhih Guan, and Anna Blumental-Perry
- Subjects
Chemistry ,Retrograde signaling ,Cigarette smoke ,Mitochondrion ,Function (biology) ,Cell biology - Published
- 2020
20. Author response: PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation
- Author
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Zhaofeng Gao, Dawid Krokowski, Christine McDonald, Tristan J de Jesus, Xing-Huang Gao, David A. Buchner, Ganes C. Sen, Raul Jobava, Evelyn Chukwurah, Greeshma Ray, Calvin U. Cotton, Kenneth T. Farabaugh, Parameswaran Ramakrishnan, Bo-Jhih Guan, Michael S. Kilberg, Maria Hatzoglou, Jing Wu, Ovidio Bussolati, Massimiliano G. Bianchi, and Michelle Suzanne Longworth
- Subjects
Osmotic shock ,Chemistry ,medicine ,Inflammation ,medicine.symptom ,Pact ,Protein kinase R ,Cell biology ,Intensity (physics) - Published
- 2020
21. RITA requires eIF2α-dependent modulation of mRNA translation for its anti-cancer activity
- Author
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Johannes Ristau, Shuo Liang, Galina Selivanova, Ivan Topisirovic, Sylvain Peuget, Vincent van Hoef, Jiawei Zhu, Ola Larsson, Maria Hatzoglou, and Bo-Jhih Guan
- Subjects
0301 basic medicine ,Cancer Research ,Eukaryotic Initiation Factor-2 ,Immunology ,Cell ,Antineoplastic Agents ,Apoptosis ,Breast Neoplasms ,Drug development ,Article ,law.invention ,eIF-2 Kinase ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,law ,Protein biosynthesis ,medicine ,Humans ,RNA, Messenger ,Phosphorylation ,lcsh:QH573-671 ,Regulation of gene expression ,lcsh:Cytology ,Chemistry ,Translation (biology) ,Cell Biology ,Neoplasm Proteins ,3. Good health ,Cell biology ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,medicine.anatomical_structure ,Protein Biosynthesis ,030220 oncology & carcinogenesis ,MCF-7 Cells ,Suppressor ,Female ,Tumor Suppressor Protein p53 ,Signal transduction ,Reactive Oxygen Species ,Signal Transduction - Abstract
Tumor protein 53 (p53, encoded by the TP53 gene) is a key tumor suppressor regulating cell fates in response to internal and external stresses. As TP53 is mutated or silenced in a majority of tumors, reactivation of p53 by small molecules represents a promising strategy in cancer therapeutics. One such agent is RITA (reactivation of p53 and induction of tumor cell apoptosis), which restores p53 expression in cells with hyperactive HDM2 and induces apoptosis. Yet, mechanisms underlying the anticancer activity of RITA are incompletely understood. Here we show that RITA suppresses mRNA translation independently of p53 by inducing eIF2α phosphorylation. Surprisingly, reactivation of p53 following RITA treatment is critically dependent on eIF2α phosphorylation. Moreover, inhibition of eIF2α phosphorylation attenuates pro-apoptotic and anti-neoplastic effects of RITA, while inducing phosphorylation of eIF2α enhances the anticancer activity of RITA. Collectively, these findings demonstrate that the translational machinery plays a major role in determining the antineoplastic activity of RITA, and suggest that combining p53 activators and translation modulators may be beneficial.
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- 2019
22. GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells
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Maria Hatzoglou, Padmanabhan P. Pattabiraman, Anna Blumental-Perry, Brian Storrie, Dawid Krokowski, Xing-Huang Gao, Eric Pearlman, Ting Wei Mu, Yuke Zheng, Jing Wu, Raul Jobava, Xiao Jing Di, Martin D. Snider, Shijie Liu, and Bo-Jhih Guan
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0301 basic medicine ,Osmosis ,Osmotic shock ,Amino Acid Transport System A ,Medical Physiology ,Phosphatase ,Blotting, Western ,Protein Serine-Threonine Kinases ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,symbols.namesake ,Protein Phosphatase 1 ,Extracellular ,2.1 Biological and endogenous factors ,Integrated stress response ,Humans ,ISR ,Aetiology ,Amino Acids ,Eye Disease and Disorders of Vision ,lcsh:QH301-705.5 ,SNAT2 ,amino acid transport ,Blotting ,Chemistry ,Reverse Transcriptase Polymerase Chain Reaction ,Endoplasmic reticulum ,Protein phosphatase 1 ,Golgi apparatus ,Transport protein ,Cell biology ,Protein Transport ,030104 developmental biology ,lcsh:Biology (General) ,hyperosmotic stress ,GADD34 ,symbols ,Biochemistry and Cell Biology ,Western ,Golgi fragmentation - Abstract
Summary: GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR) pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER)-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome. : Here, Krokowski et al. show that GADD34/PP1 protects the microtubule network, prevents Golgi fragmentation, and preserves protein trafficking independent of its action in the integrated stress response (ISR). In osmoadaptation, GADD34 facilitates trans-Golgi-mediated processing of the endoplasmic reticulum (ER)-synthesized amino acid transporter SNAT2, which in turn increases amino acid uptake. Keywords: SNAT2, GADD34, hyperosmotic stress, amino acid transport, Golgi fragmentation, ISR
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- 2017
23. Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress
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Di Hu, Yuanhui Mao, Aleksandra Trifunovic, Dawid Krokowski, Donny D. Licatalosi, Jing Wu, Xin Erica Shu, Youwei Zhang, Maria Hatzoglou, Eckhard Jankowsky, Bo-Jhih Guan, Leah L. Zagore, Ivan Topisirovic, Saba Valadkhan, Zhaofeng Gao, Chien-Wen Chen, Andrew C. Hsieh, Xin Qi, William C. Merrick, Raul Jobava, Evelyn Chukwurah, and Shu-Bing Qian
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Osmotic shock ,Codon, Initiator ,Biology ,Mitochondrion ,Article ,Mitochondrial Proteins ,Mice ,Adenosine Triphosphate ,Eukaryotic translation ,Osmotic Pressure ,Animals ,Humans ,Integrated stress response ,Fragmentation (cell biology) ,Molecular Biology ,PI3K/AKT/mTOR pathway ,Cell Proliferation ,ATF4 ,Translation (biology) ,Cell Biology ,Fibroblasts ,Adaptation, Physiological ,Mitochondria ,Cell biology ,Kinetics ,HEK293 Cells ,Protein Biosynthesis ,Ribosomes ,Signal Transduction - Abstract
To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.
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- 2021
24. IL-4 up-regulates cyclooxygenase-1 expression in macrophages
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Ashley E. Shay, Joseph F. Urban, Bo-Jhih Guan, Vivek Narayan, K. Sandeep Prabhu, and Bastihalli T. Diwakar
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Male ,0301 basic medicine ,Immunology ,Macrophage polarization ,Bone Marrow Cells ,AMP-Activated Protein Kinases ,Ligands ,Biochemistry ,Immunomodulation ,03 medical and health sciences ,Animals ,Humans ,Enzyme Inhibitors ,Receptor ,Protein kinase A ,Molecular Biology ,Mechanistic target of rapamycin ,Cells, Cultured ,Interleukin 4 ,Tissue homeostasis ,Strongylida Infections ,biology ,Prostaglandin D2 ,Macrophages ,Models, Immunological ,Membrane Proteins ,Interleukin ,AMPK ,Cell Biology ,Macrophage Activation ,Metformin ,Recombinant Proteins ,Cell biology ,Enzyme Activation ,Mice, Inbred C57BL ,Luminescent Proteins ,HEK293 Cells ,030104 developmental biology ,Enzyme Induction ,Cyclooxygenase 1 ,biology.protein ,Interleukin-4 ,Nippostrongylus - Abstract
Macrophages use various cell-surface receptors to sense their environment and undergo polarized responses. The cytokines, interleukin (IL)-4 and IL-13, released from T-helper type 2 (Th2) cells, drive macrophage polarization toward an alternatively activated phenotype (M2). This phenotype is associated with the expression of potent pro-resolving mediators, such as the prostaglandin (PG) D2-derived cyclopentenone metabolite, 15d-PGJ2, produced by the cyclooxygenase (Ptgs; Cox) pathway. Interestingly, IL-4 treatment of bone marrow-derived macrophages (BMDMs) significantly down-regulates Cox-2 protein expression, whereas Cox-1 levels are significantly increased. This phenomenon not only challenges the dogma that Cox-1 is only developmentally regulated, but also demonstrates a novel mechanism in which IL-4-dependent regulation of Cox-1 involves the activation of the mechanistic target of rapamycin complex (mTORC). Using specific chemical inhibitors, we demonstrate here that IL-4-dependent Cox-1 up-regulation occurs at the post-transcriptional level via the Fes-Akt-mTORC axis. Activation of AMP-activated protein kinase (AMPK) by metformin, inhibition of mTORC by torin 1, or CRISPR/Cas9-mediated genetic knock-out of tuberous sclerosis complex-2 (Tsc2) blocked the IL-4-dependent expression of Cox-1 and the ability of macrophages to polarize to M2. However, use of 15d-PGJ2 partially rescued the effects of AMPK activation, suggesting the importance of Cox-1 in macrophage polarization as also observed in a model of gastrointestinal helminth clearance. In summary, these findings suggest a new paradigm where IL-4-dependent up-regulation of Cox-1 expression may play a key role in tissue homeostasis and wound healing during Th2-mediated immune responses, such as parasitic infections.
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- 2017
25. Metabolic adaptation of skeletal muscle to hyperammonemia drives the beneficial effects of l-leucine in cirrhosis
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Maria Hatzoglou, Dawid Krokowski, Bo-Jhih Guan, Gangarao Davuluri, Srinivasan Dasarathy, Samjhana Thapaliya, Avinash Kumar, and Dharmvir Singh
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Liver Cirrhosis ,0301 basic medicine ,Sarcopenia ,medicine.medical_specialty ,Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Leucine ,Internal medicine ,medicine ,Animals ,Hyperammonemia ,Humans ,Myocyte ,Integrated stress response ,Phosphorylation ,Muscle, Skeletal ,Hepatology ,Myogenesis ,Muscles ,Skeletal muscle ,medicine.disease ,Lactulose ,Rats ,Glutamine ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,Hepatic Encephalopathy ,030211 gastroenterology & hepatology - Abstract
Background & Aims Increased skeletal muscle ammonia uptake with loss of muscle mass adversely affects clinical outcomes in cirrhosis. Hyperammonemia causes reduced protein synthesis and sarcopenia but the cellular responses to impaired proteostasis and molecular mechanism of l-leucine induced adaptation to ammonia induced stress were determined. Methods Response to activation of amino acid deficiency sensor, GCN2, in the skeletal muscle from cirrhotic patients and the portacaval anastomosis (PCA) rat were quantified. During hyperammonemia and l-leucine supplementation, protein synthesis, phosphorylation of eIF2α, mTORC1 signaling, l-leucine transport and response to l-leucine supplementation were quantified. Adaptation to cellular stress via ATF4 and its target GADD34 were also determined. Results Activation of the eIF2α kinase GCN2 and impaired mTORC1 signaling were observed in skeletal muscle from cirrhotic patients and PCA rats. Ammonia activated GCN2 mediated eIF2α phosphorylation (eIF2α-P) and impaired mTORC1 signaling that inhibit protein synthesis in myotubes and MEFs. Adaptation to ammonia induced stress did not involve translational reprogramming by activation transcription factor 4 (ATF4) dependent induction of the eIF2α-P phosphatase subunit GADD34. Instead, ammonia increased expression of the leucine/glutamine exchanger SLC7A5, l-leucine uptake and intracellular l-leucine levels, the latter not being sufficient to rescue the inhibition of protein synthesis, due to potentially enhanced mitochondrial sequestration of l-leucine. l-leucine supplementation rescued protein synthesis inhibition caused by hyperammonemia. Conclusions Response to hyperammonemia is reminiscent of the cellular response to amino acid starvation, but lacks the adaptive ATF4 dependent integrated stress response (ISR). Instead, hyperammonemia-induced l-leucine uptake was an adaptive response to the GCN2-mediated decreased protein synthesis. Lay summary Sarcopenia or skeletal muscle loss is the most frequent complication in cirrhosis but there are no treatments because the cause(s) of muscle loss in liver disease are not known. Results from laboratory experiments in animals and muscle cells were validated in human patients with cirrhosis to show that ammonia plays a key role in causing muscle loss in patients with cirrhosis. We identified a novel stress response to ammonia in the muscle that decreases muscle protein content that can be reversed by supplementation with the amino acid l-leucine.
- Published
- 2016
26. PACT-mediated PKR activation acts as a hyperosmotic stress intensity sensor weakening osmoadaptation and enhancing inflammation
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Tristan J de Jesus, Calvin U. Cotton, Maria Hatzoglou, Zhaofeng Gao, Michael S. Kilberg, Massimiliano G. Bianchi, Xing-Huang Gao, David A. Buchner, Jing Wu, Ovidio Bussolati, Bo-Jhih Guan, Ganes C. Sen, Raul Jobava, Evelyn Chukwurah, Greeshma Ray, Christine McDonald, Kenneth T. Farabaugh, Dawid Krokowski, Michelle Suzanne Longworth, and Parameswaran Ramakrishnan
- Subjects
0301 basic medicine ,Hyperosmotic stress ,Mouse ,Mice ,eIF-2 Kinase ,0302 clinical medicine ,Immunology and Inflammation ,Gene expression ,NF-κB p65 ,Biology (General) ,PACT ,Chemistry ,Kinase ,General Neuroscience ,NF-kappa B ,RNA-Binding Proteins ,General Medicine ,PKR ,Adaptation, Physiological ,Cell biology ,030220 oncology & carcinogenesis ,Medicine ,RNA Interference ,medicine.symptom ,Signal Transduction ,Research Article ,QH301-705.5 ,Science ,Inflammation ,Pact ,General Biochemistry, Genetics and Molecular Biology ,Proinflammatory cytokine ,Cell Line ,03 medical and health sciences ,Osmotic Pressure ,medicine ,Animals ,Humans ,Transcription factor ,NF-κB c-Rel ,General Immunology and Microbiology ,Genetics and Genomics ,Protein kinase R ,Proto-Oncogene Proteins c-rel ,030104 developmental biology ,Gene Expression Regulation ,TonEBP ,Carrier Proteins ,Homeostasis - Abstract
The inability of cells to adapt to increased environmental tonicity can lead to inflammatory gene expression and pathogenesis. The Rel family of transcription factors TonEBP and NF-κB p65 play critical roles in the switch from osmoadaptive homeostasis to inflammation, respectively. Here we identified PACT-mediated PKR kinase activation as a marker of the termination of adaptation and initiation of inflammation in Mus musculus embryonic fibroblasts. We found that high stress-induced PACT-PKR activation inhibits the interaction between NF-κB c-Rel and TonEBP essential for the increased expression of TonEBP-dependent osmoprotective genes. This resulted in enhanced formation of TonEBP/NF-κB p65 complexes and enhanced proinflammatory gene expression. These data demonstrate a novel role of c-Rel in the adaptive response to hyperosmotic stress, which is inhibited via a PACT/PKR-dependent dimer redistribution of the Rel family transcription factors. Our results suggest that inhibiting PACT-PKR signaling may prove a novel target for alleviating stress-induced inflammatory diseases., eLife digest Cells are sensitive to changes in their environment. For example, maintaining normal salt levels in the blood, also called tonicity, is essential for the health of individual cells and the organism as a whole. Tonicity controls the movement of water in and out of the cell: high levels of salt inside the cell draw water in, while high levels of salt outside the cell draw water out. If salt levels in the environment surrounding the cells become too high, too much water will be drawn out, causing the cells to shrink. Changes in tonicity can cause the cell to become stressed. Initially, cells adapt to this stress by switching on sets of genes that help restore fluid balance and allow the cell to regain its normal shape and size. If the increase in tonicity exceeds tolerable stress levels and harms the cell, this initiates an inflammatory response which ultimately leads to cell death. However, it remained unclear how cells switch from adapting to responding with inflammation. Now, Farabaugh et al. have used an experimental system which mimics high salt to identify the mechanism that allows cells to switch between these two responses. The experiments showed that when salt levels are too high, cells switch on a stress sensing protein called PACT, which activates another protein called PKR. When PACT was deleted from mouse cells, this led to a decrease in the activity of inflammatory genes, and prevented the cells from self-destructing. Other proteins that are involved in the adaptive and inflammatory response are the NF-κB family of proteins and TonEBP. Farabaugh et al. found that under low intensity stress, when salt levels outside the cell are slightly too high, a family member of NF-κB works with TonEBP to switch on adaptive genes. But, if salt levels continue to rise, PACT activates and turns on PKR. This blocks the interaction between NF-κB and TonEBP, allowing another family member of NF-κB to interact with TonEBP instead. This switches the adaptive response off and the inflammatory response on. There are many diseases that involve changes in tonicity, including diabetes, cancer, inflammatory bowel disease, and dry eye syndrome. Understanding the proteins involved in the adaptive and inflammatory response could lead to the development of drugs that help to protect cells from stress-induced damage.
- Published
- 2019
27. Translational control of breast cancer plasticity
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Christos Patsis, Michael Jewer, Laura Lee, Kristofferson Tandoc, Indrani Dutta, Lynne-Marie Postovit, Gabrielle M. Siegers, Mackenzie Coatham, Julia Schueler, Antonis E. Koromilas, Ivan Topisirovic, Jiahui Liu, Dylan Dieters-Castator, James Uniacke, Andrea Brumwell, Guihua Zhang, Bo-Jhih Guan, Maria Hatzoglou, Zhihua Xu, Daniela F. Quail, Krista Vincent, and Scott D. Findlay
- Subjects
Gene isoform ,Homeobox protein NANOG ,Cancer cell ,Cancer research ,medicine ,Translational Activation ,Biology ,medicine.disease ,Phenotype ,Reprogramming ,ISRIB ,Metastasis - Abstract
Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5’ Untranslated Regions (5’UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. This leads to accumulation of corresponding proteins which induce plasticity and “fate-switching” toward stem-cell like phenotypes. Surprisingly, we observed that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem cell-like phenotypes. Strikingly, these effects can be overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB). Collectively, our findings unravel a hitherto unappreciated mechanism of induction of plasticity of breast cancer cells, and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.
- Published
- 2019
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28. Angiogenin-Cleaved tRNA Halves Interact with Cytochrome c, Protecting Cells from Apoptosis during Osmotic Stress
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Naresh Babu V. Sepuri, Marc Parisien, Xing-Huang Gao, Madhavi Gorla, Yiyuan Yuan, Marianne Pusztai-Carey, Andrea Putnam, Raul Jobava, Zhaoyang John Feng, Maria Hatzoglou, Guo-fu Hu, Dawid Krokowski, Tao Pan, Eckhard Jankowsky, Mridusmita Saikia, and Bo-Jhih Guan
- Subjects
Osmotic shock ,Angiogenin ,Cell Survival ,Apoptosis ,Electrophoretic Mobility Shift Assay ,Mitochondrion ,Biology ,environment and public health ,Mice ,RNA, Transfer ,Osmotic Pressure ,Apoptosomes ,Animals ,Molecular Biology ,Cells, Cultured ,Ribonucleoprotein ,Neurons ,RNA Cleavage ,Base Sequence ,Caspase 3 ,Sequence Analysis, RNA ,Cytochrome c ,Cytochromes c ,RNA ,Ribonuclease, Pancreatic ,Articles ,Cell Biology ,Fibroblasts ,Caspase 9 ,Mitochondria ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Apoptotic Protease-Activating Factor 1 ,Ribonucleoproteins ,Biochemistry ,embryonic structures ,cardiovascular system ,biology.protein ,Apoptosome - Abstract
Adaptation to changes in extracellular tonicity is essential for cell survival. However, severe or chronic hyperosmotic stress induces apoptosis, which involves cytochrome c (Cyt c) release from mitochondria and subsequent apoptosome formation. Here, we show that angiogenin-induced accumulation of tRNA halves (or tiRNAs) is accompanied by increased survival in hyperosmotically stressed mouse embryonic fibroblasts. Treatment of cells with angiogenin inhibits stress-induced formation of the apoptosome and increases the interaction of small RNAs with released Cyt c in a ribonucleoprotein (Cyt c-RNP) complex. Next-generation sequencing of RNA isolated from the Cyt c-RNP complex reveals that 20 tiRNAs are highly enriched in the Cyt c-RNP complex. Preferred components of this complex are 5′ and 3′ tiRNAs of specific isodecoders within a family of isoacceptors. We also demonstrate that Cyt c binds tiRNAs in vitro, and the pool of Cyt c-interacting RNAs binds tighter than individual tiRNAs. Finally, we show that angiogenin treatment of primary cortical neurons exposed to hyperosmotic stress also decreases apoptosis. Our findings reveal a connection between angiogenin-generated tiRNAs and cell survival in response to hyperosmotic stress and suggest a novel cellular complex involving Cyt c and tiRNAs that inhibits apoptosome formation and activity.
- Published
- 2014
29. Reselection of a Genomic Upstream Open Reading Frame in Mouse Hepatitis Coronavirus 5'-Untranslated-Region Mutants
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Bo-Jhih Guan, Yi-Hsin Fan, David A. Brian, Hung-Yi Wu, and Yu Pin Su
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Untranslated region ,Five prime untranslated region ,viruses ,Mutant ,Immunology ,Codon, Initiator ,Genome, Viral ,Biology ,medicine.disease_cause ,Microbiology ,Cell Line ,Rodent Diseases ,Mice ,Open Reading Frames ,Virology ,Upstream open reading frame ,medicine ,Animals ,Point Mutation ,Coronavirus ,Genetics ,Point mutation ,Molecular biology ,Stop codon ,Genome Replication and Regulation of Viral Gene Expression ,Open reading frame ,Protein Biosynthesis ,Insect Science ,Mutation ,5' Untranslated Regions ,Coronavirus Infections - Abstract
An AUG-initiated upstream open reading frame (uORF) encoding a potential polypeptide of 3 to 13 amino acids (aa) is found within the 5′ untranslated region (UTR) of >75% of coronavirus genomes based on 38 reference strains. Potential CUG-initiated uORFs are also found in many strains. The AUG-initiated uORF is presumably translated following genomic 5′-end cap-dependent ribosomal scanning, but its function is unknown. Here, in a reverse-genetics study with mouse hepatitis coronavirus, the following were observed. (i) When the uORF AUG-initiating codon was replaced with a UAG stop codon along with a U112A mutation to maintain a uORF-harboring stem-loop 4 structure, an unimpaired virus with wild-type (WT) growth kinetics was recovered. However, reversion was found at all mutated sites within five virus passages. (ii) When the uORF was fused with genomic (main) ORF1 by converting three in-frame stop codons to nonstop codons, a uORF-ORF1 fusion protein was made, and virus replicated at WT levels. However, a frameshifting G insertion at virus passage 7 established a slightly 5′-extended original uORF. (iii) When uAUG-eliminating deletions of 20, 30, or 51 nucleotides (nt) were made within stem-loop 4, viable but debilitated virus was recovered. However, a C80U mutation in the first mutant and an A77G mutation in the second appeared by passage 10, which generated alternate uORFs that correlated with restored WT growth kinetics. In vitro , the uORF-disrupting nondeletion mutants showed enhanced translation of the downstream ORF1 compared with the WT. These results together suggest that the uORF represses ORF1 translation yet plays a beneficial but nonessential role in coronavirus replication in cell culture.
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- 2013
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30. A Self-defeating Anabolic Program Leads to β-Cell Apoptosis in Endoplasmic Reticulum Stress-induced Diabetes via Regulation of Amino Acid Flux
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Jaeseok Han, Martin D. Snider, Elena Bevilacqua, Stefan Bröer, Randal J. Kaufman, Celvie L. Yuan, Marek Tchórzewski, Antonis E. Koromilas, Mridusmita Saikia, Ovidio Bussolati, Colleen M. Croniger, Michelle Puchowicz, Scot R. Kimball, Dawid Krokowski, Peter Arvan, Tao Pan, Mithu Majumder, Bo-Jhih Guan, and Maria Hatzoglou
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Male ,Transcriptional Activation ,Amino Acid Transport Systems ,Cell Survival ,Eukaryotic Initiation Factor-2 ,Apoptosis ,Biology ,Biochemistry ,Amino Acyl-tRNA Synthetases ,Mice ,RNA, Transfer ,Insulin-Secreting Cells ,Protein biosynthesis ,Animals ,Humans ,Gene Regulation ,Chronic stress ,Amino acid transporter ,Amino Acids ,Phosphorylation ,Molecular Biology ,chemistry.chemical_classification ,Endoplasmic reticulum ,Cell Biology ,Endoplasmic Reticulum Stress ,Activating Transcription Factor 4 ,Cell biology ,Amino acid ,Mice, Inbred C57BL ,Glutamine ,HEK293 Cells ,Diabetes Mellitus, Type 2 ,chemistry ,Protein Biosynthesis ,Unfolded protein response ,Leucine ,Protein Processing, Post-Translational - Abstract
Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing β-cells in type 2 diabetes mellitus. β-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in β-cells in vivo. Paradoxically, chronic ER stress in β-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in β-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.
- Published
- 2013
31. Genome-wide Identification and Quantitative Analysis of Cleaved tRNA Fragments Induced by Cellular Stress
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Guo-fu Hu, Pavel Ivanov, Tao Pan, Paul A. Anderson, Marc Parisien, Bo-Jhih Guan, Maria Hatzoglou, Dawid Krokowski, and Mridusmita Saikia
- Subjects
Angiogenin ,Arsenites ,Eukaryotic Initiation Factor-2 ,Hypertonic Solutions ,Cleavage (embryo) ,Biochemistry ,Recombinant Angiogenin ,Substrate Specificity ,Mice ,RNA, Transfer ,Stress, Physiological ,Anticodon ,Protein biosynthesis ,Animals ,RNA, Messenger ,Ribonuclease ,Phosphorylation ,Molecular Biology ,Oligonucleotide Array Sequence Analysis ,Cell Nucleus ,Genome ,Base Sequence ,biology ,RNA ,Ribonuclease, Pancreatic ,Cell Biology ,stomatognathic diseases ,Oxidative Stress ,Gene Knockdown Techniques ,Protein Biosynthesis ,Transfer RNA ,biology.protein ,Nucleic Acid Conformation - Abstract
Certain stress conditions can induce cleavage of tRNAs around the anticodon loop via the use of the ribonuclease angiogenin. The cellular factors that regulate tRNA cleavage are not well known. In this study we used normal and eIF2α phosphorylation-deficient mouse embryonic fibroblasts and applied a microarray-based methodology to identify and compare tRNA cleavage patterns in response to hypertonic stress, oxidative stress (arsenite), and treatment with recombinant angiogenin. In all three scenarios mouse embryonic fibroblasts deficient in eIF2α phosphorylation showed a higher accumulation of tRNA fragments including those derived from initiator-tRNA(Met). We have shown that tRNA cleavage is regulated by the availability of angiogenin, its substrate (tRNA), the levels of the angiogenin inhibitor RNH1, and the rates of protein synthesis. These conclusions are supported by the following findings: (i) exogenous treatment with angiogenin or knockdown of RNH1 increased tRNA cleavage; (ii) tRNA fragment accumulation was higher during oxidative stress than hypertonic stress, in agreement with a dramatic decrease of RNH1 levels during oxidative stress; and (iii) a positive correlation was observed between angiogenin-mediated tRNA cleavage and global protein synthesis rates. Identification of the stress-specific tRNA cleavage mechanisms and patterns will provide insights into the role of tRNA fragments in signaling pathways and stress-related disorders.
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- 2012
32. Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress
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Xing-Huang Gao, Dawid Krokowski, Maria Hatzoglou, Massimiliano G. Bianchi, Bo-Jhih Guan, Kenneth T. Farabaugh, Andrew Schuster, Raul Jobava, Jing Wu, Edward D. Chan, Madhusudan Dey, Parameswaran Ramakrishnan, Mithu Majumder, Michelle Suzanne Longworth, and Antonis E. Koromilas
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0301 basic medicine ,Osmotic shock ,Nitrosation ,Nitric Oxide Synthase Type II ,Inflammation ,Apoptosis ,Biology ,Nitric Oxide ,Nitric oxide ,03 medical and health sciences ,chemistry.chemical_compound ,eIF-2 Kinase ,Osmotic Pressure ,medicine ,Extracellular ,Animals ,RNA, Messenger ,Phosphorylation ,Molecular Biology ,RNA, Double-Stranded ,Osmotic concentration ,Transcription Factor RelA ,Cell Biology ,Colitis ,Protein kinase R ,Molecular biology ,Nitric oxide synthase ,Enzyme Activation ,Mice, Inbred C57BL ,030104 developmental biology ,Enterocytes ,Phenotype ,chemistry ,biology.protein ,medicine.symptom ,Research Article - Abstract
High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA-binding domain. In turn, PKR stimulates nuclear accumulation of nuclear factor κB (NF-κB) p65 species phosphorylated at serine-536, which is paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and IL-1β. The PKR-mediated hyperinduction of iNOS decreases cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and posttranslational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo. Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress.
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- 2016
33. Oncogenic PIK3CA mutations reprogram glutamine metabolism in colorectal cancer
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Dawid Krokowski, Maria Hatzoglou, Bo Cao, Guo-Fang Zhang, Ronald A. Conlon, Thomas LaFramboise, Kenneth W. Kinzler, Bohan Dong, Joseph Willis, Claire Xu, Sanford D. Markowitz, Stephen P. Fink, Henri Brunengraber, Xiujing Feng, Yardena Samuels, Zhenghe John Wang, Qingling Li, Chao Wang, Yujun Hao, Victor E. Velculescu, Min Xiang, Bo-Jhih Guan, Yan Xu, Zhicheng Jin, Neal J. Meropol, and Bert Vogelstein
- Subjects
0301 basic medicine ,Glutamine ,Mutant ,General Physics and Astronomy ,medicine.disease_cause ,Mice ,Adenosine Triphosphate ,Enzyme Inhibitors ,Regulation of gene expression ,Mutation ,Multidisciplinary ,Aminooxyacetic Acid ,Tumor Burden ,3. Good health ,Gene Expression Regulation, Neoplastic ,Ketoglutaric Acids ,Female ,Signal transduction ,Colorectal Neoplasms ,HT29 Cells ,Signal Transduction ,endocrine system ,Class I Phosphatidylinositol 3-Kinases ,Science ,Citric Acid Cycle ,Mice, Nude ,Antineoplastic Agents ,Adenocarcinoma ,Protein Serine-Threonine Kinases ,P110α ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Cell Line, Tumor ,medicine ,Animals ,Humans ,neoplasms ,Transaminases ,Pyruvate Dehydrogenase Acetyl-Transferring Kinase ,General Chemistry ,HCT116 Cells ,Activating Transcription Factor 4 ,Xenograft Model Antitumor Assays ,Molecular biology ,Citric acid cycle ,030104 developmental biology ,Cancer cell - Abstract
Cancer cells often require glutamine for growth, thereby distinguishing them from most normal cells. Here we show that PIK3CA mutations reprogram glutamine metabolism by upregulating glutamate pyruvate transaminase 2 (GPT2) in colorectal cancer (CRC) cells, making them more dependent on glutamine. Compared with isogenic wild-type (WT) cells, PIK3CA mutant CRCs convert substantially more glutamine to α-ketoglutarate to replenish the tricarboxylic acid cycle and generate ATP. Mutant p110α upregulates GPT2 gene expression through an AKT-independent, PDK1–RSK2–ATF4 signalling axis. Moreover, aminooxyacetate, which inhibits the enzymatic activity of aminotransferases including GPT2, suppresses xenograft tumour growth of CRCs with PIK3CA mutations, but not with WT PIK3CA. Together, these data establish oncogenic PIK3CA mutations as a cause of glutamine dependency in CRCs and suggest that targeting glutamine metabolism may be an effective approach to treat CRC patients harbouring PIK3CA mutations., Cancer cells rely on glutamine to replenish the TCA cycle. Here, the authors show that oncogenic PIK3CA mutations drive this metabolic rewiring in colorectal cancer cells by up-regulating glutamate pyruvate transaminase expression, thus increasing sensitivity to glutamine starvation.
- Published
- 2016
34. Author response: Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response
- Author
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Marc Parisien, Ruma Banerjee, Mithu Majumder, Ilya Bederman, Gurkan Bebek, Belinda Willard, Benlian Wang, Xing-Huang Gao, Charles R. Evans, Charles L. Hoppel, Maria Hatzoglou, Paul L. Fox, Ming Liu, Peter Arvan, Dawid Krokowski, Stanton L. Gerson, Bo-Jhih Guan, Omer Kabil, and Luda Diatchenko
- Subjects
Metabolic reprogramming ,Integrated stress response ,Biology ,Protein sulfhydration ,Cell biology - Published
- 2015
35. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response
- Author
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Luda Diatchenko, Xing-Huang Gao, Charles R. Evans, Ming Liu, Charles L. Hoppel, Bo-Jhih Guan, Ilya Bederman, Gurkan Bebek, Belinda Willard, Omer Kabil, Stanton L. Gerson, Marc Parisien, Peter Arvan, Paul L. Fox, Mithu Majumder, Dawid Krokowski, Benlian Wang, Ruma Banerjee, and Maria Hatzoglou
- Subjects
quantitative proteomics ,Mouse ,Proteome ,QH301-705.5 ,Science ,Quantitative proteomics ,hydrogen sulfide ,Cystathionine gamma-lyase (CSE) ,Biology ,Proteomics ,Integrated Stress Response ,Biochemistry ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Stress, Physiological ,protein sulfhydration ,Integrated stress response ,metabolic reprogramming ,Animals ,Cysteine ,Biology (General) ,030304 developmental biology ,0303 health sciences ,General Immunology and Microbiology ,General Neuroscience ,Endoplasmic reticulum ,Computational Biology ,General Medicine ,Cell Biology ,Protein sulfhydration ,Mice, Inbred C57BL ,Gene Expression Regulation ,Medicine ,Flux (metabolism) ,Reprogramming ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery ,Metabolic Networks and Pathways ,Research Article - Abstract
The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. DOI: http://dx.doi.org/10.7554/eLife.10067.001, eLife digest Proteins play essential roles in almost every aspect of a cell’s life, and also contribute to the structure and function of body tissues and organs. Cells and tissues adapt to their continuously changing environments by regulating the activity of their proteins. For example, proteins that are not fully active immediately after they are built instead require further ‘posttranslational’ modifications to become active. Amino acids are the building blocks of proteins, and cysteine amino acids are frequent sites of posttranslational modifications because they are particularly chemically reactive. Under certain conditions inside the cell, the sulfur atom in a cysteine can bond with chemical group containing a second sulfur atom plus a hydrogen atom. This process, which is known as sulfhydration, can be triggered by the presence of the gas molecule, hydrogen sulfide (H2S). The levels of hydrogen sulfide are highly regulated in the body, and it has been suggested to play a role in aging, environmental stress and many diseases. However, it is not clear whether sulfhydration plays a major role in disease conditions by modifying protein activity. Efforts to address this question have been limited by a lack of methods that can measure the extent of sulfhydration of proteins. However, Gao et al. have now devised such a method. The approach takes steps to avoid false-positive and false-negative results, and can identify changes in the sulfhydration of cysteines across the entire complement of proteins produced by a cell, tissue or organ. Gao et al. then used this new method to show that a master regulator of transcription (i.e. a protein that regulates the expression of many genes) causes large-scale changes in cysteine sulfhydration. These large-scale changes resulted in the reprogramming of the cell’s energy metabolism, and further experiments showed that hydrogen sulfide accumulation influences sulfhydration, protein activity and signaling pathways. The development of this new method may now lead to additional discoveries into the role of hydrogen sulfide as a signaling molecule. DOI: http://dx.doi.org/10.7554/eLife.10067.002
- Published
- 2015
36. A Unique ISR Program Determines Cellular Responses to Chronic Stress
- Author
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Maria Hatzoglou, Dawid Krokowski, Orna Elroy-Stein, Anthony Wynshaw-Boris, Leoš Shivaya Valášek, Raul Jobava, Anton A. Komar, Vincent van Hoef, Xing-Huang Gao, Antonis E. Koromilas, Marie Cargnello, Ola Larsson, William C. Merrick, Scot R. Kimball, Ivan Topisirovic, and Bo-Jhih Guan
- Subjects
0301 basic medicine ,Time Factors ,Transcription, Genetic ,Eukaryotic Initiation Factor-3 ,Biology ,Transfection ,Article ,Mice ,Open Reading Frames ,eIF-2 Kinase ,03 medical and health sciences ,Animals ,Humans ,Integrated stress response ,Chronic stress ,RNA, Messenger ,Molecular Biology ,eIF2 ,Endoplasmic reticulum ,Cell Biology ,Fibroblasts ,Cellular Reprogramming ,Endoplasmic Reticulum Stress ,Cell biology ,HEK293 Cells ,Phenotype ,030104 developmental biology ,Proteostasis ,Protein Biosynthesis ,eIF2B ,Unfolded protein response ,biology.protein ,RNA Interference ,Reprogramming ,Signal Transduction - Abstract
The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits "foamy cell" development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.
- Published
- 2017
37. L-type Calcium Channel Blockers Enhance Trafficking and Function of Epilepsy-associated α1(D219N) Subunits of GABA(A) Receptors
- Author
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Maria Hatzoglou, Ting Wei Mu, Dong Yun Han, Yajuan Wang, and Bo-Jhih Guan
- Subjects
Models, Molecular ,Calcium Channels, L-Type ,Calnexin ,Protein subunit ,Pharmacology ,Biology ,Biochemistry ,GABAA-rho receptor ,Cell Line ,medicine ,Humans ,L-type calcium channel ,Protein Interaction Maps ,Receptor ,Neurons ,Epilepsy ,Endoplasmic reticulum ,General Medicine ,Endoplasmic Reticulum-Associated Degradation ,Calcium Channel Blockers ,Receptors, GABA-A ,Cell biology ,Protein Subunits ,Protein Transport ,HEK293 Cells ,Verapamil ,Molecular Medicine ,Calreticulin ,medicine.drug ,Cys-loop receptors - Abstract
Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory ion channels in the mammalian central nervous system and play an essential role in regulating inhibition-excitation balance in neural circuits. The α1 subunit harboring the D219N mutation of GABAA receptors was reported to be retained in the endoplasmic reticulum (ER) and traffic inefficiently to the plasma membrane, leading to a loss of function of α1(D219N) subunits and thus idiopathic generalized epilepsy (IGE). We present the use of small molecule proteostasis regulators to enhance the forward trafficking of α1(D219N) subunits to restore their function. We showed that treatment with verapamil (4 μM, 24 h), an L-type calcium channel blocker, substantially increases the α1(D219N) subunit cell surface level in both HEK293 cells and neuronal SH-SY5Y cells and remarkably restores the GABA-induced maximal chloride current in HEK293 cells expressing α1(D219N)β2γ2 receptors to a level that is comparable to wild type receptors. Our drug mechanism study revealed that verapamil treatment promotes the ER to Golgi trafficking of the α1(D219N) subunits post-translationally. To achieve that, verapamil treatment enhances the interaction between the α1(D219N) subunit and β2 subunit and prevents the aggregation of the mutant protein by shifting the protein from the detergent-insoluble fractions to detergent-soluble fractions. By combining (35)S pulse-chase labeling and MG-132 inhibition experiments, we demonstrated that verapamil treatment does not inhibit the ER-associated degradation of the α1(D219N) subunit. In addition, its effect does not involve a dynamin-1 dependent endocytosis. To gain further mechanistic insight, we showed that verapamil increases the interaction between the mutant protein and calnexin and calreticulin, two major lectin chaperones in the ER. Moreover, calnexin binding promotes the forward trafficking of the mutant subunit. Taken together, our data indicate that verapamil treatment enhances the calnexin-assisted forward trafficking and subunit assembly, which leads to substantially enhanced functional surface expression of the mutant receptors. Since verapamil is an FDA-approved drug that crosses blood-brain barrier and has been used as an additional medication for some epilepsies, our findings suggest that verapamil holds great promise to be developed to ameliorate IGE resulting from α1(D219N) subunit trafficking deficiency.
- Published
- 2015
38. Coordinated Regulation of the Neutral Amino Acid Transporter SNAT2 and the Protein Phosphatase Subunit GADD34 Promotes Adaptation to Increased Extracellular Osmolarity*
- Author
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Maria Hatzoglou, Martin D. Snider, Dawid Krokowski, Jing Wu, Kenneth T. Farabaugh, Ovidio Bussolati, Bo-Jhih Guan, Massimiliano G. Bianchi, Mithu Majumder, and Raul Jobava
- Subjects
Amino Acid Transport System A ,Cell Survival ,Phosphatase ,Response element ,Eukaryotic Initiation Factor-2 ,Activating Transcription Factor 4 ,Biology ,Biochemistry ,Cell Line ,Mice ,Osmotic Pressure ,Protein Phosphatase 1 ,Protein biosynthesis ,Transcriptional regulation ,Animals ,Humans ,Gene Regulation ,Phosphorylation ,Promoter Regions, Genetic ,Molecular Biology ,eIF2 ,Protein phosphatase 1 ,Cell Biology ,HEK293 Cells ,Gene Expression Regulation ,HeLa Cells - Abstract
Cells respond to shrinkage induced by increased extracellular osmolarity via programmed changes in gene transcription and mRNA translation. The immediate response to this stress includes the induction of expression of the neutral amino acid transporter SNAT2. Increased SNAT2-mediated uptake of neutral amino acids is an essential adaptive mechanism for restoring cell volume. In contrast, stress-induced phosphorylation of the α subunit of the translation initiation factor eIF2 (eIF2α) can promote apoptosis. Here we show that the response to mild hyperosmotic stress involves regulation of the phosphorylation of eIF2α by increased levels of GADD34, a regulatory subunit of protein phosphatase 1 (PP1). The induction of GADD34 was dependent on transcriptional control by the c-Jun-binding cAMP response element in the GADD34 gene promoter and posttranscriptional stabilization of its mRNA. This mechanism differs from the regulation of GADD34 expression by other stresses that involve activating transcription factor 4 (ATF4). ATF4 was not translated during hyperosmotic stress despite an increase in eIF2α phosphorylation. The SNAT2-mediated increase in amino acid uptake was enhanced by increased GADD34 levels in a manner involving decreased eIF2α phosphorylation. It is proposed that the induction of the SNAT2/GADD34 axis enhances cell survival by promoting the immediate adaptive response to stress.
- Published
- 2015
39. Translational control of PML contributes to TNFα-induced apoptosis of MCF7 breast cancer cells and decreased angiogenesis in HUVECs
- Author
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Kuo Sheng Hsu, H. Y. Kao, M. Lam, M. Hatzoglou, D. Guan, Xiwen Cheng, and Bo-Jhih Guan
- Subjects
0301 basic medicine ,Angiogenesis ,viruses ,Neovascularization, Physiologic ,Apoptosis ,Breast Neoplasms ,HL-60 Cells ,Internal Ribosome Entry Sites ,Promyelocytic Leukemia Protein ,Protein Serine-Threonine Kinases ,medicine.disease_cause ,p38 Mitogen-Activated Protein Kinases ,03 medical and health sciences ,Promyelocytic leukemia protein ,Matrix Metalloproteinase 10 ,Cell Movement ,medicine ,Human Umbilical Vein Endothelial Cells ,Humans ,Nuclear protein ,Protein kinase A ,Molecular Biology ,Chemokine CCL2 ,Cell Proliferation ,Original Paper ,biology ,Tumor Necrosis Factor-alpha ,Tumor Suppressor Proteins ,Intracellular Signaling Peptides and Proteins ,virus diseases ,Nuclear Proteins ,Cell Biology ,Virology ,3. Good health ,Cell biology ,Gene Expression Regulation, Neoplastic ,Internal ribosome entry site ,030104 developmental biology ,Protein Biosynthesis ,Cancer cell ,biology.protein ,MCF-7 Cells ,Tumor necrosis factor alpha ,Carcinogenesis ,5' Untranslated Regions ,Transcription Factors - Abstract
The tumor suppressor protein promyelocytic leukemia (PML) is a key regulator of inflammatory responses and tumorigenesis and functions through the assembly of subnuclear structures known as PML nuclear bodies (NBs). The inflammation-related cytokine tumor necrosis factor-α (TNFα) is known to induce PML protein accumulation and PML NB formation that mediate TNFα-induced cell death in cancer cells and inhibition of migration and capillary tube formation in endothelial cells (ECs). In this study, we uncover a novel mechanism of PML gene regulation in which the p38 MAPK and its downstream kinase MAP kinase-activated protein kinase 1 (MNK1) mediate TNFα-induced PML protein accumulation and PML NB formation. The mechanism includes the presence of an internal ribosome entry site (IRES) found within the well-conserved 100 nucleotides upstream of the PML initiation codon. The activity of the PML IRES is induced by TNFα in a manner that involves MNK1 activation. It is proposed that the p38-MNK1-PML network regulates TNFα-induced apoptosis in breast cancer cells and TNFα-mediated inhibition of migration and capillary tube formation in ECs.
- Published
- 2014
40. ER stress inhibitor attenuates hearing loss and hair cell death in Cdh23erl/erl mutant mice
- Author
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Ruben Stepanyan, Shami Entenman, Maria Hatzoglou, Ulrich Müller, Min Xu, Juan Hu, Bo Li, Qing Yin Zheng, Bo-Jhih Guan, Luke Apisa, and Heping Yu
- Subjects
0301 basic medicine ,Cancer Research ,medicine.medical_specialty ,Hearing loss ,Eukaryotic Initiation Factor-2 ,Immunology ,Down-Regulation ,Audiology ,Biology ,Salubrinal ,eIF-2 Kinase ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Heat shock protein ,Hair Cells, Auditory ,otorhinolaryngologic diseases ,medicine ,Animals ,Phosphorylation ,Hearing Loss ,Endoplasmic Reticulum Chaperone BiP ,Heat-Shock Proteins ,Endoplasmic reticulum ,Thiourea ,Cell Biology ,Cadherins ,Endoplasmic Reticulum Stress ,Mice, Mutant Strains ,3. Good health ,Cell biology ,Hair Cells, Auditory, Outer ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Cinnamates ,Apoptosis ,Mutation ,Unfolded Protein Response ,Unfolded protein response ,Original Article ,Hair cell ,medicine.symptom ,Signal transduction ,Transcription Factor CHOP - Abstract
Hearing loss is one of the most common sensory impairments in humans. Mouse mutant models helped us to better understand the mechanisms of hearing loss. Recently, we have discovered that the erlong (erl) mutation of the cadherin23 (Cdh23) gene leads to hearing loss due to hair cell apoptosis. In this study, we aimed to reveal the molecular pathways upstream to apoptosis in hair cells to exploit more effective therapeutics than an anti-apoptosis strategy. Our results suggest that endoplasmic reticulum (ER) stress is the earliest molecular event leading to the apoptosis of hair cells and hearing loss in erl mice. We also report that the ER stress inhibitor, Salubrinal (Sal), could delay the progression of hearing loss and preserve hair cells. Our results provide evidence that therapies targeting signaling pathways in ER stress development prevent hair cell apoptosis at an early stage and lead to better outcomes than those targeting downstream factors, such as tip-link degeneration and apoptosis.
- Published
- 2016
41. Genetic Evidence of a Long-Range RNA-RNA Interaction between the Genomic 5′ Untranslated Region and the Nonstructural Protein 1 Coding Region in Murine and Bovine Coronaviruses
- Author
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Bo-Jhih Guan, Hung-Yi Wu, David A. Brian, and Yu Pin Su
- Subjects
Untranslated region ,Five prime untranslated region ,Genotype ,viruses ,Immunology ,Molecular Sequence Data ,Genome, Viral ,Biology ,Viral Nonstructural Proteins ,medicine.disease_cause ,Virus Replication ,Microbiology ,Cell Line ,Mice ,Open Reading Frames ,Virology ,Cricetinae ,medicine ,Coding region ,Animals ,Humans ,Nucleotide Motifs ,Base Pairing ,Coronavirus ,Genetics ,Coronavirus, Bovine ,NSP1 ,Murine hepatitis virus ,Base Sequence ,Inverted Repeat Sequences ,Nucleic acid sequence ,RNA ,virus diseases ,biology.organism_classification ,Genome Replication and Regulation of Viral Gene Expression ,Phenotype ,Insect Science ,Mutation ,Mutagenesis, Site-Directed ,Nucleic Acid Conformation ,RNA, Viral ,Cattle ,5' Untranslated Regions ,Sequence Alignment ,Betacoronavirus - Abstract
Higher-order RNA structures in the 5′ untranslated regions (UTRs) of the mouse hepatitis coronavirus (MHV) and bovine coronavirus (BCoV), separate species in the betacoronavirus genus, appear to be largely conserved despite an ∼36% nucleotide sequence divergence. In a previous study, each of three 5′-end-proximal cis -acting stem-loop domains in the BCoV genome, I/II, III, and IV, yielded near-wild-type (wt) MHV phenotypes when used by reverse genetics to replace its counterpart in the MHV genome. Replacement with the BCoV 32-nucleotide (nt) inter-stem-loop fourth domain between stem-loops III and IV, however, required blind cell passaging for virus recovery. Here, we describe suppressor mutations within the transplanted BCoV 32-nt domain that along with appearance of potential base pairings identify an RNA-RNA interaction between this domain and a 32-nt region ∼200 nt downstream within the nonstructural protein 1 (Nsp1)-coding region. Mfold and phylogenetic covariation patterns among similarly grouped betacoronaviruses support this interaction, as does cotransplantation of the BCoV 5′ UTR and its downstream base-pairing domain. Interestingly, cotransplantation of the BCoV 5′ UTR and BCoV Nsp1 coding region directly yielded an MHV wt-like phenotype, which demonstrates a cognate interaction between these two BCoV regions, which in the MHV genome act in a fully interspecies-compliant manner. Surprisingly, the 30-nt inter-stem-loop domain in the MHV genome can be deleted and viral progeny, although debilitated, are still produced. These results together identify a previously undescribed long-range RNA-RNA interaction between the 5′ UTR and Nsp1 coding region in MHV-like and BCoV-like betacoronaviruses that is cis acting for viral fitness but is not absolutely required for viral replication in cell culture.
- Published
- 2012
42. An Optimal cis-Replication Stem-Loop IV in the 5′ Untranslated Region of the Mouse Coronavirus Genome Extends 16 Nucleotides into Open Reading Frame 1▿
- Author
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Hung-Yi Wu, David A. Brian, and Bo-Jhih Guan
- Subjects
Untranslated region ,Five prime untranslated region ,Immunology ,Viral Plaque Assay ,Biology ,medicine.disease_cause ,Virus Replication ,Microbiology ,Terminal loop ,Cell Line ,Open Reading Frames ,Virology ,medicine ,Animals ,Bovine coronavirus ,Coronavirus ,Genetics ,Coronavirus, Bovine ,Recombination, Genetic ,Murine hepatitis virus ,Microbial Viability ,Stem-loop ,Genome Replication and Regulation of Viral Gene Expression ,Open reading frame ,Viral replication ,Insect Science ,RNA, Viral ,5' Untranslated Regions - Abstract
The 288-nucleotide (nt) 3′ untranslated region (UTR) in the genome of the bovine coronavirus (BCoV) and 339-nt 3′ UTR in the severe acute respiratory syndrome (SARS) coronavirus (SCoV) can each replace the 301-nt 3′ UTR in the mouse hepatitis coronavirus (MHV) for virus replication, thus demonstrating common 3′ cis -replication signals. Here, we show that replacing the 209-nt MHV 5′ UTR with the ∼63%-sequence-identical 210-nt BCoV 5′ UTR by reverse genetics does not yield viable virus, suggesting 5′ end signals are more stringent or possibly are not strictly 5′ UTR confined. To identify potential smaller, 5′-common signals, each of three stem-loop (SL) signaling domains and one inter-stem-loop domain from the BCoV 5′ UTR was tested by replacing its counterpart in the MHV genome. The SLI/II domain (nucleotides 1 to 84) and SLIII domain (nucleotides 85 to 141) each immediately enabled near-wild-type (wt) MHV-like progeny, thus behaving similarly to comparable 5′-proximal regions of the SCoV 5′ UTR as shown by others. The inter-stem-loop domain (nt 142 to 173 between SLs III and IV) enabled small plaques only after genetic adaptation. The SLIV domain (nt 174 to 210) required a 16-nt extension into BCoV open reading frame 1 (ORF1) for apparent stabilization of a longer BCoV SLIV (nt 174 to 226) and optimal virus replication. Surprisingly, pleiomorphic SLIV structures, including a terminal loop deletion, were found among debilitated progeny from intra-SLIV chimeras. The results show the inter-stem-loop domain to be a potential novel species-specific cis -replication element and that cis -acting SLIV in the viral genome extends into ORF1 in a manner that stabilizes its lower stem and is thus not 5′ UTR confined.
- Published
- 2011
43. Aquatic birnavirus infection activates the transcription factor NF-kappaB via tyrosine kinase signalling leading to cell death
- Author
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Bo-Jhih Guan, Nina Santi, Jen-Leih Wu, Øystein Evensen, Jiann Ruey Hong, and Guor Mour Her
- Subjects
Time Factors ,Cell Survival ,Veterinary (miscellaneous) ,Apoptosis ,Protein tyrosine phosphatase ,Aquatic Science ,Mitogen-activated protein kinase kinase ,Cysteine Proteinase Inhibitors ,Receptor tyrosine kinase ,Cell Line ,Fish Diseases ,Salmon ,Birnaviridae ,Animals ,Protein Kinase Inhibitors ,biology ,Cell Death ,NF-kappa B ,JAK-STAT signaling pathway ,Protein-Tyrosine Kinases ,Birnaviridae Infections ,Molecular biology ,Genistein ,Cell biology ,biology.protein ,Janus kinase ,Tyrosine kinase ,Oligopeptides ,Platelet-derived growth factor receptor ,Proto-oncogene tyrosine-protein kinase Src ,Signal Transduction - Abstract
Our previous studies found that infectious pancreatic necrosis virus (IPNV) induces host apoptotic cell death, possibly through a newly synthesized protein trigger. Here, we examine whether IPNV infection can induce NF-kappaB activation through tyrosine kinase signalling of CHSE-214 cell death (host cell death). Using the electrophoretic mobility shift assay (EMSA) to detect transcription factor activation, we found that NF-kappaB is apparently activated 6-8 h post-IPNV infection. Using genistein (100 microg mL(-1); a tyrosine kinase inhibitor) to determine whether NF-kappaB activation requires tyrosine kinase activation, we found genistein blocks NF-kappaB activation at 8 h post-infection (p.i), and either enhances cell viability up to 50% at 12 h p.i. or blocks DNA fragmentation at 24 h p.i. Furthermore, the proteasome inhibitors PSI-I and PSI-II (both at 40 microm) also effectively blocked the NF-kappaB activation as well as stimulating a 30% increase in cell viability (30% decrease in apoptosis) at 8 and 12 h p.i. Taken together our data suggest that IPNV may induce NF-kappaB activation through tyrosine kinase signalling, which may be associated with induction of apoptosis.
- Published
- 2008
44. Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress.
- Author
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Farabaugh, Kenneth T., Majumder, Mithu, Bo-Jhih Guan, Jobava, Raul, Jing Wu, Dawid Krokowski, Xing-Huang Gao, Schuster, Andrew, Longworth, Michelle, Chan, Edward D., Massimiliano Bianchi, Dey, Madhusudan, Koromilas, Antonis E., Ramakrishnan, Parameswaran, and Hatzoglou, Maria
- Subjects
PROTEIN kinases ,INFLAMMATION ,OSMOSIS ,RNA-binding proteins ,OSMOLAR concentration ,PHENOTYPES - Abstract
High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA binding domain. In turn, PKR stimulated nuclear accumulation of nuclear factor-κB (NF-κB) p65 species phosphorylated at serine-536, which was paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes including inducible nitric oxide synthase (iNOS), IL-6, and IL-1β. The PKR mediated-hyperinduction of iNOS decreased cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and post-translational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo. Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
45. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the Integrated Stress Response.
- Author
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Xing-Huang Gao, Krokowski, Dawid, Bo-Jhih Guan, Hatzoglou, Maria, Gerson, Stanton L., Bederman, Ilya, Majumder, Mithu, Hoppel, Charles L., Parisien, Marc, Diatchenko, Luda, Kabil, Omer, Banerjee, Ruma, Willard, Belinda, Wang, Benlian, Bebek, Gurkan, Evans, Charles R., Ming Liu, Arvan, Peter, and Fox, Paul L.
- Subjects
CYSTEINE ,ENDOPLASMIC reticulum ,PANCREATIC acinar cells - Abstract
The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H
2 S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the Integrated Stress Response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. [ABSTRACT FROM AUTHOR]- Published
- 2015
- Full Text
- View/download PDF
46. Angiogenin-Cleaved tRNA Halves Interact with Cytochrome c, Protecting Cells from Apoptosis during Osmotic Stress.
- Author
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Saikia, Mridusmita, Jobava, Raul, Parisien, Marc, Putnam, Andrea, Krokowski, Dawid, Xing-Huang Gao, Bo-Jhih Guan, Yiyuan Yuan, Jankowsky, Eckhard, Zhaoyang Feng, Guo-fu Hu, Pusztai-Carey, Marianne, Gorla, Madhavi, Sepuri, Naresh Babu V., Tao Pan, and Hatzoglou, Maria
- Subjects
CYTOCHROME c ,NUCLEOTIDE sequence ,OSMOSIS ,APOPTOSIS ,FIBROBLASTS ,ANGIOGENIN - Abstract
Adaptation to changes in extracellular tonicity is essential for cell survival. However, severe or chronic hyperosmotic stress induces apoptosis, which involves cytochrome c (Cyt c) release from mitochondria and subsequent apoptosome formation. Here, we show that angiogenin-induced accumulation of tRNA halves (or tiRNAs) is accompanied by increased survival in hyperosmotically stressed mouse embryonic fibroblasts. Treatment of cells with angiogenin inhibits stress-induced formation of the apoptosome and increases the interaction of small RNAs with released Cyt c in a ribonucleoprotein (Cyt c-RNP) complex. Nextgeneration sequencing of RNA isolated from the Cyt c-RNP complex reveals that 20 tiRNAs are highly enriched in the Cyt c-RNP complex. Preferred components of this complex are 5' and 3' tiRNAs of specific isodecoders within a family of isoacceptors. We also demonstrate that Cyt c binds tiRNAs in vitro, and the pool of Cyt c-interacting RNAs binds tighter than individual tiRNAs. Finally, we show that angiogenin treatment of primary cortical neurons exposed to hyperosmotic stress also decreases apoptosis. Our findings reveal a connection between angiogenin-generated tiRNAs and cell survival in response to hyperosmotic stress and suggest a novel cellular complex involving Cyt c and tiRNAs that inhibits apoptosome formation and activity. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
47. Translational Control during Endoplasmic Reticulum Stress beyond Phosphorylation of the Translation Initiation Factor eIF2α.
- Author
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Bo-Jhih Guan, Krokowski, Dawid, Majumder, Mithu, Schmotzer, Christine L., Kimball, Scot R., Merrick, William C., Koromilas, Antonis E., and Hatzoglou, Maria
- Subjects
- *
PROTEIN folding , *ENDOPLASMIC reticulum , *GENE expression , *PHOSPHORYLATION , *RAPAMYCIN - Abstract
The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes stress to which an unfolded protein response is activated to render cell survival or apoptosis (chronic stress). Transcriptional and translational reprogramming is tightly regulated during the unfolded protein response to ensure specific gene expression. The master regulator of this response is the PERK/eIF2α/ATF4 signaling where eIF2α is phosphorylated (eIF2α-P) by the kinase PERK. This signal leads to global translational shutdown, but it also enables translation of the transcription factor ATF4 mRNA. We showed recently that ATF4 induces an anabolic program through the up-regulation of selected amino acid transporters and aminoacyl-tRNA synthetases. Paradoxically, this anabolic program led cells to apoptosis during chronic ER stress in a manner that involved recovery from stress-induced protein synthesis inhibition. By using eIF2α-P-deficient cells as an experimental system, we identified a communicating network of signaling pathways that contribute to the inhibition of protein synthesis during chronic ER stress. This eIF2α-P-independent network includes (i) inhibition of mammalian target of rapamycin kinase protein complex 1 (mTORC1)-targeted protein phosphorylation, (ii) inhibited translation of a selective group of 5'-terminal oligopyrimidine mRNAs (encoding proteins involved in the translation machinery and translationally controlled by mTORC1 signaling), and (iii) inhibited translation of non-5'-terminal oligopyrimidine ribosomal protein mRNAs and ribosomal RNA biogenesis. We propose that the PERK/eIF2α-P/ATF4 signaling acts as a brake in the decline of protein synthesis during chronic ER stress by positively regulating signaling downstream of the mTORC1 activity. These studies advance our knowledge on the complexity of the communicating signaling pathways in controlling protein synthesis rates during chronic stress. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
48. Reselection of a Genomic Upstream Open Reading Frame in Mouse Hepatitis Coronavirus 5'-Untranslated-Region Mutants.
- Author
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Hung-Yi Wu, Bo-Jhih Guan, Yu-Pin Su, Yi-Hsin Fan, and Brian, David A.
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OPEN reading frames (Genetics) , *HEPATITIS , *CORONAVIRUSES , *GENETIC mutation , *GENETIC code , *POLYPEPTIDES , *VIRAL genomes , *LABORATORY mice - Abstract
An AUG-initiated upstream open reading frame (uORF) encoding a potential polypeptide of 3 to 13 amino acids (aa) is found within the 5= untranslated region (UTR) of>75% of coronavirus genomes based on 38 reference strains. Potential CUG-initiated uORFs are also found in many strains. The AUG-initiated uORF is presumably translated following genomic 5=-end cap-dependent ribosomal scanning, but its function is unknown. Here, in a reverse-genetics study with mouse hepatitis coronavirus, the following were observed. (i) When the uORF AUG-initiating codon was replaced with a UAG stop codon along with a U112A mutation to maintain a uORF-harboring stem-loop 4 structure, an unimpaired virus with wild-type (WT) growth kinetics was recovered. However, reversion was found at all mutated sites within five virus passages. (ii) When the uORF was fused with genomic (main) ORF1 by converting three in-frame stop codons to nonstop codons, a uORF-ORF1 fusion protein was made, and virus replicated at WT levels. However, a frameshifting G insertion at virus passage 7 established a slightly 5=-extended original uORF. (iii) When uAUG-eliminating deletions of 20, 30, or 51 nucleotides (nt) were made within stem-loop 4, viable but debilitated virus was recovered. However, a C80U mutation in the first mutant and an A77G mutation in the second appeared by passage 10, which generated alternate uORFs that correlated with restored WT growth kinetics. In vitro, the uORF-disrupting nondeletion mutants showed enhanced translation of the downstream ORF1 compared with the WT. These results together suggest that the uORF represses ORF1 translation yet plays a beneficial but nonessential role in coronavirus replication in cell culture. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
49. Bovine Coronavirus Nonstructural Protein 1 (p28) Is an RNA Binding Protein That Binds Terminal Genomic cis-Replication Elements.
- Author
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Gustin, Kortney M., Bo-Jhih Guan, Dziduszko, Agnieszka, and Brian, David A.
- Subjects
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CORONAVIRUSES , *CARRIER proteins , *ANIMAL diseases , *VIRAL proteins , *ESCHERICHIA coli - Abstract
Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5'-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of ∼60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5' untranslated region (UTR)- and one 3' UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5' UTR with ∼2.5 μM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
50. IL-4 up-regulates cyclooxygenase-1 expression in macrophages.
- Author
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Shay, Ashley E., Diwakar, Bastihalli T., Bo-Jhih Guan, Narayan, Vivek, Urban Jr., Joseph F., and Prabhu, K. Sandeep
- Subjects
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MACROPHAGES , *CYCLOOXYGENASES , *CYTOKINES , *INTERLEUKIN-1 , *HOMEOSTASIS - Abstract
Macrophages use various cell-surface receptors to sense their environment and undergo polarized responses. The cytokines, interleukin (IL)-4 and IL-13, released from T-helper type 2 (Th2) cells, drive macrophage polarization toward an alternatively activated phenotype (M2). This phenotype is associated with the expression of potent pro-resolving mediators, such as the prostaglandin (PG) D2-derived cyclopentenone metabolite, 15d-PGJ2, produced by the cyclooxygenase (Ptgs; Cox) pathway. Interestingly, IL-4 treatment of bone marrow-derived macrophages (BMDMs) significantly down-regulates Cox-2 protein expression, whereas Cox-1 levels are significantly increased. This phenomenon not only challenges the dogma that Cox-1 is only developmentally regulated, but also demonstrates a novel mechanism in which IL-4-dependent regulation of Cox-1 involves the activation of the mechanistic target of rapamycin complex (mTORC). Using specific chemical inhibitors, we demonstrate here that IL-4-dependent Cox-1 up-regulation occurs at the post-transcriptional level via the Fes-Akt-mTORC axis. Activation of AMP-activated protein kinase (AMPK) by metformin, inhibition of mTORC by torin 1, or CRISPR/Cas9-mediated genetic knock-out of tuberous sclerosis complex-2 (Tsc2) blocked the IL-4-dependent expression of Cox-1 and the ability of macrophages to polarize to M2. However, use of 15d-PGJ2 partially rescued the effects of AMPK activation, suggesting the importance of Cox-1 in macrophage polarization as also observed in a model of gastrointestinal helminth clearance. In summary, these findings suggest a new paradigm where IL-4-dependent up-regulation of Cox-1 expression may play a key role in tissue homeostasis and wound healing during Th2-mediated immune responses, such as parasitic infections. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
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