Stress-responsive Transcription Research Articles

Spatial Analysis of NQO1 in Non-Small Cell Lung Cancer Shows Its Expression Is Independent of NRF1 and NRF2 in the Tumor Microenvironment.

Nuclear factor erythroid 2-related factor 1 (NFE2L1, NRF1) and nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2) are distinct oxidative stress response transcription factors, both of which have been shown to perform cytoprotective functions, modulating cell stress response and homeostasis. NAD(P)H:quinone oxidoreductase (NQO1) is a mutual downstream antioxidant gene target that catalyzes the two-electron reduction of an array of substrates, protecting against reactive oxygen species (ROS) generation. NQO1 is upregulated in non-small cell lung cancer (NSCLC) and is proposed as a predictive biomarker and therapeutic target. Antioxidant protein expression of immune cells within the NSCLC tumor microenvironment (TME) remains undetermined and may affect immune cell effector functions and survival outcomes. Multiplex immunofluorescence was performed to examine the co-localization of NQO1, NRF1 and NRF2 within the tumor and TME of 162 chemotherapy-naïve, early-stage NSCLC patients treated by primary surgical resection. This study demonstrates that NQO1 protein expression is high in normal, tumor-adjacent tissue and that NQO1 expression varies depending on the cell type. Inter and intra-patient heterogenous NQO1 expression was observed in lung cancer. Co-expression analysis showed NQO1 is independent of NRF1 and NRF2 in tumors. Density-based co-expression analysis demonstrated NRF1 and NRF2 double-positive expression in cancer cells is associated with improved overall survival.

Three stress-responsive NAC transcription factors, Pp-SNACs, differentially and synergistically regulate abiotic stress in pear

NAC proteins contribute to regulate diverse plant developmental processes and tolerances to biotic and abiotic stresses. In pear, limited reports are available about the transcriptional regulations of PpNACs during abiotic stress signaling. The transcriptome of Pyrus ussuriensis in response to cold stress (4 °C) for 5 h and 12 h revealed some differentially expressed NAC genes. We found three putative NAC genes, PpNAC88, PpNAC92, and PpNAC130, from subgroup-3D (stress responsive NAC subgroup in pear) that were differentially expressed during cold stress in pear. At the same time, RNA-seq of Pyrus betulaefolia under salt stress also revealed high RPKM values of these Pp-NACs. Further, these PpNACs, PpNAC88/92/130, were named Pp-SNACs due to their phylogenetic group with well-known SNACs (stress-responsive NACs) in other crops. qRT-PCR revealed that all Pp-SNACs were significantly and differentially expressed in response to cold and salt stresses, similar to stress-responsive genes, PpCOR47/15A/RD29A/KIN. High transcript abundances of all PpSNACs along with the PpABF2/ABF3/RD29A/COR15A (ABA and stress-responsive genes) were also observed during ABA treatment to both pear calli and explants. Furthermore, luciferase and Y1H assays revealed the transcriptional regulations of Pp-SNACs on PpCOR47/15A/RD29A promoters, while PpNAC88 and PpNAC130 both, in-vivo and in-vitro, can regulate the promoter of PpCOR15A. Conversely, we also found that PpNAC88 and PpNAC130 also make protein complex, and the combination of PpNAC88 and PpNAC130 synergistically regulates the PpCOR15A promoter, which may highlight multidirectional regulations of these Pp-SNACs during abiotic stress in the pear.

The Pex3-mediated peroxisome biogenesis plays a critical role in metabolic biosynthesis, stress response, and pathogenicity in Alternaria alternata

Peroxisomes are microbodies involved in the metabolism of fatty acids and hydrogen peroxide (H2O2) in eukaryotes. In the current study, an AaPex3 gene encoding a peroxisome membrane protein was demonstrated to be required for peroxisome biogenesis and resistance to peroxides and superoxide-generating compounds. Deleting AaPex3 affected the expression of the genes encoding the NADPH oxidase (NoxA) and the Yap1 stress-responsive transcription regulator, both of which have been implicated in ROS resistance. The AaPex3-mediated peroxisome biogenesis negatively affected resistance to singlet oxygen-generating compounds, 2-chloro-5-hydroxypyridine (CHP), and 2,3,5-triiodobenzoic acid (TIBA), novel phenotypes associated with peroxisomes. Nile red staining revealed that ΔAaPex3 accumulated more lipid bodies than the wild type. ΔAaPex3 conidia had thinner cell walls than the wild type, suggesting the involvement of AaPex3 in maintaining cell wall integrity. Genetic evidence has also demonstrated that the AaPex3-mediated peroxisome biogenesis is required for conidiogenesis, conidia germination, siderophore biosynthesis, toxin production, and virulence. Biotin or lipids could restore ΔAaPex3 growth in axenic culture and on the surface of citrus leaves. In contrast, co-application of ΔAaPex3 with biotin and oleic acid on citrus leaves failed to induce necrotic lesions. Our results revealed the multifaceted functions of peroxisomes in the phytopathogenic fungus.

An integrated transcriptome mapping the regulatory network of coding and long non-coding RNAs provides a genomics resource in chickpea

Large-scale transcriptome analysis can provide a systems-level understanding of biological processes. To accelerate functional genomic studies in chickpea, we perform a comprehensive transcriptome analysis to generate full-length transcriptome and expression atlas of protein-coding genes (PCGs) and long non-coding RNAs (lncRNAs) from 32 different tissues/organs via deep sequencing. The high-depth RNA-seq dataset reveal expression dynamics and tissue-specificity along with associated biological functions of PCGs and lncRNAs during development. The coexpression network analysis reveal modules associated with a particular tissue or a set of related tissues. The components of transcriptional regulatory networks (TRNs), including transcription factors, their cognate cis-regulatory motifs, and target PCGs/lncRNAs that determine developmental programs of different tissues/organs, are identified. Several candidate tissue-specific and abiotic stress-responsive transcripts associated with quantitative trait loci that determine important agronomic traits are also identified. These results provide an important resource to advance functional/translational genomic and genetic studies during chickpea development and environmental conditions.

Peptide ligand-mediated trade-off between plant growth and stress response.

Deciding whether to grow or to divert energy to stress responses is a major physiological trade-off for plants surviving in fluctuating environments. We show that three leucine-rich repeat receptor kinases (LRR-RKs) act as direct ligand-perceiving receptors for PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY)-family peptides and mediate switching between two opposing pathways. By contrast to known LRR-RKs, which activate signaling upon ligand binding, PSY receptors (PSYRs) activate the expression of various genes encoding stress response transcription factors upon depletion of the ligands. Loss of PSYRs results in defects in plant tolerance to both biotic and abiotic stresses. This ligand-deprivation-dependent activation system potentially enables plants to exert tuned regulation of stress responses in the tissues proximal to metabolically dysfunctional damaged sites where ligand production is impaired.

Yeast Smy2 and its human homologs GIGYF1 and -2 regulate Cdc48/VCP function during transcription stress

The "last resort" pathway results in ubiquitylation and degradation of RNA polymerase II in response to transcription stress and is governed by factors such as Def1 in yeast. Here, we show that the SMY2 gene acts as a multi-copy suppressor of DEF1 deletion and functions at multiple steps of the last resort pathway. We also provide genetic and biochemical evidence from disparate cellular processes that Smy2 works more broadly as a hitherto overlooked regulator of Cdc48 function. Similarly, the Smy2 homologs GIGYF1 and -2 affect the transcription stress response in human cells and regulate the function of the Cdc48 homolog VCP/p97, presently being explored as a target for cancer therapy. Indeed, we show that the apoptosis-inducing effect of VCP inhibitors NMS-873 and CB-5083 is GIGYF1/2 dependent.

Heterologous Overexpression of ZmHDZIV13 Enhanced Drought and Salt Tolerance in Arabidopsis and Tobacco

The homeodomain leucine zipper (HD-Zip) IV transcription factor is indispensable in the response of plants to abiotic stress. Systematic studies have been carried out in Arabidopsis, rice and other species from which a series of stress resistance-related genes have been isolated. However, the function of the HD-Zip IV protein in maize is not clear. In this study, we cloned the HD-Zip IV gene ZmHDZIV13 and identified its function in the stress response. Our phylogenetic analysis showed that ZmHDZIV13 and AtHDG11 had high homology and might have similar functions. The heterologous overexpression of ZmHDZIV13 in Arabidopsis resulted in sensitivity to abscisic acid (ABA), salt tolerance during germination and drought tolerance in seedlings. Under drought stress, the transgenic Arabidopsis showed stronger drought resistance than the wild-type (control). The malondialdehyde content of ZmHDZIV13 transgenic plants was lower than that of the control, and the relative water content and proline content were significantly higher than those of the control. After the drought was relieved, the expression levels of stress-related genes were up-regulated in transgenic Arabidopsis. These results show that ZmHDZIV13, as a stress-responsive transcription factor, plays a role in the positive regulation of abiotic stress tolerance and can regulate an ABA-dependent signaling pathway to regulate drought response in plants.

ALKBH9C, a potential RNA m6 A demethylase, regulates the response of Arabidopsis to abiotic stresses and abscisic acid.

Although several studies have shown that AlkB homolog (ALKBH) proteins are potential RNA demethylases (referred to as 'erasers'), biological functions of only a few ALKBH proteins have been characterized to date. In this study, we determined the function of ALKBH9C (At4g36090) in seed germination and seedling growth of Arabidopsis thaliana in response to abiotic stress and abscisic acid (ABA). Seed germination of the alkbh9c mutant was delayed in response to salt, drought, coldand ABA. Moreover, seedling growth of the mutant was repressed under salt stress or ABA but enhanced under drought conditions. Notably, the stress-responsive phenotypes were associated with the altered expression of several m6 A-modified transcripts related to salt, droughtor ABA response. Global m6 A levels were increased in the alkbh9c mutant, and ALKBH9C bound to m6 A-modified RNAs and had in vitro m6 A demethylase activity, suggesting its potential role as an m6 A eraser. The m6 A levels in several stress-responsive genes were increased in the alkbh9c mutant, and the stability of m6 A-modified transcripts was altered in the mutant. Collectively, our results suggest that m6 A eraser ALKBH9C is crucial for seed germination and seedling growth of Arabidopsis in response to abiotic stresses or ABA via affecting the stability of stress-responsive transcripts.

ZmNAC074, a maize stress-responsive NAC transcription factor, confers heat stress tolerance in transgenic Arabidopsis.

The harsh environment such as high temperature greatly limits the growth, development and production of crops worldwide. NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play key regulatory roles in abiotic stress responses of plants. However, the functional roles of NAC TFs in heat stress response of maize remain elusive. In our present study, we identified and isolated a stress-responsive NAC transcription factor gene in maize, designated as ZmNAC074 and orthologous with rice OsNTL3. Further studies revealed that ZmNAC074 may encode a membrane-bound transcription factor (MTF) of NAC family in maize, which is comprised of 517 amino acid residues with a transmembrane domain at the C-terminus. Moreover, ZmNAC074 was highly expressed and induced by various abiotic stresses in maize seedlings, especially in leaf tissues under heat stress. Through generating ZmNAC074 transgenic plants, phenotypic and physiological analyses further displayed that overexpression of ZmNAC074 in transgenic Arabidopsis confers enhanced heat stress tolerance significantly through modulating the accumulation of a variety of stress metabolites, including reactive oxygen species (ROS), antioxidants, malondialdehyde (MDA), proline, soluble protein, chlorophyll and carotenoid. Further, quantitative real-time PCR analysis showed that the expression levels of most ROS scavenging and HSR- and UPR-associated genes in transgenic Arabidopsis were significantly up-regulated under heat stress treatments, suggesting that ZmNAC074 may encode a positive regulator that activates the expression of ROS-scavenging genes and HSR- and UPR-associated genes to enhance plant thermotolerance under heat stress conditions. Overall, our present study suggests that ZmNAC074 may play a crucial role in conferring heat stress tolerance in plants, providing a key candidate regulatory gene for heat stress tolerance regulation and genetic improvement in maize as well as in other crops.

RGS11-CaMKII complex mediated redox control attenuates chemotherapy-induced cardiac fibrosis

Dose limiting cardiotoxicity remains a major limiting factor in the clinical use of several cancer chemotherapeutics including anthracyclines and the antimetabolite 5-fluorouracil (5-FU). Prior work has demonstrated that chemotherapeutics increase expression of R7 family regulator of G protein signaling (RGS) protein-binding partner Gβ5, which drives myocyte cytotoxicity. However, though several R7 family members are expressed in heart, the exact role of each protein in chemotherapy driven heart damage remains unclear. Here, we demonstrate that RGS11, downregulated in the human heart following chemotherapy exposure, possesses potent anti-apoptotic actions, in direct opposition to the actions of fellow R7 family member RGS6. RGS11 forms a direct complex with the apoptotic kinase CaMKII and stress responsive transcription factor ATF3 and acts to counterbalance the ability of CaMKII and ATF3 to trigger oxidative stress, mitochondrial dysfunction, cell death, and release of the cardiokine neuregulin-1 (NRG1), which mediates pathological intercommunication between myocytes and endothelial cells. Doxorubicin triggers RGS11 depletion in the murine myocardium, and cardiac-specific OE of RGS11 decreases doxorubicin-induced fibrosis, myocyte hypertrophy, apoptosis, oxidative stress, and cell loss and aids in the maintenance of left ventricular function. Conversely, RGS11 knockdown in heart promotes cardiac fibrosis associated with CaMKII activation and ATF3/NRG1 induction. Indeed, inhibition of CaMKII largely prevents the fibrotic remodeling resulting from cardiac RGS11 depletion underscoring the functional importance of the RGS11-CaMKII interaction in the pathogenesis of cardiac fibrosis. These data describe an entirely new role for RGS11 in heart and identify RGS11 as a potential new target for amelioration of chemotherapy-induced cardiotoxicity.

Tissue-specific transcriptomic analysis uncovers potential roles of natural antisense transcripts in Arabidopsis heat stress response.

Natural antisense transcripts (NATs) are an important class of non-coding ribonucleic acids (RNAs) that have been shown to regulate gene expression. Using strand-specific RNA sequencing, 36,317 NAT pairs were identified, and 5,536 were specifically expressed under heat stress. We found distinct expression patterns between vegetative and reproductive tissues for both coding genes and genes encoding NATs. Genes for heat-responsive NATs are associated with relatively high levels of H3K4me3 and low levels of H3K27me2/3. On the other hand, small RNAs are significantly enriched in sequence overlapping regions of NAT pairs, and a large number of heat-responsive NATs pairs serve as potential precursors of nat-siRNAs. Collectively, our results suggest epigenetic modifications and small RNAs play important roles in the regulation of NAT expression, and highlight the potential significance of heat-inducible NATs.

Morpho-physiological responses and expression analysis of MYB94 transcription factor gene in four groundnut cultivars under drought and salinity stress

Aim: The present study aimed to evaluate the impact of salinity stress on morpho-physiological parameters and relative expression of the MYB94 transcription factor gene among four widely cultivated groundnut cultivars. Methodology: A comparative analysis of morpho-physiological parameters was studied in four groundnut cultivars (Kadiri-Amaravati, Kadiri-Lepakshi, Kadiri-6, and Kadiri-9) subjected to different concentrations of NaCl prepared in half-strength Arnon’s nutrient growth medium. The relative expression analysis of a stress responsive transcription factor gene, MYB94 was studied in four groundnut cultivars subjected to drought and NaCl stress by qRT PCR. Results: NaCl stress resulted in a significant increase in proline content, MDA content in cultivar Kadiri-9 compared to other cultivar studied. On the contrary, plant growth, shoot dry weight, root dry weight, leaf relative water content, cell membrane injury decreased to lesser extent in cultivar Kadiri-9 than Kadiri-Amaravati, Kadiri-Lepakshi, Kadiri-6 under salinity stress. qRT-PCR based relative expression analysis of the MYB94 gene revealed a significant increase of transcript expression levels in cultivar Kadiri-9 compared to other three cultivars under both drought and salinity stress. Interpretation: Groundnut cultivar Kadiri-9 showed better tolerance to NaCl stress as evidenced from enhanced proline content, better relative water content and lesser extent of membrane damage when compared with other groundnut cultivars under given NaCl stress treatments. In addition, relative expression of MYB94 gene was significantly higher due to NaCl stress as compared to drought stress conditions in cultivar Kadiri-9 suggested that the MYB94 could be involved in salt stress tolerance. Key words: Groundnut, MYB94 transcription factor, qRT-PCR, Salinity stress

Model yeast as a versatile tool to examine the antioxidant and anti-ageing potential of flavonoids, extracted from medicinal plants.

The demand for the production of herbal extracts for cosmetics, food, and health supplements, known as plant-based medicine, is rising globally. Incorporating herbal extracts could help to create higher value products due to the functional properties of bioactive compounds. Because the phytochemical composition could vary depending on the processing methods, a simple bioassay of herbal bioactive compounds is an important screening method for the purposes of functional characterization and quality assurance. As a simplified eukaryotic model, yeast serves as a versatile tool to examine functional property of bioactive compounds and to gain better understanding of fundamental cellular processes, because they share similarities with the processes in humans. In fact, aging is a well-conserved phenomenon between yeast and humans, making yeast a powerful genetic tool to examine functional properties of key compounds obtained from plant extracts. This study aimed to apply a well-established model yeast, Saccharomyces cerevisiae, to examine the antioxidant and anti-aging potential of flavonoids, extracted from medicinal plants, and to gain insight into yeast cell adaptation to oxidative stress. Some natural quercetin analogs, including morin, kaempferol, aromadendrin, and steppogenin, protected yeast cells against oxidative stress induced by acetic acid, as shown by decreased cell sensitivity. There was also a reduction in intracellular reactive oxygen species following acetic acid treatment. Using the chronological aging assay, quercetin, morin, and steppogenin could extend the lifespan of wild-type S. cerevisiae by 15%–25%. Consistent with the fact that oxidative stress is a key factor to aging, acetic acid resistance was associated with increased gene expression of TOR1, which encodes a key growth signaling kinase, and MSN2 and MSN4, which encode stress-responsive transcription factors. The addition of the antioxidant morin could counteract this increased expression, suggesting a possible modulatory role in cell signaling and the stress response of yeast. Therefore, yeast represents a versatile model organism and rapid screening tool to discover potentially rejuvenescent molecules with anti-aging and anti-oxidant potential from natural resources and to advance knowledge in the molecular study of stress and aging.

AMPK and NRF2: Interactive players in the same team for cellular homeostasis?

NRF2 (Nuclear factor E2 p45-related factor 2) is a stress responsive transcription factor lending cells resilience against oxidative, xenobiotic, and also nutrient or proteotoxic insults. AMPK (AMP-activated kinase), considered as prime regulator of cellular energy homeostasis, not only tunes metabolism to provide the cell at any time with sufficient ATP or building blocks, but also controls redox balance and inflammation. Due to observed overlapping cellular responses upon AMPK or NRF2 activation and common stressors impinging on both AMPK and NRF2 signaling, it is plausible to assume that AMPK and NRF2 signaling may interdepend and cooperate to readjust cellular homeostasis. After a short introduction of the two players this narrative review paints the current picture on how AMPK and NRF2 signaling might interact on the molecular level, and highlights their possible crosstalk in selected examples of pathophysiology or bioactivity of drugs and phytochemicals.

Impact of Antioxidant Feed and Growth Manipulation on the Redox Regulation of Atlantic Salmon Smolts.

Accumulating evidence indicates a close relationship between oxidative stress and growth rate in fish. However, the underlying mechanisms of this relationship remain unclear. This study evaluated the combined effect of dietary antioxidants and growth hormone (GH) on the liver and the muscle redox status of Atlantic salmon. There were two sequential experimental phases (EP) termed EP1 and EP2, each lasting for 6 weeks. In EP1, Atlantic salmon were fed either low-(L, 230 mg/kg ascorbic acid (Asc), 120 mg/kg α-tocopherol (α-TOH)), or high-(H, 380 mg/kg Asc, 210 mg/kg α-TOH)vitamin diets. The vitamins were supplemented as stable forms and the feeding was continued in EP2. In EP2, half of the fish were implanted with 3 μL per g body weight of recombinant bovine GH (Posilac®, 1 mg rbGH g BW−1) suspended in sesame oil, while the other half were held in different tanks and sham-implanted with similar volumes of the sesame oil vehicle. Here, we show that increasing high levels of vitamin C and E (diet H) increased their content in muscle and liver during EP1. GH implantation decreased vitamin C and E levels in both liver and muscle but increased malondialdehyde (MDA) levels only in the liver. GH also affected many genes and pathways of antioxidant enzymes and the redox balance. Among the most consistent were the upregulation of genes coding for the NADPH oxidase family (NOXs) and downregulation of the oxidative stress response transcription factor, nuclear factor-erythroid 2-related factor 2 (nrf2), and its downstream target genes in the liver. We verified that GH increases the growth rate until the end of the trail and induces an oxidative effect in the liver and muscle of Atlantic salmon. Dietary antioxidants do lower oxidative stress but have no effect on the growth rate. The present study is intended as a starting point to understand the potential interactions between growth and redox signaling in fish.

Structural and Functional Studies of S-(2-Carboxyethyl)-L-Cysteine and S-(2-Carboxyethyl)-l-Cysteine Sulfoxide.

Insecticidal non-proteinogenic amino acid S-(2-carboxyethyl)-L-cysteine (β-CEC) and its assumed metabolite, S-(2-carboxyethyl)-l-cysteine sulfoxide (β-CECO), are present abundantly in a number of plants of the legume family. In humans, these amino acids may occur as a result of exposure to environmental acrylonitrile or acrylamide, and due to consumption of the legumes. The β-CEC molecule is a homolog of S-carboxymethyl-l-cysteine (carbocisteine, CMC), a clinically employed antioxidant and mucolytic drug. We report here detailed structural data for β-CEC and β-CECO, as well as results of in vitro studies evaluating cytotoxicity and the protective potential of the amino acids in renal tubular epithelial cells (RTECs) equipped with reporters for activity of seven stress-responsive transcription factors. In RTECs, β-CEC and the sulfoxide were not acutely cytotoxic, but activated the antioxidant Nrf2 pathway. β-CEC, but not the sulfoxide, induced the amino acid stress signaling, which could be moderated by cysteine, methionine, histidine, and tryptophan. β-CEC enhanced the cytotoxic effects of arsenic, cadmium, lead, and mercury, but inhibited the cytotoxic stress induced by cisplatin, oxaliplatin, and CuO nanoparticles and acted as an antioxidant in a copper-dependent oxidative DNA degradation assay. In these experiments, the structure and activities of β-CEC closely resembled those of CMC. Our data suggest that β-CEC may act as a mild activator of the cytoprotective pathways and as a protector from platinum drugs and environmental copper cytotoxicity.

Artificially stimulating retrotransposon activity increases mortality and accelerates a subset of aging phenotypes in Drosophila.

Transposable elements (TEs) are mobile sequences of DNA that can become transcriptionally active as an animal ages. Whether TE activity is simply a by-product of heterochromatin breakdown or can contribute toward the aging process is not known. Here, we place the TE gypsy under the control of the UAS GAL4 system to model TE activation during aging. We find that increased TE activity shortens the life span of male Drosophila melanogaster. The effect is only apparent in middle-aged animals. The increase in mortality is not seen in young animals. An intact reverse transcriptase is necessary for the decrease in life span, implicating a DNA-mediated process in the effect. The decline in life span in the active gypsy flies is accompanied by the acceleration of a subset of aging phenotypes. TE activity increases sensitivity to oxidative stress and promotes a decline in circadian rhythmicity. The overexpression of the Forkhead-box O family (FOXO) stress response transcription factor can partially rescue the detrimental effects of increased TE activity on life span. Our results provide evidence that active TEs can behave as effectors in the aging process and suggest a potential novel role for dFOXO in its promotion of longevity in D. melanogaster.

Bromoacetic acid induces neurogenic injury in the chicken brain by activating oxidative stress and NF-κB inflammatory pathway

The bromoacetic acid (BAA) is one of the most teratogenic and neurotoxic disinfection byproducts. Birds take environmental water as their habitat and are inevitably affected by BAA in the environment. However, the neurotoxicity caused by BAA in birds has not been reported and the mechanism remains unclear. In this study, we chose chickens as the avian model to explore the effects of different concentrations of BAA on the brain tissues. Here, we selected the 3 μg/L dose of BAA detected in Tai Lake basin as a reference, and designed 1-, 100-, and 1000-fold of the environmental exposure dose as the experimental doses to explore the neurotoxicity of BAA in birds. Results showed that BAA increased the number of pyknotic nuclear neurons, deformed vascular sheaths, and glial cells in the brain. BAA inhibited the activity of antioxidant enzymes and the expression of antioxidant genes. With the increase of BAA concentration, the oxidative stress-responsive transcription factor NF-κB was activated. Furthermore, BAA remarkably changed the expression of lipid metabolism related genes (i.e., acc, gpat, hmgr, pparα, cpt1, and ampkα). Importantly, BAA decreased the mRNA and protein expression levels of autophagy-related genes (i.e., atg5, ulk1, beclin1, and lc3). Meantime, BAA increased the mRNA and protein levels of apoptotic and pro-apoptotic genes, such as p53, bax, cytochrome c, caspase-9, and caspase-3. Overall, our study provided new insights into the potential neurotoxic effects of BAA in birds, which was important for the clinical monitoring and prevention of BAA.

Effect of a Radical Mutation in Plastidic Starch Phosphorylase PHO1a on Potato Growth and Cold Stress Response

The plant response to stresses includes changes in starch metabolism regulated by a complex catalytic network, in which plastidic starch phosphorylase PHO1a is one of the key players. In this study, we used the CRISPR-Cas9 system to edit the PHO1a gene in four potato (Solanum tuberosum L.) cultivars, which resulted in the introduction of a radical mutation, G261V, into the PHO1a functional domain. The mutants had altered morphology and differed from wild-type plants in starch content in the roots and leaves. Exposure to cold stress revealed the differential response of parental and transgenic plants in terms of starch content and the expression of genes coding for β-amylases, amylase inhibitors, and stress-responsive MADS-domain transcription factors. These results suggest that the G261V mutation causes changes in the functional activity of PHO1a, which in turn affect the coordinated operation of starch catabolism enzymes both under normal and cold stress conditions, possibly through differential expression of MADS-domain transcription factors. Our results highlight a critical regulatory role of PHO1a in starch metabolism, root and shoot development, and stress response in potatoes.

Elucidation of an mTORC2-PKC-NRF2 pathway that sustains the ATF4 stress response and identification of Sirt5 as a key ATF4 effector

Proliferating cancer cells are dependent on glutamine metabolism for survival when challenged with oxidative stresses caused by reactive oxygen species, hypoxia, nutrient deprivation and matrix detachment. ATF4, a key stress responsive transcription factor, is essential for cancer cells to sustain glutamine metabolism when challenged with these various types of stress. While it is well documented how the ATF4 transcript is translated into protein as a stress response, an important question concerns how the ATF4 message levels are sustained to enable cancer cells to survive the challenges of nutrient deprivation and damaging reactive oxygen species. Here, we now identify the pathway in triple negative breast cancer cells that provides a sustained ATF4 response and enables their survival when encountering these challenges. This signaling pathway starts with mTORC2, which upon sensing cellular stresses arising from glutamine deprivation or an acute inhibition of glutamine metabolism, initiates a cascade of events that triggers an increase in ATF4 transcription. Surprisingly, this signaling pathway is not dependent on AKT activation, but rather requires the mTORC2 target, PKC, which activates the transcription factor Nrf2 that then induces ATF4 expression. Additionally, we identify a sirtuin family member, the NAD+-dependent de-succinylase Sirt5, as a key transcriptional target for ATF4 that promotes cancer cell survival during metabolic stress. Sirt5 plays fundamental roles in supporting cancer cell metabolism by regulating various enzymatic activities and by protecting an enzyme essential for glutaminolysis, glutaminase C (GAC), from degradation. We demonstrate that ectopic expression of Sirt5 compensates for knockdowns of ATF4 in cells exposed to glutamine deprivation-induced stress. These findings provide important new insights into the signaling cues that lead to sustained ATF4 expression as a general stress-induced regulator of glutamine metabolism, as well as highlight Sirt5 an essential effector of the ATF4 response to metabolic stress.