Activation Of Stress Response Research Articles

Abstract 4959: TIC10/ONC201 activates ClpP to degrade ALAS1 while PG3 does not degrade ALAS1 in pathway to HRI, ATF4 and CHOP activation leading to tumor cell death

Abstract TRAIL-inducing compound TIC10/ONC201 binds and activates mitochondrial protease ClpP leading to integrated stress response (ISR) and ATF4 transcription factor activation. Our lab previously reported that ONC201 activates HRI (heme-regulated inhibitor) kinase but does not signal eIF2-alpha phosphorylation through PERK or GCN2. However, the connection between ClpP activation of HRI remains unknown. PG3, a prodigiosin analog that bypasses a defective p53 pathway potently induces ATF4 and pro-apoptotic PUMA. Our data indicate that PG3 activates ATF4 through ISR via HRI as knockdown of HRI but not PKR blocked upregulation of ATF4 and CHOP in several tumor cell lines. We have been exploring how ClpP activation leads to HRI and ISR activation, and the mechanism by which PG3 leads to HRI activation. We find that activation of HRI by PG3 or ONC201 is not through the OMA1/DELE1◊HRI activation pathway. Moreover, ROS and NO are not responsible for ONC201- or PG3-induced HRI activation. ALAS1 (5'-aminolevulinate synthase 1) catalyzes the first rate-limiting step in heme (Iron-protoporphyrin) biosynthesis. ONC201 treatment leads to potent downregulation and inhibition of ALAS1, indicating that ONC201 inhibits heme biosynthesis. It is well known that reduced heme results in activation of the HRI kinase. We show that an inhibitor of heme biosynthesis or knockdown of ALAS1 results in HRI activation, while knockdown of HRI blocks upregulation of ATF4 by ONC201. Knockdown of ClpP rescues ONC201-induced downregulation of ALAS1 which blocks ONC201-induced upregulation of CHOP. Our studies identify a novel link between ClpP activation induced by ONC201 treatment and ATF4 upregulation, via the ClpP/ALAS1◊HRI◊ATF4/CHOP pathway. However, PG3 does not inhibit ALAS1, reduce its expression or therefore signal through ClpP. PG3 treatment did not lead to degradation of ALAS1, indicating that PG3 does not activate ClpP as ALAS1 is a known ClpP direct substrate. We are further investigating the signaling pathway that leads to PG3-induced activation of HRI. Our results suggest that different small molecule inducers of the ISR such as ONC201 and PG3 can achieve an anti-tumor effect through different pathways involving kinase HRI ultimately leading to ATF4/CHOP activation and tumor cell death. Citation Format: Xiaobing Tian, Wafik S. El-Deiry. TIC10/ONC201 activates ClpP to degrade ALAS1 while PG3 does not degrade ALAS1 in pathway to HRI, ATF4 and CHOP activation leading to tumor cell death. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4959.

Abstract 4842: Stress-induced mitochondrial adaptations in the polyaneuploid cancer cell state

Abstract Metastatic cancer is responsible for 90% of cancer deaths and is incurable due to resistance to all systemic anticancer therapies. While the classic model of therapy resistance is described by the presence of pre-existing resistant clones from tumor cell heterogeneity, our lab proposes the polyaneuploid cancer cell (PACC) state as a survival mechanism that cancer cells access when exposed to environmental stress such as hypoxia and chemotherapy. Our preliminary data has shown that cells that enter the PACC state exhibit repeated whole genome doubling, an increase in cell size, and proliferative arrest. In addition, we observed that a subpopulation of PACCs can give rise to non-polyploid, proliferative progeny after release from chemotherapy, modeling cancer recurrence. We hypothesize that the PACC state drives therapeutic resistance and elimination of this adaptive state is key in tackling the incurability of cancer. Mitochondria are essential organelles that have multifaceted roles in the cellular stress response and drug resistance. They integrate metabolic pathways for ATP generation and macromolecule biosynthesis, regulate apoptotic cell death, and activate stress response mechanisms in presence of drug treatment. We found that cells in the PACC state increase mitochondrial biogenesis and live cell imaging with MitoTraker dyes indicate that they also have higher activity per mitochondria when compared to parental cells. Cells that accessed the PACC state also had higher levels of TCA cycle metabolites including citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, and malate, suggesting increased mitochondrial function. Future work includes analysis of mitochondrial morphology via live cell confocal microscopy and of mitochondrial function via metabolic flux analysis on glucose and glutamine metabolism of cells in the PACC state. Our eventual goal is to characterize how cells in the PACC state alter their mitochondrial structure and function to survive therapy. Identifying vulnerabilities of the PACC phenotype will enable new therapeutic approaches to overcome therapy resistance in cancer. Citation Format: Melvin Li, Laurie G. Kostecka, Sarah R. Amend, Kenneth J. Pienta. Stress-induced mitochondrial adaptations in the polyaneuploid cancer cell state. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4842.

Single-cell transcriptome atlas of spontaneous dry age-related macular degeneration in macaques

Age-related macular degeneration is the leading cause of irreversible visual impairment in the elderly. It manifests in two forms, wet and dry. However, the mechanisms underlying spontaneous dry age-related macular degeneration (SD-AMD) remain unclear. Herein, we constructed a single-cell retinal transcription atlas in aged non-human primates with SD-AMD. Retinal tissues affected by SD-AMD exhibited a more degenerative and dysfunctional transcriptomic landscape, with global activation of the oxidative stress response and apoptotic signaling pathway. We found two distinct Müller glia subtypes in normal aged and SD-AMD macaques, one exhibiting a photoreceptor-like transcriptome and the other exhibiting a typical Müller glia transcriptome. As SD-AMD progressed, the proportion of photoreceptor-like Müller glial cells decreased, and photoreceptor-function-associated genes were downregulated, indicating weaker Müller glia potential to transit into photoreceptor-like functional states. Microglial cells showed activated features, and the complement system was activated during disease pathogenesis. We also found that the disruption of iron homeostasis and ferroptosis could promote SD-AMD pathogenesis in neural cells. Further experimentation revealed that a ferroptosis inhibitor exerted a profound rescuing effect in SD-AMD mouse models. Based on these results, our study introduces a path toward understanding the pathogenesis of SD-AMD in a non-human primate model at single-cell resolution.

Acute Ozone-Induced Transcriptional Changes in Markers of Oxidative Stress and Glucocorticoid Signaling in the Rat Hippocampus and Hypothalamus Are Sex-Specific.

Exposure to a prototypic air pollutant ozone (O3) has been associated with the activation of neuroendocrine stress response along with neural changes in oxidative stress (OS), inflammation, and Alzheimer's disease-like pathologies in susceptible animal models. We hypothesized that neural oxidative and transcriptional changes induced by O3 in stress responsive regions are sex-dependent. Male and female adult Long-Evans rats were exposed to filtered air or O3 for two consecutive days (0.8 ppm, 4 h/day) and brain regions were flash-frozen. Activities of cerebellar OS parameters and mitochondrial complex I, II, and IV enzymes were assessed to confirm prior findings. We assessed transcriptional changes in hypothalamus (HYP) and hippocampus (HIP) for markers of OS, microglial activity and glucocorticoid signaling using qPCR. Although there were no O3 or sex-related differences in the cerebellar activities of OS and mitochondrial enzymes, the levels of protein carbonyls and complex II activities were higher in females regardless of O3. There were no statistical differences in baseline expression of genes related to OS (Cat, Dhcr24, Foxm1, Gpx1, Gss, Nfe2l2, Sod1) except for lower HYP Sod1 expression in air-exposed females than males, and higher HIP Gss expression in O3-exposed females relative to matched males. Microglial marker Aif1 expression was higher in O3-exposed females relative to males; O3 inhibited Itgam only in males. The expression of Bdnf in HIP and HYP was inhibited by O3 in both sexes. Genes related to glucocorticoid signaling (Fkbp4, Fkbp5, Hsp90aa1, Hspa4, nr3c1, nr3c2) showed sex-specific effects due to O3 exposure. Baseline expression of HIP Fkbp4 was higher in females relative to males. O3 inhibited Nr3c1 in female HIP and male HYP, but Nr3c2 was inhibited in male HYP. Fkbp4 expression was higher in O3-exposed females when compared to matched males, whereas Fkbp5 was expressed at higher levels in both brain regions of males and females. These results indicate that sex-specific brain region responses to O3 might, in part, be caused by OS and regulation of glucocorticoid signaling.

Kinetic proteomics identifies targeted changes in liver metabolism and the ribo-interactome by dietary sulfur amino acid restriction

Dietary sulfur amino acid restriction (SAAR) protects against diet-induced obesity, extends healthspan, and coincides with an overall reduction in hepatic protein synthesis. To explore the underpinnings of SAAR-induced slowed growth and its impact on liver metabolism and proteostasis, we resolved changes in hepatic mRNA and protein abundances and compared synthesis rates of individual liver proteins. To achieve this, adult male mice were provided deuterium-labeled drinking water while freely consuming either a regular-fat or high-fat diet that was SAA restricted. Livers from these mice and their respective dietary controls were used to conduct transcriptomic, proteomic, and kinetic proteomic analyses. We found that remodeling of the transcriptome by SAAR was largely agnostic to dietary fat content. Shared signatures included activation of the integrated stress response alongside alterations in metabolic processes impacting lipids, fatty acids, and amino acids. Changes to the proteome correlated poorly with the transcriptome, and yet, functional clustering of kinetic proteomic changes in the liver during SAAR revealed that the management of fatty acids and amino acids were altered to support central metabolism and redox balance. Dietary SAAR also strongly influenced the synthesis rates of ribosomal proteins and ribosome-interacting proteins regardless of dietary fat. Taken together, dietary SAAR alters the transcriptome and proteome in the liver to safely manage increased fatty acid flux and energy use and couples this with targeted changes in the ribo-interactome to support proteostasis and slowed growth.

Molecular insights into Escherichia coli Cpx envelope stress response activation by the sensor lipoprotein NlpE.

Bacterial two-component signal transduction systems provide sensory inputs for appropriately adapting gene expression. These systems rely on a histidine kinase that phosphorylates a response regulator which alters gene expression. Several two-component systems include additional sensory components that can activate the histidine kinase. In Escherichia coli, the lipoprotein NlpE was identified as a sensor for the Cpx cell envelope stress response. It has remained unclear how NlpE signals to Cpx in the periplasm. In this study, we used a combination of genetics, biochemistry, and AlphaFold2 complex modeling to uncover the molecular details of how NlpE triggers the Cpx response through an interaction with the CpxA histidine kinase. Remarkably, only a short loop of NlpE is required to activate the Cpx response. A single substitution in this loop inactivates NlpE signaling to Cpx and abolishes an in vivo biochemical NlpE:CpxA interaction. An independent AlphaFold multimer prediction supported a role for the loop and predicted an interaction interface at CpxA. Mutations in this CpxA region specifically blind the histidine kinase to NlpE activation but preserve the ability to respond to other cell envelope stressors. Hence, our work additionally reveals a previously unrecognized complexity in signal integration by the CpxA periplasmic sensor domain.

Enterohemorrhagic Escherichia coli responds to gut microbiota metabolites by altering metabolism and activating stress responses

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC’s ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC’s response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

Identifying QTLs Related to Grain Filling Using a Doubled Haploid Rice (Oryza sativa L.) Population

Grain filling is an important trait of rice that affects the yield of grain-oriented crop species with sink capacity-related traits. Here, we used a doubled haploid (DH) population derived from a cross between 93-11 (P1, indica) and Milyang352 (P2, japonica) to investigate quantitative traits loci (QTLs) controlling grain filling in rice employing the Kompetitive allele-specific PCR (KASP) markers. The mapping population was grown under early-, normal-, and late-cultivation periods. The phenotypic evaluation revealed that spikelet number per panicle positively correlated with the grain-filling ratio under early cultivation conditions. Notably, three significant QTLs associated with the control of grain filling, qFG3, qFG5-1, and qFG5-2, were identified. Genes harbored by these QTLs are linked with diverse biological processes and molecular functions. Likewise, genes associated with abiotic stress response and transcription factor activity and redox homeostasis were detected. Genes such as MYB, WRKY60, and OsSh1 encoding transcription factor, β-catenin, and the tubulin FtsZ, as well as those encoding cytochrome P450, would play a forefront role in controlling grain filling under early cultivation conditions. Our results suggest that qFG3-related genes could mediate the transition between grain filling and abiotic stress response mechanisms. Fine-mapping these QTLs would help identify putative candidate genes for downstream functional characterization.

Identification of key immune and stress related genes and pathways by comparative analysis of the gene expression profile under multiple environmental stressors in pacific white shrimp (Litopenaeus vannamei)

Water salinity, pH, and nitrite concentration are considered environmental factors affecting the growth rate, survival, health, and physiological conditions of aquatic animals. The identification of key genes that are involved in the response to environmental stressors is essential for controlling stress in aquatic animals and sustainable aquaculture. In this study, RNA sequencing was performed to identify the differentially expressed genes (DEGs) and biological pathways that are involved in the response of the hepatopancreas to environmental stressors, including low salinity stress, nitrite stress, low pH stress, and high pH stress. The DEGs were enriched in biological pathways related to immune response, energy metabolism, oxidative stress response, hemostasis, and enzymatic activity of the hepatopancreas. In addition to the identification of DEGs related to each stressor, some DEGs were found to be expressed among all groups. The most important overlapping DEGs under multiple stressors were juvenile hormone esterase-like protein 2 (JHE-like), myosin light chain, C-type lectin 2, myosin-9-like, anti-lipopolysaccharide factor 1 (ALF-1), peroxisomal acyl-coenzyme An oxidase 1-like (ACX1), hepatic lectin-like, venom phosphodiesterase 2-like, hemolymph clottable protein-like (CP), cathepsin L, and Ras-like protein 2. The results of the present study provide additional information regarding the transcriptional response of the hepatopancreas to low salinity, nitrite, low pH, and high pH stress. Moreover, the discovery of several overlapping DEGs among different stressors provided a better understanding of the molecular function of the hepatopancreas.

Genetic modifiers modulate phenotypic expression of tafazzin deficiency in a mouse model of Barth syndrome.

Barth syndrome is an X-linked disorder caused by loss-of-function mutations in Tafazzin (TAZ), an acyltransferase that catalyzes remodeling of cardiolipin, a signature phospholipid of the inner mitochondrial membrane. Patients develop cardiac and skeletal muscle weakness, growth delay and neutropenia, although phenotypic expression varies considerably between patients. Taz knockout mice recapitulate many of the hallmark features of the disease. We used mouse genetics to test the hypothesis that genetic modifiers alter the phenotypic manifestations of Taz inactivation. We crossed TazKO/X females in the C57BL6/J inbred strain to males from eight inbred strains and evaluated the phenotypes of first-generation (F1) TazKO/Y progeny, compared to TazWT/Y littermates. We observed that genetic background strongly impacted phenotypic expression. C57BL6/J and CAST/EiJ[F1] TazKO/Y mice developed severe cardiomyopathy, whereas A/J[F1] TazKO/Y mice had normal heart function. C57BL6/J and WSB/EiJ[F1] TazKO/Y mice had severely reduced treadmill endurance, whereas endurance was normal in A/J[F1] and CAST/EiJ[F1] TazKO/Y mice. In all genetic backgrounds, cardiolipin showed similar abnormalities in knockout mice, and transcriptomic and metabolomic investigations identified signatures of mitochondrial uncoupling and activation of the integrated stress response. TazKO/Y cardiac mitochondria were small, clustered and had reduced cristae density in knockouts in severely affected genetic backgrounds but were relatively preserved in the permissive A/J[F1] strain. Gene expression and mitophagy measurements were consistent with reduced mitophagy in knockout mice in genetic backgrounds intolerant of Taz mutation. Our data demonstrate that genetic modifiers powerfully modulate phenotypic expression of Taz loss-of-function and act downstream of cardiolipin, possibly by altering mitochondrial quality control.

Endoplasmic Reticulum Stress in Renal Cell Carcinoma

The endoplasmic reticulum is an organelle exerting crucial functions in protein production, metabolism homeostasis and cell signaling. Endoplasmic reticulum stress occurs when cells are damaged and the capacity of this organelle to perform its normal functions is reduced. Subsequently, specific signaling cascades, together forming the so-called unfolded protein response, are activated and deeply impact cell fate. In normal renal cells, these molecular pathways strive to either resolve cell injury or activate cell death, depending on the extent of cell damage. Therefore, the activation of the endoplasmic reticulum stress pathway was suggested as an interesting therapeutic strategy for pathologies such as cancer. However, renal cancer cells are known to hijack these stress mechanisms and exploit them to their advantage in order to promote their survival through rewiring of their metabolism, activation of oxidative stress responses, autophagy, inhibition of apoptosis and senescence. Recent data strongly suggest that a certain threshold of endoplasmic reticulum stress activation needs to be attained in cancer cells in order to shift endoplasmic reticulum stress responses from a pro-survival to a pro-apoptotic outcome. Several endoplasmic reticulum stress pharmacological modulators of interest for therapeutic purposes are already available, but only a handful were tested in the case of renal carcinoma, and their effects in an in vivo setting remain poorly known. This review discusses the relevance of endoplasmic reticulum stress activation or suppression in renal cancer cell progression and the therapeutic potential of targeting this cellular process for this cancer.

Impact of a megacity on the water quality of a tropical estuary assessed by a combination of chemical analysis and in-vitro bioassays

Tropical estuaries are threatened by rapid urbanization, which leads to the spread of thousands of micropollutants and poses an environmental risk to such sensitive aqueous ecosystems. In the present study, a combination of chemical and bioanalytical water characterization was applied to investigate the impact of Ho Chi Minh megacity (HCMC, 9.2 million inhabitants in 2021) on the Saigon River and its estuary and provide a comprehensive water quality assessment. Water samples were collected along a 140-km stretch integrating the river-estuary continuum from upstream HCMC down to the estuary mouth in the East Sea. Additional water samples were collected at the mouth of the four main canals of the city center. Chemical analysis was performed targeting up to 217 micropollutants (pharmaceuticals, plasticizers, PFASs, flame retardants, hormones, pesticides). Bioanalysis was performed using six in-vitro bioassays for hormone receptor-mediated effects, xenobiotic metabolism pathways and oxidative stress response, respectively, all accompanied by cytotoxicity measurement. A total of 120 micropollutants were detected and displayed high variability along the river continuum with total concentration ranging from 0.25 to 78 μg L−1. Among them, 59 micropollutants were ubiquitous (detection frequency ≥ 80 %). An attenuation was observed in concentration and effect profiles towards the estuary. The urban canals were identified as major sources of micropollutants and bioactivity to the river, and one canal (Bến Nghé) exceeded the effect-based trigger values derived for estrogenicity and xenobiotic metabolism. Iceberg modelling apportioned the contribution of the quantified and the unknown chemicals to the measured effects. Diuron, metolachlor, chlorpyrifos, daidzein, genistein, climbazole, mebendazole and telmisartan were identified as main risk drivers of the oxidative stress response and xenobiotic metabolism pathway activation. Our study reinforced the need for improved wastewater management and deeper evaluations of the occurrence and fate of micropollutants in urbanized tropical estuarine environments.

Adaptation of Proteome and Metabolism in Different Haplotypes of Rhodosporidium toruloides during Cu(I) and Cu(II) Stress

Rhodosporidium toruloides is a carotenogenic, oleogenic yeast that is able to grow in diverse environments. In this study, the proteomic and metabolic responses to copper stress in the two haplotypes IFO0559 and IFO0880 were assessed. 0.5 mM Cu(I) extended the lag phase of both strains significantly, while only a small effect was observed for Cu(II) treatment. Other carotenogenic yeasts such as Rhodotorula mucilaginosa are known to accumulate high amounts of carotenoids as a response to oxidative stress, posed by excess copper ion activity. However, no significant increase in carotenoid accumulation for both haplotypes of R. toruloides after 144 h of 0.5 mM Cu(I) or Cu(II) stress was observed. Yet, an increase in lipid production was detected, when exposed to Cu(II), additionally, proteins related to fatty acid biosynthesis were detected in increased amounts under stress conditions. Proteomic analysis revealed that besides the activation of the enzymatic oxidative stress response, excess copper affected iron-sulfur and zinc-containing proteins and caused proteomic adaptation indicative of copper ion accumulation in the vacuole, mitochondria, and Golgi apparatus.

Cardiac Myocyte-Specific Overexpression of FASTKD1 Prevents Ventricular Rupture After Myocardial Infarction.

Background The mitochondrial mRNA-binding protein FASTKD1 (Fas-activatedserine/threonine[FAST] kinase domain-containing protein 1) protects myocytes from oxidative stress in vitro. However, the role of FASTKD1 in the myocardium in vivo is unknown. Therefore, we developed cardiac-specific FASTKD1 transgenic mice to test the effects of this protein on experimental myocardial infarction (MI). Methods and Results Transgenic mouse lines with cardiac myocyte-specific overexpression of FASTKD1 to varying degrees were generated. These mice displayed normal cardiac morphological features and function at the gross and microscopic levels. Isolated cardiac mitochondria from all transgenic mouse lines showed normal mitochondrial function, ATP levels, and permeability transition pore activity. Male nontransgenic and transgenic mice from the highest-expressing line were subjected to 8 weeks of permanent coronary ligation. Of nontransgenic mice, 40% underwent left ventricular free wall rupture within 7 days of MI compared with 0% of FASTKD1-overexpressing mice. At 3 days after MI, FASTKD1 overexpression did not alter infarct size. However, increased FASTKD1 resulted in decreased neutrophil and increased macrophage infiltration, elevated levels of the extracellular matrix component periostin, and enhanced antioxidant capacity compared with control mice. In contrast, markers of mitochondrial fusion/fission and apoptosis remained unaltered. Instead, transcriptomic analyses indicated activation of the integrated stress response in the FASTKD1 transgenic hearts. Conclusions Cardiac-specific overexpression of FASTKD1 results in viable mice displaying normal cardiac morphological features and function. However, these mice are resistant to MI-induced cardiac rupture and display altered inflammatory, extracellular matrix, and antioxidant responses following MI. Moreover, these protective effects were associated with enhanced activation of the integrated stress response.

Hypothalamic Oxytocin Neuron Activation Attenuates Intermittent Hypoxia-Induced Hypertension and Cardiac Dysfunction in an Animal Model of Sleep Apnea.

Obstructive sleep apnea is a prevalent and poorly treated cardiovascular disease that leads to hypertension and autonomic imbalance. Recent studies that restore cardiac parasympathetic tone using selective activation of hypothalamic oxytocin neurons have shown beneficial cardiovascular outcomes in animal models of cardiovascular disease. This study aimed to determine if chemogenetic activation of hypothalamic oxytocin neurons in animals with existing obstructive sleep apnea-induced hypertension would reverse or blunt the progression of autonomic and cardiovascular dysfunction. Two groups of rats were exposed to chronic intermittent hypoxia (CIH), a model of obstructive sleep apnea, for 4 weeks to induce hypertension. During an additional 4 weeks of exposure to CIH, 1 group was treated with selective activation of hypothalamic oxytocin neurons while the other group was untreated. Hypertensive animals exposed to CIH and treated with daily hypothalamic oxytocin neuron activation had lower blood pressure, faster heart rate recovery times after exercise, and improved indices of cardiac function compared with untreated hypertensive animals. Microarray analysis suggested that, compared with treated animals, untreated animals had gene expression profiles associated with cellular stress response activation, hypoxia-inducible factor stabilization, and myocardial extracellular matrix remodeling and fibrosis. In animals already presenting with CIH-induced hypertension, chronic activation of hypothalamic oxytocin neurons blunted the progression of hypertension and conferred cardioprotection after an additional 4 weeks of CIH exposure. These results have significant clinical translation for the treatment of cardiovascular disease in patients with obstructive sleep apnea.

Cardiomyocyte infection by Trypanosoma cruzi promotes innate immune response and glycolysis activation.

Chagas cardiomyopathy, a disease caused by Trypanosoma cruzi (T. cruzi) infection, is a major contributor to heart failure in Latin America. There are significant gaps in our understanding of the mechanism for infection of human cardiomyocytes, the pathways activated during the acute phase of the disease, and the molecular changes that lead to the progression of cardiomyopathy. To investigate the effects of T. cruzi on human cardiomyocytes during infection, we infected induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) with the parasite and analyzed cellular, molecular, and metabolic responses at 3 hours, 24 hours, and 48 hours post infection (hpi) using transcriptomics (RNAseq), proteomics (LC-MS), and metabolomics (GC-MS and Seahorse) analyses. Analyses of multiomic data revealed that cardiomyocyte infection caused a rapid increase in genes and proteins related to activation innate and adaptive immune systems and pathways, including alpha and gamma interferons, HIF-1α signaling, and glycolysis. These responses resemble prototypic responses observed in pathogen-activated immune cells. Infection also caused an activation of glycolysis that was dependent on HIF-1α signaling. Using gene editing and pharmacological inhibitors, we found that T. cruzi uptake was mediated in part by the glucose-facilitated transporter GLUT4 and that the attenuation of glycolysis, HIF-1α activation, or GLUT4 expression decreased T. cruzi infection. In contrast, pre-activation of pro-inflammatory immune responses with LPS resulted in increased infection rates. These findings suggest that T. cruzi exploits a HIF-1α-dependent, cardiomyocyte-intrinsic stress-response activation of glycolysis to promote intracellular infection and replication. These chronic immuno-metabolic responses by cardiomyocytes promote dysfunction, cell death, and the emergence of cardiomyopathy.

DELE1 oligomers revealed by cryo-EM structure promote the integrated stress response activation.

Mitochondria are dynamic organelles that must continually adapt and respond to cellular stress, and perturbations in mitochondrial function trigger the integrated stress response (ISR). Recent studies demonstrated that mitochondrial stress can be relayed from the mitochondrial interior to the cytosol by the release of a C-terminal proteolytic fragment of DELE1 that binds to the HRI kinase to initiate integrate stress response signaling. Here, we report the cryo-electron microscopy structure of the active, C-terminal cleavage product of human DELE1 at ∼3.8 Å resolution, revealing that DELE1 assembles into a high-order oligomer. We confirmed that this C-terminal fragment of DELE1 also assembles into large oligomers in cells. Structurally, the oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of distinct hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. Further, we show that assembly-impaired DELE1 mutants are compromised in their ability to induce ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.

Cadmium treatment induces endoplasmic reticulum stress and unfolded protein response in Arabidopsisthaliana

We report about the response of Arabidopsis thaliana to chronic and temporary Cd2+ stress, and the Cd2+ induced activation of ER stress and unfolded protein response (UPR). Cd2+-induced UPR proceeds mainly through the bZIP60 arm, which in turn activates relevant ER stress marker genes such as BiP3, CNX, PDI5 and ERdj3B in a concentration- (chronic stress) or time- (temporary stress) dependent manner. A more severe Cd-stress triggers programmed cell death (PCD) through the activation of the NAC089 transcription factor. Toxic effects of Cd2+ exposure are reduced in the Atbzip28/bzip60 double mutant in terms of primary root length and fresh shoot weight, likely due to reduced UPR and PCD activation. We also hypothesised that the enhanced Cd2+ tolerance of the Atbzip28/bzip60 double mutant is due to an increase in brassinosteroids signaling, since the amount of the brassinosteroid insensitive1 receptor (BRI1) protein decreases under Cd2+ stress only in Wt plants. These data highlight the complexity of the UPR pathway, since the ER stress response is strictly related to the type of the treatment applied and the multifaceted connections of ER signaling. The reduced sensing of Cd2+ stress in plants with UPR defects can be used as a novel strategy for phytoremediation.

Truncated ring-A amaryllidaceae alkaloid modulates the host cell integrated stress response, exhibiting antiviral activity to HSV-1 and SARSCoV-2

The total synthesis of four novel mono-methoxy and hydroxyl substituted ring-A dihydronarciclasine derivatives enabled identification of the 7-hydroxyl derivative as a potent and selective antiviral agent targeting SARSCoV-2 and HSV-1. The concentration of this small molecule that inhibited HSV-1 infection by 50% (IC50), determined by using induced pluripotent stem cells (iPCS)-derived brain organ organoids generated from two iPCS lines, was estimated to be 0.504 µM and 0.209 µM. No significant reduction in organoid viability was observed at concentrations up to 50 mM. Genomic expression analyses revealed a significant effect on host-cell innate immunity, revealing activation of the integrated stress response via PERK kinase upregulation, phosphorylation of eukaryotic initiation factor 2α (eIF2α) and type I IFN, as factors potentiating multiple host-defense mechanisms against viral infection. Following infection of mouse eyes with HSV-1, treatment with the compound dramatically reduced HSV-1 shedding in vivo.

Propofol Regulates ER Stress to Inhibit Tumour Growth and Sensitize Osteosarcoma to Doxorubicin.

Osteosarcoma is the most common malignant bone tumour affecting children and young adults. The antitumour role of propofol, a widely used intravenous sedative-hypnotic agent, has been recently reported in different cancer types. In this study, we aimed to assess the role of propofol on osteosarcoma and explore the possible mechanisms. Propofol of increasing concentrations (2.5, 5, 10, and 20 μg/ml) was used to treat the MG63 and 143B cells for 72 hours, and the CCK8 assay was applied to evaluate the tumour cell proliferation. Tumour cell migration and invasion were assessed with the transwell assay. The tumour cells were also treated with doxorubicin single agent or in combination with propofol to explore their synergic role. Differential expressed genes after propofol treatment were obtained and functionally assessed with bioinformatic tools. Expression of ER stress markers CHOP, p-eIF2α, and XBP1s was evaluated to validate the activation of ER stress response with western blot and qRT-PCR. The statistical analyses were performed with R v4.2.1. Propofol treatment led to significant growth inhibition in MG63 and 143B cells in a dose-dependent manner (p < 0.05). Osteosarcoma migration (MG63 91.4 (82-102) vs. 56.8 (49-65), p < 0.05; 143B 96.6 (77-104) vs. 45.4 (28-54), p < 0.05) and invasion (MG63 68.6 (61-80) vs. 32 (25-39), p < 0.05; 143B 90.6 (72-100) vs. 39.2 (26-55), p < 0.05) were reduced after propofol treatment. Doxorubicin sensitivity was increased after propofol treatment compared with the control group (p < 0.05). Bioinformatic analysis showed significant functional enrichment in ER stress response after propofol treatment. Upregulation of CHOP, p-eIF2α, and XBP1s was detected in MG63 and 143B secondary to propofol treatment. In conclusion, we found that propofol treatment suppressed osteosarcoma proliferation and invasion and had a synergic role with doxorubicin by inducing ER stress. Our findings provided a novel option in osteosarcoma therapy.