Cellular Stress Response Mechanisms Research Articles

Complementary Omics Strategies to Dissect p53 Signaling Networks Under Nutrient Stress

Signaling trough p53 is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

Expression of activating transcription factor 5 (ATF5) is mediated by microRNA-520b-3p under diverse cellular stress in cancer cells.

Cellular stress response mechanisms normally function to enhance survival and allow for cells to return to homeostasis following an adverse event. Cancer cells often co-opt these same mechanisms as a means to evade apoptosis and mitigate a state of constant cellular stress. Activating transcription factor 5 (ATF5) is upregulated under diverse stress conditions and is overexpressed in a variety of cancers. It was demonstrated ATF5 is a survival factor in transformed, but not normal cells. However, the regulation of ATF5 is not fully understood. The purpose of the present study was to investigate miRNA regulation at the 3’ untranslated region (UTR) of ATF5, with the goal of demonstrating a reversal of the upregulation of ATF5 induced under diverse cellular stress in cancer cells. A multifactorial approach using in silico analysis was employed to identify miRNAs 433-3p, 520b-3p, and 129-5p as potential regulators of ATF5, based on their predicted binding sites over the span of the ATF5 3’ UTR. Luciferase reporter assay data validated all three miRNA candidates by demonstrating direct binding to the target ATF5 3’. However, functional studies revealed miR-520b-3p as the sole candidate able to reverse the upregulation of ATF5 protein under diverse cellular stress. Additionally, miR-520b-3p levels were inversely related to ATF5 mRNA under endoplasmic reticulum stress and amino acid deprivation. This is the first evidence that regulation at the 3’ UTR is involved in modulating ATF5 levels under cellular stress and suggests an important role for miRNA-520b-3p in the regulation of ATF5.

Mitochondrial turnover and homeostasis in ageing and neurodegeneration.

Ageing is driven by the inexorable and stochastic accumulation of damage in biomolecules vital for proper cellular function. Although this process is fundamentally haphazard and uncontrollable, genetic and extrinsic factors influence senescent decline and ageing. Numerous gene mutations and treatments have been shown to extend the lifespan of organisms ranging from the unicellular Saccharomycescerevisiae to primates. Most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to counter both intrinsic stress and extrinsic stress. Mitochondria, the main energy hub of the cell, are highly dynamic organelles, playing essential roles in cell physiology. Mitochondrial function impinges on several signalling pathways modulating cellular metabolism, survival and healthspan. Maintenance of mitochondrial function and energy homeostasis requires both generation of new healthy mitochondria and elimination of the dysfunctional ones. Here, we survey the mechanisms regulating mitochondrial number in cells, with particular emphasis on neurons. We, further, discuss recent findings implicating perturbation of mitochondrial homeostasis in cellular senescence and organismal ageing as well as in age-associated neurodegenerative diseases.

Mitochondrial Quality Control Governed by Ubiquitin.

Mitochondria are essential organelles important for energy production, proliferation, and cell death. Biogenesis, homeostasis, and degradation of this organelle are tightly controlled to match cellular needs and counteract chronic stress conditions. Despite providing their own DNA, the vast majority of mitochondrial proteins are encoded in the nucleus, synthesized by cytosolic ribosomes, and subsequently imported into different mitochondrial compartments. The integrity of the mitochondrial proteome is permanently challenged by defects in folding, transport, and turnover of mitochondrial proteins. Therefore, damaged proteins are constantly sequestered from the outer mitochondrial membrane and targeted for proteasomal degradation in the cytosol via mitochondrial-associated degradation (MAD). Recent studies identified specialized quality control mechanisms important to decrease mislocalized proteins, which affect the mitochondrial import machinery. Interestingly, central factors of these ubiquitin-dependent pathways are shared with the ER-associated degradation (ERAD) machinery, indicating close collaboration between both tubular organelles. Here, we summarize recently described cellular stress response mechanisms, which are triggered by defects in mitochondrial protein import and quality control. Moreover, we discuss how ubiquitin-dependent degradation is integrated with cytosolic stress responses, particularly focused on the crosstalk between MAD and ERAD.

Hsp70 and the Unfolded Protein Response as a Challenging Drug Target and an Inspiration for Probe Molecule Development.

The unfolded protein response (UPR) is a cellular stress response mechanism that is critical for cell survival. Pharmacological modulation of the ATPase activity of the chaperone Hsp70 can trigger UPR-mediated cell death, thus removing pathogenic cells in human malignancies, or, alternatively, stimulate survival, thereby preventing apoptosis in neuronal cells and slowing the progress of inflammation, neurodegeneration, and aging. This Viewpoint highlights the complexity of the protein homeostasis network and discusses different approaches for modulating Hsp70 activity, including the use of a chemical reaction development-inspired library of Hsp70 agonists and antagonists.

Evolution of cellular stress response mechanisms.

The cellular stress response (CSR) is pervasive to all domains of life. It has shaped the interaction between organisms and their environment since the origin of the first cell. Although the CSR has been subject to a myriad of nuanced modifications in the various branches of life present today, its core features remain preserved. The scientific literature covering the CSR is enormous and the broad scope of this brief overview was challenging. However, it is critical to conceptually understand how cells respond to stress in a holistic sense and to point out how fundamental aspects of the CSR framework are integrated. It was necessary to be extremely selective and not feasible to even mention many interesting and important developments in this expansive field. The purpose of this overview is to sketch out general and emerging CSR concepts with an emphasis on the initial cellular strain resulting from stress (macromolecular damage) and the evolutionarily most highly conserved elements of the CSR. Examples emphasize fish and aquatic invertebrates to highlight what is known in organisms beyond mammals, yeast, and other common models. Nonetheless, select pioneering studies using canonical models are also considered and the concepts discussed are applicable to all cells. More detail on important aspects of the CSR in aquatic animals is provided in the accompanying articles of this special issue.

Mechanistic understanding of cellular responses to genomic stress.

Within the past half century we have learned of multiple pathways for repairing damaged DNA, based upon the intrinsic redundancy of information in its complementary double strands. Mechanistic details of these pathways have provided insights into environmental and endogenous threats to genomic stability. Studies on bacterial responses to ultraviolet light led to the discovery of excision repair, as well as the inducible SOS response to DNA damage. Similar responses in eukaryotes promote upregulation of error-prone translesion DNA polymerases. Recent advances in this burgeoning field include duplex DNA sequencing to provide strikingly accurate profiling of mutational signatures, analyses of gene expression patterns in single cells, CRISPR/Cas9 to generate changes at precise genomic positions, novel roles for RNA in gene expression and DNA repair, phase-separated aqueous environments for specialized cellular transactions, and DNA lesions as epigenetic signals for gene expression. The Environmental Mutagenesis and Genomics Society (EMGS), through the broad range of expertise in its membership, stands at the crossroad of basic understanding of mechanisms for genomic maintenance and the field of genetic toxicology, with the need for regulation of exposures to toxic substances. Our future challenges include devising strategies and technologies to identify individuals who are susceptible to specific genomic stresses, along with basic research on the underlying mechanisms of cellular stress responses that promote disease-causing mutations. As the science moves forward it should also be a responsibility for the EMGS to expand its outreach programs for the enlightenment and benefit of all humans and the biosphere. Environ. Mol. Mutagen. 61:25-33, 2020. © 2019 Wiley Periodicals, Inc.

Combination therapies enhance immunoregulatory properties of MIAMI cells

BackgroundMesenchymal stromal cells (MSCs), adult stromal cells most commonly isolated from bone marrow (BM), are being increasingly utilized in various therapeutic applications including tissue repair via immunomodulation, which is recognized as one of their most relevant mechanism of action. The promise of MSC-based therapies is somewhat hindered by their apparent modest clinical benefits, highlighting the need for approaches that would increase the efficacy of such therapies. Manipulation of cellular stress-response mechanism(s) such as autophagy, a catabolic stress-response mechanism, with small molecules prior to or during MSC injection could improve MSCs’ therapeutic efficacy. Unfortunately, limited information exists on how manipulation of autophagy affects MSCs’ response to inflammation and subsequent immunoregulatory properties.MethodsIn this study, we exposed BM-MSC precursor cells, “marrow-isolated adult multilineage inducible” (MIAMI) cells, to autophagy modulators tamoxifen (TX) or chloroquine (CQ), together with IFN-γ. Exposed cells then underwent RNA sequencing (RNAseq) to determine the effects of TX or CQ co-treatments on cellular response to IFN-γ at a molecular level. Furthermore, we evaluated their immunoregulatory capacity using activated CD4+ T cells by analyzing T cell activation marker CD25 and the percentage of proliferating T cells after co-culturing the cells with MIAMI cells treated or not with TX or CQ.ResultsRNAseq data indicate that the co-treatments alter both mRNA and protein levels of key genes responsible for MSCs’ immune-regulatory properties. Interestingly, TX and CQ also altered some of the microRNAs targeting such key genes. In addition, while IFN-γ treatment alone increased the surface expression of PD-L1 and secretion of IDO, this increase was further enhanced with TX. An improvement in MIAMI cells’ ability to decrease the activation and proliferation of T cells was also observed with TX, and to a lesser extent, CQ co-treatments.ConclusionAltogether, this work suggests that both TX and CQ have a potential to enhance MIAMI cells’ immunoregulatory properties. However, this enhancement is more pronounced with TX co-treatment.

Nrf2 dysfunction and impaired cellular resilience to oxidative stressors in the aged vasculature: from increased cellular senescence to the pathogenesis of age-related vascular diseases.

Aging is associated with increased oxidative stress in vascular endothelial and smooth muscle cells, which contribute to the development of a wide range of diseases affecting the circulatory system in older adults. There is growing evidence that in addition to increased production of reactive oxygen species (ROS), aging critically impairs pathways determining cellular resilience to oxidative stressors. In young organisms, the evolutionarily conserved nuclear factor-erythroid-2-related factor 2 (Nrf2)-mediated antioxidant response pathway maintains cellular reduction-oxidation homeostasis and promotes a youthful cellular phenotype by regulating the transcription of an array of cytoprotective (antioxidant, pro-survival, anti-inflammatory and macromolecular damage repair) genes. A critical mechanism by which increased ROS production and Nrf2 dysfunction promote vascular aging and exacerbate pathogenesis of age-related vascular diseases is induction of cellular senescence, an evolutionarily conserved cellular stress response mechanism. Senescent cells cease dividing and undergo distinctive phenotypic alterations, contributing to impairment of angiogenic processes, chronic sterile inflammation, remodeling of the extracellular matrix, and barrier dysfunction. Herein, we review mechanisms contributing to dysregulation of Nrf2-driven cytoprotective responses in the aged vasculature and discuss the multifaceted role of Nrf2 dysfunction in the genesis of age-related pathologies affecting the circulatory system, including its role in induction of cellular senescence. Therapeutic strategies that restore Nrf2 signaling and improve vascular resilience in aging are explored to reduce cardiovascular mortality and morbidity in older adults.

Cell line-dependent increase in cellular quercetin accumulation upon stress induced by valinomycin and lipopolysaccharide, but not by TNF-α.

As the interface between the luminal and internal environment, the intestinal epithelium is strongly exposed to food-related, host-related and microbial stress. Furthermore, the endothelial stress response plays an important role in vascular disease development, which may be improved upon consumption of dietary bioactives such as polyphenols. The impact of the latter, however, is largely individual-dependent and effects are, in most cases, only observed under mild diseased conditions. Here, it is hypothesized that the individual's stressor levels may contribute to this variable response. To this end, the impact of the stressors (i) valinomycin (as model for cereulide, food-related microbial metabolite), (ii) TNF-α (host-related) and (iii) lipopolysaccharide (gram-negative bacterial cell related) on flavonoid accumulation was investigated in several intestinal and endothelial cell lines. Flow cytometry, confocal microscopy and an in-house developed, robust and high-throughput spectrofluorometric method, showed that quercetin accumulated in all tested cell lines in a dose-dependent manner. Upon stress induced by valinomycin and to a lesser extent by lipopolysaccharide, but not by TNF-α, an increased quercetin accumulation was observed in proliferating intestinal and endothelial cells and not in differentiated intestinal or quiescent endothelial cells. Therefore, flavonoid accumulation may be a potential cellular stress response mechanism which strongly depends on the applied stressor, flavonoid, cell line and even growth conditions. This opens perspectives for further understanding the mechanisms by which cellular stress may shape the individual's response to bioactive compounds.

Scaling laws of cell-fate responses to transient stress

Analysis and modelling of dose-survival curves of cells and tissues are often used to assess therapeutic efficacy or environmental risks, much less to infer the intracellular regulatory mechanisms of cellular stress response. However, systematic measurements of how cell survival depends on the time profile of stress, such as exposure duration, provide practical means to decipher the homeostatic dynamics of stress-response regulatory networks. In this paper, we propose a dynamical framework to theoretically address the relationship between cell fate response to a transient stress and the underlying regulatory feedback mechanisms. A simple network topology that couples a homeostatic negative feedback and a death-triggering positive feedback is shown to display four response regimes for which the iso-effect relationships between duration and intensity are captured by specific power laws. These distinct response regimes define several windows of stress duration for which lethality is not merely proportional to the product of intensity and duration, and, thus, for which cells are either more tolerant or more vulnerable to a given dose. Overall, this study highlights the differential roles of feedback strength, timescale and nonlinearity in promoting survivability to particular stress profiles, providing a valuable framework for a comparative analysis of diverse stress-specific regulatory networks.

PS1360 LONG NON‐CODING RNA NEAT1 TARGETING TRIGGERS ANTI‐TUMOR ACTIVITY AND RESULTS IN CHEMO‐SENSITIZING EFFECT IN MULTIPLE MYELOMA CELLS

Background:Multiple myeloma (MM) is a fatal malignant proliferation of antibody‐secreting bone marrow plasma cells (PCs) characterized by high genomic instability. The discovery of long non‐coding RNAs (lncRNAs), for which the biological role and therapeutic potential remain open questions, has added a further layer of complexity to the pathobiology of the disease.NEAT1 is a lncRNA located at 11q13, transcribed in two different isoforms, retained in the nucleus where it forms the core structural component of the paraspeckle sub‐organelles.Aims:Our recent work reported a significant overexpression of NEAT1 in MM as compared to normal control and its putative involvement in different mechanisms of cellular stress response. Based on these data, the present study was aimed at characterizing the biological role of NEAT1 in MM cells in order to evaluate whether its targeting could represent a promising strategy for novel anti‐MM therapeutic options.Methods:qRT‐PCR has been used to evaluate NEAT1 expression level in a panel of different hematological cell lines by the use of two distinct couples of primers, able to amplify either both isoforms or selectively the long variant of NEAT1. To investigate the specific role played by NEAT1 in MM, we silenced NEAT1 in NCI‐H929 and AMO‐1 cell lines by using the gimnotyc delivery of LNA‐gapmeR; efficiency of silencing was evaluated by qRT‐PCR and RNA‐FISH. Proliferation, cell cycle and apoptosis were investigated by BrdU incorporation assay and FACS analysis, respectively. Gene expression profiling upon NEAT1 silencing was investigated by GeneChip® Human Gene 2.0 ST array by the use of Affimetrix platform.Results:Our study demonstrated that NEAT1 expression level is significantly higher in MM cells with respect to other hematological malignancies. NEAT1 silencing was able to antagonize MM cell proliferation, as suggested by a significant decrease in the number of growing cells, and to affect cell cycle with an increased percentage of sub G0/G1 phase events. Furthermore, NEAT1 deprivation was able to trigger apoptosis in vitro as revealed by the increase of Annexin+/7AAD+ cells and of cleaved PARP, Casp3 and Casp7 fractions. Induction of apoptosis and reduction of cell viability upon NEAT1 silencing were also confirmed on purified CD138+ MM primary cells. By transcriptome analyses, we found that, in both NCI‐H929 and AMO‐1, NEAT1 targeting down‐regulated genes involved in DNA repair processes. Finally, we found that NEAT1 silencing had a synergistic effect in MM cells when combined with proteasome inhibitors. Furthermore, our data indicated an anti‐proliferative and pro‐apoptotic effect of NEAT1 silencing in vivo in xenograft mouse model.Summary/Conclusion:Overall, our study investigating the pathogenic role of NEAT1 in MM provided novel important insights concerning the NEAT1‐dependent regulation of MM growth and proliferation. We also provided preliminary evidence that a LNA‐gapmeR NEAT1‐targeting strategy may represent a potential therapeutic approach for MM treatment.

The role of sphingolipid metabolism in lifespan and healthspan extension in C. elegans

Multiple advancements in research have led to a better understanding of the aging process. However, many metabolic pathways that promote healthy aging are still unknown. Here, we investigate the bioactive sphingolipid metabolism pathway and its roles in regulating lifespan and healthspan, which is the average period of one's life in good health. Sphingolipids are involved in cellular signaling and known to promote cell proliferation, secretion, stress response and survival. The sphingolipid pathway is composed of sphingomyelin, ceramide, sphingosine and sphingosine‐1‐phosphate (S1P). Ceramide synthases (CER synthases) mediate the conversion of ceramide to sphingosine while sphingosine kinase (SPHK) converts sphingosine to S1P. Using C. elegans, we previously found that mutations in sphk‐1/SPHK and hyl‐2/CER synthase result in decreased lifespan, suggesting that sphingolipids play a role in lifespan extension. However, it is unknown whether the sphingolipid genes act in known cellular pathways that mediate aging. Therefore, we tested the genetic interactions between sphingolipid enzymes and known models of aging, including the insulin like signaling (ILS) pathway that has been linked to lifespan extension. For this, lifespan analyses were performed on daf‐16/FOXO, which has a shortened lifespan phenotype, coupled with knockdown of sphingolipid metabolism genes. DAF‐16 is a downstream transcription factor target of DAF‐2/insulin‐like growth factor receptor and has important roles in mediating the gene expression of stress response. We observed that daf‐16 is epistatic to sphingolipid mutants for lifespan phenotypes, as indicated by similar short lifespan phenotypes of double knockout of daf‐16;hyl‐2 and daf‐16;sphk‐1compared to mutants with a single knockout of daf‐16. Thus, sphingolipids may act upstream of DAF‐16 to mediate stress response genes and lifespan. Future experiments will analyze transcriptional reporters for DAF‐16, and known stress response genes SOD‐3/superoxide dismutase, to determine whether cellular stress response mechanisms occurs normally in hyl‐2 and sphk‐1mutants under induced oxidative stress. An analysis of stress response will further enhance our understanding of the interaction between sphingolipids and known pathways that promote lifespan and healthspan.Support or Funding InformationResearch reported in this publication was supported by the National Institute On Aging of the National Institutes of Health under Award Number R15AG052933.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proteomic changes in oocytes after in vitro maturation in lipotoxic conditions are different from those in cumulus cells

Maternal lipolytic metabolic disorders result in a lipotoxic microenvironment in the ovarian follicular fluid (FF) which deteriorates oocyte quality. Although cellular stress response mechanisms are well defined in somatic cells, they remain largely unexplored in oocytes, which have distinct organelle structure and nuclear transcription patterns. Here we used shotgun proteomic analyses to study cellular responses of bovine oocytes and cumulus cells (CCs) after in vitro maturation under lipotoxic conditions; in the presence of pathophysiological palmitic acid (PA) concentration as a model. Differentially regulated proteins (DRPs) were mainly localized in the endoplasmic reticulum, mitochondria and nuclei of CCs and oocytes, however the DRPs and their direction of change were cell-type specific. Proteomic changes in PA-exposed CCs were predominantly pro-apoptotic unfolded protein responses (UPRs), mitochondrial and metabolic dysfunctions, and apoptotic pathways. This was also functionally confirmed. Interestingly, although the oocytes were enclosed by CCs during PA exposure, elevated cellular stress levels were also evident. However, pro-survival UPRs, redox regulatory and compensatory metabolic mechanisms were prominent despite evidence of mitochondrial dysfunction, oxidative stress, and reduced subsequent embryo development. The data provides a unique insight that enriches the understanding of the cellular stress responses in metabolically-compromised oocytes and forms a fundamental base to identify new targets for fertility treatments as discussed within.

Circadian control of stress granules by oscillating EIF2\u03b1

Stress granule formation is important for stress response in normal cells and could lead to chemotherapy resistance in cancer cells. Aberrant stress granule dynamics are also known to disrupt proteostasis, affect RNA metabolism, and contribute to neuronal cell death. Meanwhile, circadian abnormality is an aging-related risk factor for cancer and neurodegeneration. Whether stress granule dynamics are circadian regulated is entirely unknown. Here we show that the formation of stress granules varied by zeitgeber time in mouse liver. Moreover, altering circadian regulation by silencing the core circadian gene Bmal1 in a cell line expressing an endogenous GFP-tagged G3BP1 significantly increased stress granule dynamics, while the overexpression of Bmal1 decreased them. Surprisingly, increased stress granule dynamics and formation by transient decrease of BMAL1 coincided with increased resistance to stress-induced cell death. The circadian regulation of stress granules was mediated by oscillating eIF2α expression. At zeitgeber time when BMAL1 and eIF2α were at nadir, reduction of unphosphorylated eIF2α could significantly alter the ratio of phosphorylated/total eIF2α and quickly lead to increased formation of stress granules. Therefore, diurnal oscillating eIF2α connects the circadian cue to a cellular stress response mechanism that is vital for both neurodegeneration and cancer.

Large scale study of anti-sense regulation by differential network analysis

BackgroundSystems biology aims to analyse regulation mechanisms into the cell. By mapping interactions observed in different situations, differential network analysis has shown its power to reveal specific cellular responses or specific dysfunctional regulations. In this work, we propose to explore on a large scale the role of natural anti-sense transcription on gene regulation mechanisms, and we focus our study on apple (Malus domestica) in the context of fruit ripening in cold storage.ResultsWe present a differential functional analysis of the sense and anti-sense transcriptomic data that reveals functional terms linked to the ripening process. To develop our differential network analysis, we introduce our inference method of an Extended Core Network; this method is inspired by C3NET, but extends the notion of significant interactions. By comparing two extended core networks, one inferred with sense data and the other one inferred with sense and anti-sense data, our differential analysis is first performed on a local view and reveals AS-impacted genes, genes that have important interactions impacted by anti-sense transcription. The motifs surrounding AS-impacted genes gather transcripts with functions mostly consistent with the biological context of the data used and the method allows us to identify new actors involved in ripening and cold acclimation pathways and to decipher their interactions. Then from a more global view, we compute minimal sub-networks that connect the AS-impacted genes using Steiner trees. Those Steiner trees allow us to study the rewiring of the AS-impacted genes in the network with anti-sense actors.ConclusionAnti-sense transcription is usually ignored in transcriptomic studies. The large-scale differential analysis of apple data that we propose reveals that anti-sense regulation may have an important impact in several cellular stress response mechanisms. Our data mining process enables to highlight specific interactions that deserve further experimental investigations.

Nonspecific stress response to temperature increase in Gammarus lacustris Sars with respect to oxygen-limited thermal tolerance concept.

The previously undescribed dynamics of the heat shock protein HSP70 and subsequent lipid peroxidation products have been assessed alongside lactate dehydrogenase activity for Gammarus lacustris Sars, an amphipod species from the saltwater Lake Shira (Republic of Khakassia). Individuals were exposed to a gradual temperature increase of 1 °C/hour (total exposure duration of 26 hours) starting from the mean annual temperature of their habitat (7 °C) up to 33 °C. A complex of biochemical reactions occurred when saltwater G. lactustris was exposed to the gradual changes in temperature. This was characterized by a decrease in lactate dehydrogenase activity and the launching of lipid peroxidation. The HSP70 level did not change significantly during the entire experiment. In agreement with the concept of oxygen-limited thermal tolerance, an accumulation of the most toxic lipid peroxides (triene conjugates and Schiff bases) in phospholipids occurred at the same time and temperature as the accumulation of lactate. The main criterion overriding the temperature threshold was, therefore, the transition to anaerobiosis, confirmed by the elevated lactate levels as observed in our previous associated study, and by the development of cellular stress, which was expressed by an accumulation of lipid peroxidation products. An earlier hypothesis, based on freshwater individuals of the same species, has been confirmed whereby the increased thermotolerance of G. lacustris from the saltwater lake was caused by differences in energy metabolism and energy supply of nonspecific cellular stress-response mechanisms. With the development of global climate change, these reactions could be advantageous for saltwater G. lacustris. The studied biochemical reactions can be used as biomarkers for the stress status of aquatic organisms when their habitat temperature changes.

Effect of Aronia melanocarpa Extract on Lifespan and Stress Resistance in C. elegans

Oxidative stress caused by exposure to reactive oxygen species (ROS) induces widespread damage within the cell. ROS have damaging effects on the structure of DNA, proteins, and lipids, which contributes to the pathology of numerous diseases such as Parkinson's, Alzheimer's, and cancer. Cellular stress response mechanisms serve to mitigate the effects of oxidative stress by using antioxidant compounds to neutralize ROS. Black chokeberry (Aronia melanocarpa) is considered to be an excellent source of antioxidants such as polyphenols, anthocyanins and pro‐anthocyanins. The aim of this study was to investigate whether a crude mixture of Aronia extract would increase lifespan and stress resistance in Caenorhabditis elegans. N2 Bristol strain survival and stress resistance were tested with toxicity (0 mg×mL−1, 0.1 mg×mL−1, 1 mg×mL−1, 5 mg×mL−1, and 10 mg×mL−1, A. melanocarpa) and heat stress assays. Supplementation with 0.1 mg×mL−1 Aronia significantly increased survival as compared to the control condition (p = 0.006). No significant difference in survival was observed with Aronia supplementation during heat stress (p = 0.27). In conclusion, these data suggested Aronia supplementation had a beneficial effect on worm survival. However, more research is necessary to determine its effect on survival and resistance in response to cellular heat stress.Support or Funding InformationNew investigator start‐up, McNair Scholar ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Systems-Level Feedbacks of NRF2 Controlling Autophagy upon Oxidative Stress Response

Although the primary role of autophagy-dependent cellular self-eating is cytoprotective upon various stress events (such as starvation, oxidative stress, and high temperatures), sustained autophagy might lead to cell death. A transcription factor called NRF2 (nuclear factor erythroid-related factor 2) seems to be essential in maintaining cellular homeostasis in the presence of either reactive oxygen or nitrogen species generated by internal metabolism or external exposure. Accumulating experimental evidence reveals that oxidative stress also influences the balance of the 5′ AMP-activated protein kinase (AMPK)/rapamycin (mammalian kinase target of rapamycin or mTOR) signaling pathway, thereby inducing autophagy. Based on computational modeling here we propose that the regulatory triangle of AMPK, NRF2 and mTOR guaranties a precise oxidative stress response mechanism comprising of autophagy. We suggest that under conditions of oxidative stress, AMPK is crucial for autophagy induction via mTOR down-regulation, while NRF2 fine-tunes the process of autophagy according to the level of oxidative stress. We claim that the cellular oxidative stress response mechanism achieves an incoherently amplified negative feedback loop involving NRF2, mTOR and AMPK. The mTOR-NRF2 double negative feedback generates bistability, supporting the proper separation of two alternative steady states, called autophagy-dependent survival (at low stress) and cell death (at high stress). In addition, an AMPK-mTOR-NRF2 negative feedback loop suggests an oscillatory characteristic of autophagy upon prolonged intermediate levels of oxidative stress, resulting in new rounds of autophagy stimulation until the stress events cannot be dissolved. Our results indicate that AMPK-, NRF2- and mTOR-controlled autophagy induction provides a dynamic adaptation to altering environmental conditions, assuming their new frontier in biomedicine.

Ответы эндотелиальных клеток на деформацию сдвига: механотрансдукция, клеточный стресс и адаптация

Эндотелиальные клетки, выстилающие стенки сосудов, являются одними из важнейших регуляторных элементов кровеносной системы. Непосредственно соприкасаясь с потоком крови, эти механочувствительные клетки способны детектировать свою деформацию через ее тангенциальный компонент (сдвиг) и составляющую, направленную по нормали к поверхности (растяжение). Деформация сдвига является ключевым индуктором комплекса сигнальных путей, опосредуемых тирозинкиназами, интегринами, ионными каналами, вовлекающих также мембранные липиды, гликокаликс и другие клеточные компоненты. На фоне достаточно большого количества данных о сигнальной трансдукции, в литературе меньше внимания уделено клеточной адаптации к сдвиговой деформации и сравнительно мало информации об участии генов стрессового ответа. Гидродинамические условия в определенных зонах сосудистой системы характеризуются значительной неоднородностью, что может приводить к ослаблению обратных связей, необходимых для поддержания гомеостаза в эндотелиальных клетках. Это может способствовать развитию заболеваний, например, таких, как атеросклероз. В обзоре обсуждаются новые аспекты и концепции, связанные с ответами эндотелиоцитов на сдвиговую деформацию и основные методы анализа эффектов сдвиговой деформации in vitro . Цель исследования. Обобщение современных данных о механизмах механочувствительности и механотрансдукции эндотелия. Результаты. В обзоре изложены основные механизмы механочувствительности клеток эндотелия, пути внутриклеточной передачи сигнала, рассмотрено вовлечение механизмов стрессового ответа клеток и адаптации. Обсуждаются эксперименты по изучению молекулярных основ механотрансдукции, в том числе белков и других молекул, вовлеченных в детектирование, передачу сигнала и клеточный ответ на сдвиговую деформацию.Endothelial cells lining the walls of blood vessels are one of the most important regulatory elements of the circulatory system. These mechanosensitive cells are in a direct contact with the flow of blood and able to detect deformation through its tangential component (shear) and the component directed along the normal to the surface (tension). Shear stress is the key inducer of the complex of signaling pathways mediated by tyrosine kinases, integrins, ion channels, involving also membrane lipids, glycocalyx and other cellular components. There are large amount of data on signal transduction in the literature, but less attention is paid to cellular adaptation to shear stress and there is relatively little information on the involvement of stress response genes in that process. Hydrodynamic conditions in certain zones of the vascular system are characterized by considerable heterogeneity, which can lead to weakening of feedbacks necessary for maintaining homeostasis in endothelial cells. This can contribute to the development of diseases such as atherosclerosis. This review presents new aspects and concepts related to the responses of endotheliocytes to shear stress and, in addition, highlights the basic methods of analyzing the effects of shear stress in vitro . Purpose of the study. Generalization of modern data on mechanisms of mechanosensitivity and mechanotransduction of the endothelium. Results. The review outlines the main mechanosensitivity mechanisms of endothelial cells, the pathways of intracellular signaling, the involvement of mechanisms of cellular stress response and adaptation. There are descriptions of experiments in which the molecular basis of mechanotransduction is identified, including the determination of proteins and other molecules involved in detection, signal transduction, and cellular response to shear stress.