Topoisomerase 1 facilitates nucleosome reassembly at stress genes during recovery.

Chromatin remodeling is essential to allow full development of alternative gene expression programs in response to environmental changes. In fission yeast, oxidative stress triggers massive transcriptional changes including the activation of hundreds of genes, with the participation of histone modifying complexes and chromatin remodelers. DNA transcription is associated to alterations in DNA topology, and DNA topoisomerases facilitate elongation along gene bodies. Here, we test whether the DNA topoisomerase Top1 participates in the RNA polymerase II-dependent activation of the cellular response to oxidative stress. Cells lacking Top1 are resistant to H2O2 stress. The transcriptome of Δtop1 strain was not greatly affected in the absence of stress, but activation of the anti-stress gene expression program was more sustained than in wild-type cells. Top1 associated to stress open reading frames. While the nucleosomes of stress genes are partially and transiently evicted during stress, the chromatin configuration remains open for longer times in cells lacking Top1, facilitating RNA polymerase II progression. We propose that, by removing DNA tension arising from transcription, Top1 facilitates nucleosome reassembly and works in synergy with the chromatin remodeler Hrp1 as opposing forces to transcription and to Snf22 / Hrp3 opening remodelers.

Comparing Mitochondrial Activity, Oxidative Stress Tolerance, and Longevity of Thirteen Ascomycota Yeast Species.

Aging is characterized by a number of hallmarks including loss of mitochondrial homeostasis and decay in stress tolerance, among others. Unicellular eukaryotes have been widely used to study chronological aging. As a general trait, calorie restriction and activation of mitochondrial respiration has been proposed to contribute to an elongated lifespan. Most aging-related studies have been conducted with the Crabtree-positive yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and with deletion collections deriving from these conventional yeast models. We have performed an unbiased characterization of longevity using thirteen fungi species, including S. cerevisiae and S. pombe, covering a wide range of the Ascomycota clade. We have determined their mitochondrial activity by oxygen consumption, complex IV activity, and mitochondrial redox potential, and the results derived from these three methodologies are highly overlapping. We have phenotypically compared the lifespans of the thirteen species and their capacity to tolerate oxidative stress. Longevity and elevated tolerance to hydrogen peroxide are correlated in some but not all yeasts. Mitochondrial activity per se cannot anticipate the length of the lifespan. We have classified the strains in four groups, with members of group 1 (Kluyveromyces lactis, Saccharomyces bayanus and Lodderomyces elongisporus) displaying high mitochondrial activity, elevated resistance to oxidative stress, and elongated lifespan.

Formation of Transient Protein Aggregate-like Centers Is a General Strategy Postponing Degradation of Misfolded Intermediates.

When misfolded intermediates accumulate during heat shock, the protein quality control system promotes cellular adaptation strategies. In Schizosaccharomyces pombe, thermo-sensitive proteins assemble upon stress into protein aggregate-like centers, PACs, to escape from degradation. The role of this protein deposition strategy has been elusive due to the use of different model systems and reporters, and to the addition of artificial inhibitors, which made interpretation of the results difficult. Here, we compare fission and budding yeast model systems, expressing the same misfolding reporters in experiments lacking proteasome or translation inhibitors. We demonstrate that mild heat shock triggers reversible PAC formation, with the collapse of both reporters and chaperones in a process largely mediated by chaperones. This assembly postpones proteasomal degradation of the misfolding reporters, and their Hsp104-dependent disassembly occurs during stress recovery. Severe heat shock induces formation of cytosolic PACs, but also of nuclear structures resembling nucleolar rings, NuRs, presumably to halt nuclear functions. Our study demonstrates that these distantly related yeasts use very similar strategies to adapt and survive to mild and severe heat shock and that aggregate-like formation is a general cellular scheme to postpone protein degradation and facilitate exit from stress.

Antagonistic effects of mitochondrial matrix and intermembrane space proteases on yeast aging.

BACKGROUND: In many organisms, aging is characterized by a loss of mitochondrial homeostasis. Multiple factors such as respiratory metabolism, mitochondrial fusion/fission, or mitophagy have been linked to cell longevity, but the exact impact of each one on the aging process is still unclear. RESULTS: Using the deletion mutant collection of the fission yeast Schizosaccharomyces pombe, we have developed a genome-wide screening for mutants with altered chronological lifespan. We have identified four mutants associated with proteolysis at the mitochondria that exhibit opposite effects on longevity. The analysis of the respiratory activity of these mutants revealed a positive correlation between increased respiration rate and prolonged lifespan. We also found that the phenotype of the long-lived protease mutants could not be explained by impaired mitochondrial fusion/fission activities, but it was dependent on mitophagy induction. The anti-aging role of mitophagy was supported by the effect of a mutant defective in degradation of mitochondria, which shortened lifespan of the long-lived mutants. CONCLUSIONS: Our characterization of the mitochondrial protease mutants demonstrates that mitophagy sustains the lifespan extension of long-lived mutants displaying a higher respiration potential.

Corrigendum: CXCL17 Is a Specific Diagnostic Biomarker for Severe Pandemic Influenza A(H1N1) That Predicts Poor Clinical Outcome.

[This corrects the article DOI: 10.3389/fimmu.2021.633297.].

Clinical and Immunological Factors That Distinguish COVID-19 From Pandemic Influenza A(H1N1).

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a global health threat with the potential to cause severe disease manifestations in the lungs. Although COVID-19 has been extensively characterized clinically, the factors distinguishing SARS-CoV-2 from other respiratory viruses are unknown. Here, we compared the clinical, histopathological, and immunological characteristics of patients with COVID-19 and pandemic influenza A(H1N1). We observed a higher frequency of respiratory symptoms, increased tissue injury markers, and a histological pattern of alveolar pneumonia in pandemic influenza A(H1N1) patients. Conversely, dry cough, gastrointestinal symptoms and interstitial lung pathology were observed in COVID-19 cases. Pandemic influenza A(H1N1) was characterized by higher levels of IL-1RA, TNF-α, CCL3, G-CSF, APRIL, sTNF-R1, sTNF-R2, sCD30, and sCD163. Meanwhile, COVID-19 displayed an immune profile distinguished by increased Th1 (IL-12, IFN-γ) and Th2 (IL-4, IL-5, IL-10, IL-13) cytokine levels, along with IL-1ß, IL-6, CCL11, VEGF, TWEAK, TSLP, MMP-1, and MMP-3. Our data suggest that SARS-CoV-2 induces a dysbalanced polyfunctional inflammatory response that is different from the immune response against pandemic influenza A(H1N1). Furthermore, we demonstrated the diagnostic potential of some clinical and immune factors to differentiate both diseases. These findings might be relevant for the ongoing and future influenza seasons in the Northern Hemisphere, which are historically unique due to their convergence with the COVID-19 pandemic.

CXCL17 Is a Specific Diagnostic Biomarker for Severe Pandemic Influenza A(H1N1) That Predicts Poor Clinical Outcome.

The C-X-C motif chemokine ligand 17 (CXCL17) is chemotactic for myeloid cells, exhibits bactericidal activity, and exerts anti-viral functions. This chemokine is constitutively expressed in the respiratory tract, suggesting a role in lung defenses. However, little is known about the participation of CXCL17 against relevant respiratory pathogens in humans. Here, we evaluated the serum levels and lung tissue expression pattern of CXCL17 in a cohort of patients with severe pandemic influenza A(H1N1) from Mexico City. Peripheral blood samples obtained on admission and seven days after hospitalization were processed for determinations of serum CXCL17 levels by enzyme-linked immunosorbent assay (ELISA). The expression of CXCL17 was assessed by immunohistochemistry (IHQ) in lung autopsy specimens from patients that succumbed to the disease. Serum CXCL17 levels were also analyzed in two additional comparative cohorts of coronavirus disease 2019 (COVID-19) and pulmonary tuberculosis (TB) patients. Additionally, the expression of CXCL17 was tested in lung autopsy specimens from COVID-19 patients. A total of 122 patients were enrolled in the study, from which 68 had pandemic influenza A(H1N1), 24 had COVID-19, and 30 with PTB. CXCL17 was detected in post-mortem lung specimens from patients that died of pandemic influenza A(H1N1) and COVID-19. Interestingly, serum levels of CXCL17 were increased only in patients with pandemic influenza A(H1N1), but not COVID-19 and PTB. CXCL17 not only differentiated pandemic influenza A(H1N1) from other respiratory infections but showed prognostic value for influenza-associated mortality and renal failure in machine-learning algorithms and regression analyses. Using cell culture assays, we also identified that human alveolar A549 cells and peripheral blood monocyte-derived macrophages increase their CXCL17 production capacity after influenza A(H1N1) pdm09 virus infection. Our results for the first time demonstrate an induction of CXCL17 specifically during pandemic influenza A(H1N1), but not COVID-19 and PTB in humans. These findings could be of great utility to differentiate influenza and COVID-19 and to predict poor prognosis specially at settings of high incidence of pandemic A(H1N1). Future studies on the role of CXCL17 not only in severe pandemic influenza, but also in seasonal influenza, COVID-19, and PTB are required to validate our results.

Expression of Surfactant Protein D Distinguishes Severe Pandemic Influenza A(H1N1) from Coronavirus Disease 2019.

The differentiation between influenza and coronavirus disease 2019 (COVID-19) could constitute a diagnostic challenge during the ongoing winter owing to their clinical similitude. Thus, novel biomarkers are required to enable making this distinction. Here, we evaluated whether the surfactant protein D (SP-D), a collectin produced at the alveolar epithelium with known immune properties, was useful to differentiate pandemic influenza A(H1N1) from COVID-19 in critically ill patients. Our results revealed high serum SP-D levels in patients with severe pandemic influenza but not those with COVID-19. This finding was validated in a separate cohort of mechanically ventilated patients with COVID-19 who also showed low plasma SP-D levels. However, plasma SP-D levels did not distinguish seasonal influenza from COVID-19 in mild-to-moderate disease. Finally, we found that high serum SP-D levels were associated with death and renal failure among severe pandemic influenza cases. Thus, our studies have identified SP-D as a unique biomarker expressed during severe pandemic influenza but not COVID-19.

The Hsp40 Mas5 Connects Protein Quality Control and the General Stress Response through the Thermo-sensitive Pyp1.

Upon heat shock, the fission yeast Hsp40 chaperone Mas5 drives temperature-sensitive proteins toward protein aggregate centers (PACs) to avoid their degradation until lower temperatures favor their refolding. We show here that cells lacking Mas5 are resistant to oxidative stress. Components of the general stress pathways, the MAP kinase Sty1 and the transcription factor Atf1, are suppressors of this phenotype. Strain Δmas5 expresses higher levels of Sty1- and Atf1-dependent stress genes than wild-type cells. Pyp1, the main tyrosine phosphatase maintaining Sty1 inactive in the absence of stress, is a temperature-sensitive protein that aggregates upon temperature up-shifts in a Mas5-dependent manner. In strain Δmas5, Pyp1 is sent to proteasomal degradation even in the absence of stress. We propose that Pyp1 is a thermo-sensitive phosphatase, which during heat stress coalescences into PACs in a Mas5-dependent manner, to promote full activation of the anti-stress Sty1-Atf1 cascade.

Activation of NRF2 by topical apocarotenoid treatment mitigates radiation-induced dermatitis.

Radiation therapy is a frontline treatment option for cancer patients; however, the effects of radiotherapy on non-tumor tissue (e.g. radiation-induced dermatitis) often worsen patient quality of life. Previous studies have implicated the importance of redox balance in preventing dermatitis, specifically in reference to modulation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway. Due to the cytoprotective functions of transcriptional target genes of NRF2, we investigated how modulation of NRF2 expression could affect DNA damage, oxidative stress, and cell viability in response to radiotherapy. Specifically, it was noted that NRF2 knockdown sensitized human skin keratinocytes to ionizing radiation; likewise, genetic ablation of NRF2 in vivo increased radiosensitivity of murine epidermis. Oppositely, pharmacological induction of NRF2 via the apocarotenoid bixin lowered markers of DNA damage and oxidative stress, while preserving viability in irradiated keratinocytes. Mechanistic studies indicated that topical pretreatment using bixin as an NRF2 activator antagonized initial DNA damage by raising cellular glutathione levels. Additionally, topical application of bixin prevented radiation-induced dermatitis, epidermal thickening, and oxidative stress in the skin of SKH1 mice. Overall, these data indicate that NRF2 is critical for mitigating the harmful skin toxicities associated with ionizing radiation, and that topical upregulation of NRF2 via bixin could prevent radiation-induced dermatitis.

Chaperone-Facilitated Aggregation of Thermo-Sensitive Proteins Shields Them from Degradation during Heat Stress.

Cells have developed protein quality-control strategies to manage the accumulation of misfolded substrates during heat stress. Using a soluble reporter of misfolding in fission yeast, Rho1.C17R-GFP, we demonstrate that upon mild heat shock, the reporter collapses in protein aggregate centers (PACs). They contain and/or require several chaperones, such as Hsp104, Hsp16, and the Hsp40/70 couple Mas5/Ssa2. Stress granules do not assemble at mild temperatures and, therefore, are not required for PAC formation; on the contrary, PACs may serve as nucleation centers for the assembly of stress granules. In contrast to the general belief, the dominant fate of these PACs is not degradation, and the aggregated reporter can be disassembled by chaperones and recovers native structure and activity. Using mass spectrometry, we show that thermo-unstable endogenous proteins form PACs as well. In conclusion, formation of PACs during heat shock is a chaperone-mediated adaptation strategy.

Spermidine Confers Liver Protection by Enhancing NRF2 Signaling Through a MAP1S-Mediated Noncanonical Mechanism.

Spermidine (SPD), a naturally occurring polyamine, has been recognized as a caloric restriction mimetic that confers health benefits, presumably by inducing autophagy. Recent studies have reported that oral administration of SPD protects against liver fibrosis and hepatocarcinogenesis through activation of microtubule associated protein 1S (MAP1S)-mediated autophagy. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor that mediates cellular protection by maintaining the cell's redox, metabolic, and proteostatic balance. In this study, we demonstrate that SPD is a noncanonical NRF2 inducer, and that MAP1S is a component of this noncanonical pathway of NRF2 activation. Mechanistically, MAP1S induces NRF2 signaling through two parallel mechanisms, both resulting in NRF2 stabilization: (1) MAP1S competes with Kelch-like ECH-associated protein 1 (KEAP1) for NRF2 binding through an ETGE motif, and (2) MAP1S accelerates p62-dependent degradation of KEAP1 by the autophagy pathway. We further demonstrate that SPD confers liver protection by enhancing NRF2 signaling. The importance of both NRF2 and p62-dependent autophagy in SPD-mediated liver protection was confirmed using a carbon tetrachloride-induced liver fibrosis model in wild-type, Nrf2-/- , p62-/- and Nrf2-/- ;p62-/- mice, as the protective effect of SPD was significantly reduced in NRF2 or p62 single knockout mice, and completely abolished in the double knockout mice. Conclusion: Our results demonstrate the pivotal role of NRF2 in mediating the health benefit of SPD, particularly in the context of liver pathologies.

Modulating NRF2 in Disease: Timing Is Everything.

The transcription factor nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2) is a central regulator of redox, metabolic, and protein homeostasis that intersects with many other signaling cascades. Although the understanding of the complex nature of NRF2 signaling continues to grow, there is only one therapeutic targeting NRF2 for clinical use, dimethyl fumarate, used for the treatment of multiple sclerosis. The discovery of new therapies is confounded by the fact that NRF2 levels vary significantly depending on physiological and pathological context. Thus, properly timed and targeted manipulation of the NRF2 pathway is critical in creating effective therapeutic regimens. In this review, we summarize the regulation and downstream targets of NRF2. Furthermore, we discuss the role of NRF2 in cancer, neurodegeneration, and diabetes as well as cardiovascular, kidney, and liver disease, with a special emphasis on NRF2-based therapeutics, including those that have made it into clinical trials.

RPA1 binding to NRF2 switches ARE-dependent transcriptional activation to ARE-NRE-dependent repression.

NRF2 regulates cellular redox homeostasis, metabolic balance, and proteostasis by forming a dimer with small musculoaponeurotic fibrosarcoma proteins (sMAFs) and binding to antioxidant response elements (AREs) to activate target gene transcription. In contrast, NRF2-ARE-dependent transcriptional repression is unreported. Here, we describe NRF2-mediated gene repression via a specific seven-nucleotide sequence flanking the ARE, which we term the NRF2-replication protein A1 (RPA1) element (NRE). Mechanistically, RPA1 competes with sMAF for NRF2 binding, followed by interaction of NRF2-RPA1 with the ARE-NRE and eduction of promoter activity. Genome-wide in silico and RNA-seq analyses revealed this NRF2-RPA1-ARE-NRE complex mediates negative regulation of many genes with diverse functions, indicating that this mechanism is a fundamental cellular process. Notably, repression of MYLK, which encodes the nonmuscle myosin light chain kinase, by the NRF2-RPA1-ARE-NRE complex disrupts vascular integrity in preclinical inflammatory lung injury models, illustrating the translational significance of NRF2-mediated transcriptional repression. Our findings reveal a gene-suppressive function of NRF2 and a subset of negatively regulated NRF2 target genes, underscoring the broad impact of NRF2 in physiological and pathological settings.

NRF2 and the Hallmarks of Cancer.

The transcription factor NRF2 is the master regulator of the cellular antioxidant response. Though recognized originally as a target of chemopreventive compounds that help prevent cancer and other maladies, accumulating evidence has established the NRF2 pathway as a driver of cancer progression, metastasis, and resistance to therapy. Recent studies have identified new functions for NRF2 in the regulation of metabolism and other essential cellular functions, establishing NRF2 as a truly pleiotropic transcription factor. In this review, we explore the roles of NRF2 in the hallmarks of cancer, indicating both tumor suppressive and tumor-promoting effects.

Topical Bixin Confers NRF2-Dependent Protection Against Photodamage and Hair Graying in Mouse Skin.

Environmental exposure to solar ultraviolet (UV) radiation causes acute photodamage, premature aging, and skin cancer, attributable to UV-induced genotoxic, oxidative, and inflammatory stress. The transcription factor NRF2 [nuclear factor erythroid 2 (E2)-related factor 2] is the master regulator of the cellular antioxidant response protecting skin against various environmental stressors including UV radiation and electrophilic pollutants. NRF2 in epidermal keratinocytes can be activated using natural chemopreventive compounds such as the apocarotenoid bixin, an FDA-approved food additive and cosmetic ingredient from the seeds of the achiote tree (Bixa orellana). Here, we tested the feasibility of topical use of bixin for NRF2-dependent skin photoprotection in two genetically modified mouse models [SKH1 and C57BL/6J (Nrf2+/+ versus Nrf2-/- )]. First, we observed that a bixin formulation optimized for topical NRF2 activation suppresses acute UV-induced photodamage in Nrf2+/+ but not Nrf2-/- SKH1 mice, a photoprotective effect indicated by reduced epidermal hyperproliferation and oxidative DNA damage. Secondly, it was demonstrated that topical bixin suppresses PUVA (psoralen + UVA)-induced hair graying in Nrf2+/+ but not Nrf2-/- C57BL/6J mice. Collectively, this research provides the first in vivo evidence that topical application of bixin can protect against UV-induced photodamage and PUVA-induced loss of hair pigmentation through NRF2 activation. Topical NRF2 activation using bixin may represent a novel strategy for human skin photoprotection, potentially complementing conventional sunscreen-based approaches.

Increased O-GlcNAcylation of SNAP29 Drives Arsenic-Induced Autophagic Dysfunction.

Environmental exposure to arsenic is linked to adverse health effects, including cancer and diabetes. Pleiotropic cellular effects are observed with arsenic exposure. Previously, we demonstrated that arsenic dysregulated the autophagy pathway at low, environmentally relevant concentrations. Here we show that arsenic blocks autophagy by preventing autophagosome-lysosome fusion. Specifically, arsenic disrupts formation of the STX17-SNAP29-VAMP8 SNARE complex, where SNAP29 mediates vesicle fusion through bridging STX17-containing autophagosomes to VAMP8-bearing lysosomes. Mechanistically, arsenic inhibits SNARE complex formation, at least in part, by enhancing O-GlcNAcylation of SNAP29. Transfection of O-GlcNAcylation-defective, but not wild-type, SNAP29 into clustered regularly interspaced short palindromic repeat (CRISPR)-mediated SNAP29 knockout cells abolishes arsenic-mediated autophagy inhibition. These findings reveal a mechanism by which low levels of arsenic perturb proteostasis through inhibition of SNARE complex formation, providing a possible therapeutic target for disease intervention in the more than 200 million people exposed to unsafe levels of arsenic.

Low-level arsenic causes proteotoxic stress and not oxidative stress.

Prolonged exposure to arsenic has been shown to increase the risk of developing a number of diseases, including cancer and type II diabetes. Arsenic is present throughout the environment in its inorganic forms, and the level of exposure varies greatly by geographical location. The current recommended maximum level of arsenic exposure by the EPA is 10µg/L, but levels>50-1000µg/L have been detected in some parts of Asia, the Middle East, and the Southwestern United States. One of the most important steps in developing treatment options for arsenic-linked pathologies is to understand the cellular pathways affected by low levels of arsenic. Here, we show that acute exposure to non-lethal, low-level arsenite, an environmentally relevant arsenical, inhibits the autophagy pathway. Furthermore, arsenite-induced autophagy inhibition initiates a transient, but moderate ER stress response. Significantly, low-level arsenite exposure does not exhibit an increase in oxidative stress. These findings indicate that compromised autophagy, and not enhanced oxidative stress occurs early during arsenite exposure, and that restoring the autophagy pathway and proper proteostasis could be a viable option for treating arsenic-linked diseases. As such, our study challenges the existing paradigm that oxidative stress is the main underlying cause of pathologies associated with environmental arsenic exposure.