Cytoprotective Proteins Research Articles

Senescence marker protein 30 confers neuroprotection in oxygen-glucose deprivation/reoxygenation-injured neurons through modulation of Keap1/Nrf2 signaling: Role of SMP30 in OGD/R-induced neuronal injury.

Senescence marker protein 30 (SMP30) is a senescence marker molecule and identified as a calcium regulatory protein. Currently, SMP30 has emerged as a cytoprotective protein in a wide range of cell types. However, the role of SMP30 in regulating neuronal survival during cerebral ischemia/reperfusion injury remains unclear. In the present study, we aimed to investigate the biological function and regulatory mechanism of SMP30 on neuronal survival using a cellular model induced by oxygen-glucose deprivation/reoxygenation (OGD/R). The results showed that SMP30 expression was significantly decreased by OGD/R exposure in neurons. Functional experiments demonstrated that SMP30 overexpression significantly rescued the decreased cell viability and attenuated the apoptosis and reactive oxygen species generation in OGD/R-exposed neurons. By contrast, SMP30 knockdown exhibited the opposite effect. Mechanism research revealed that SMP30 overexpression contributed to the activation of nuclear factor erythroid 2-related factor (Nrf2)/antioxidant response element (ARE) signaling associated with downregulation of Kelch-like ECH-associated protein (Keap1). Keap1 overexpression or Nrf2 silencing significantly reversed SMP30-mediated neuroprotection against OGD/R-induced injury. Overall, these findings demonstrate that SMP30 overexpression protects neurons from OGD/R-induced apoptosis and oxidative stress by enhancing Nrf2/ARE antioxidant signaling via inhibition of Keap1. These data highlight the importance of the SMP30/Keap1/Nrf2/ARE signaling axis in regulating neuronal survival during cerebral ischemia/reperfusion injury.

Conformational States of the Cytoprotective Protein Bcl-xL

Bcl-xL is a major inhibitor of apoptosis, a fundamental homeostatic process of programmed cell death that is highly conserved across evolution. Because it plays prominent roles in cancer, Bcl-xL is a major target for anticancer therapy and for studies aimed at understanding its structure and activity. Although Bcl-xL is active primarily at intracellular membranes, most studies have focused on soluble forms of the protein lacking both the membrane-anchoring C-terminal tail and the intrinsically disordered loop, and this has resulted in a fragmented view of the protein’s biological activity. Here, we describe the conformation of full-length Bcl-xL. Using NMR spectroscopy, molecular dynamics simulations, and isothermal titration calorimetry, we show how the three structural elements affect the protein’s structure, dynamics, and ligand-binding activity in both its soluble and membrane-anchored states. The combined data provide information about the molecular basis for the protein’s functionality and a view of its complex molecular mechanisms.

Abstract 412: DJ-1 Deficiency Impairs Post-Ischemic Cardiac Fatty Acid Oxidation

Background: DJ-1 is a cytoprotective protein implicated in many cellular processes. We have recently shown that DJ-1 plays a protective role in the setting of acute myocardial ischemia-reperfusion injury and heart failure. However, specific mechanisms of action remain unknown. Here, we sought to determine if DJ-1 maintains fatty acid oxidation following myocardial I/R injury via the regulation of PPARalpha transcriptional activity. Methods and Results: WT and DJ-1 KO hearts were subjected to 45 minutes of ischemia via coronary artery ligation followed by up to 72 hours of reperfusion. Initial studies revealed that PPARalpha activity (PPARalpha Transcription Factor Assay Kit; abcam) was lower in DJ-1 KO hearts under both basal (sham) and I/R conditions. Next, we performed an analysis of PPAR genes using a qPCR array (Mouse PPAR Targets RT2 Profiler PCR Array from Qiagen) that profiled the expression of 84 key genes involved in PPAR activation and response. Marked differences in genes involved in FA metabolism were evident in the hearts of DJ-1 KO mice. Further studies using qPCR validated a significant decrease in the expression of acsl1 (protein ACSL1), acsl3, acsl5, cpt1b (protein CPT1), slc25a20 (protein CACT), and cpt2. The protein expression of ACSL1, CPT1, and CACT were also decreased in the hearts of DJ-1 KO mice following I/R injury. These changes were accompanied by higher cardiac lipid content and depressed mitochondrial fatty-acid β-oxidation and ATP synthesis. Conclusion: These data demonstrate that DJ-1 plays an essential role in regulating post-I/R cardiac FAO. Future studies will garner mechanistic insight for the impaired function of PPARalpha with the loss of DJ-1.

Flavonoids Activation of the Transcription Factor Nrf2 as a Hypothesis Approach for the Prevention and Modulation of SARS-CoV-2 Infection Severity.

The Nrf2-Keap1-ARE pathway is the principal regulator of antioxidant and phase II detoxification genes. Its activation increases the expression of antioxidant and cytoprotective proteins, protecting cells against infections. Nrf2 modulates virus-induced oxidative stress, ROS generation, and disease pathogenesis, which are vital in the viral life cycle. During respiratory viral infections, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an inflammatory process, and oxidative stress of the epithelium lining cells activate the transcription factor Nrf2, which protects cells from oxidative stress and inflammation. Nrf2 reduces angiotensin-converting enzyme 2 (ACE2) receptors expression in respiratory epithelial cells. SARS-CoV2 has a high affinity for ACE2 that works as receptors for coronavirus surface spike glycoprotein, facilitating viral entry. Disease severity may also be modulated by pre-existing conditions, such as impaired immune response, obesity, and age, where decreased level of Nrf2 is a common feature. Consequently, Nrf2 activators may increase Nrf2 levels and enhance antiviral mediators’ expression, which could initiate an “antiviral state”, priming cells against viral infection. Therefore, this hypothesis paper describes the use of flavonoid supplements combined with vitamin D3 to activate Nrf2, which may be a potential target to prevent and/or decrease SARS-CoV-2 infection severity, reducing oxidative stress and inflammation, enhancing innate immunity, and downregulating ACE2 receptors.

Podocyte Integrin-β3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy.

Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. The zymogen protein C and aPC bind to podocyte integrin-β3, a subunit of integrin-αvβ3. Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-αvβ3 induces transient binding of integrin-β3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-β3via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-β3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin-β3 interaction by specifically deleting podocyte integrin-β3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.Conclusions aPC-integrin-αvβ3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-αvβ3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1

Mitochondrial quality control is mediated by the PTEN-induced kinase 1 (PINK1), a cytoprotective protein that is dysregulated in inflammatory lung injury and neurodegenerative diseases. Here, we show that a ubiquitin E3 ligase receptor component, FBXO7, targets PINK1 for its cellular disposal. FBXO7, by mediating PINK1 ubiquitylation and degradation, was sufficient to induce mitochondrial injury and inflammation in experimental pneumonia. A computational simulation-based screen led to the identification of a small molecule, BC1464, which abrogated FBXO7 and PINK1 association, leading to increased cellular PINK1 concentrations and activities, and limiting mitochondrial damage. BC1464 exerted antiinflammatory activity in human tissue explants and murine lung inflammation models. Furthermore, BC1464 conferred neuroprotection in primary cortical neurons, human neuroblastoma cells, and patient-derived cells in several culture models of Parkinson's disease. The data highlight a unique opportunity to use small molecule antagonists that disrupt PINK1 interaction with the ubiquitin apparatus to enhance mitochondrial quality, limit inflammatory injury, and maintain neuronal viability.

KEAP1, a cysteine-based sensor and a drug target for the prevention and treatment of chronic disease.

Redox imbalance and persistent inflammation are the underlying causes of most chronic diseases. Mammalian cells have evolved elaborate mechanisms for restoring redox homeostasis and resolving acute inflammatory responses. One prominent mechanism is that of inducing the expression of antioxidant, anti-inflammatory and other cytoprotective proteins, while also suppressing the production of pro-inflammatory mediators, through the activation of transcription factor nuclear factor-erythroid 2 p45-related factor 2 (NRF2). At homeostatic conditions, NRF2 is a short-lived protein, which avidly binds to Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 functions as (i) a substrate adaptor for a Cullin 3 (CUL3)-based E3 ubiquitin ligase that targets NRF2 for ubiquitination and proteasomal degradation, and (ii) a cysteine-based sensor for a myriad of physiological and pharmacological NRF2 activators. Here, we review the intricate molecular mechanisms by which KEAP1 senses electrophiles and oxidants. Chemical modification of specific cysteine sensors of KEAP1 results in loss of NRF2-repressor function and alterations in the expression of NRF2-target genes that encode large networks of diverse proteins, which collectively restore redox balance and resolve inflammation, thus ensuring a comprehensive cytoprotection. We focus on the cyclic cyanoenones, the most potent NRF2 activators, some of which are currently in clinical trials for various pathologies characterized by redox imbalance and inflammation.

Enterovirus 71 induces neural cell apoptosis and autophagy through promoting ACOX1 downregulation and ROS generation

ABSTRACT Enterovirus 71 (EV71) infection causes hand, foot, and mouth disease (HFMD), and even fatal neurological complications. However, the mechanisms underlying EV71 neurological pathogeneses are largely unknown. This study reveals a distinct mechanism by which EV71 induces apoptosis and autophagy in neural cells. EV71 non-structure protein 3D (also known as RNA-dependent RNA polymerase, RdRp) interacts with the peroxisomal protein acyl-CoA oxidase 1 (ACOX1), and contributes to ACOX1 downregulation. Further studies demonstrate that EV71 reduces peroxisome numbers. Additionally, knockdown of ACOX1 or peroxin 19 (PEX19) induces apoptosis and autophagy in neural cells including human neuroblastoma (SK-N-SH) cells and human astrocytoma (U251) cells, and EV71 infection induces neural cell death through attenuating ACOX1 production. Moreover, EV71 infection and ACOX1 knockdown facilitate reactive oxygen species (ROS) production and attenuate the cytoprotective protein deglycase (DJ-1)/Nuclear factor erythroid 2-related factor 2 (NRF2)/Heme oxygenase 1 (HO-1) pathway (DJ-1/NRF2/HO-1), which collectively result in ROS accumulation in neural cells. In conclusion, EV71 downregulates ACOX1 protein expression, reduces peroxisome numbers, enhances ROS generation, and attenuates the DJ-1/NRF2/HO-1 pathway, thereby inducing apoptosis and autophagy in neural cells. These findings provide new insights into the mechanism underlying EV71-induced neural pathogenesis, and suggest potential treatments for EV71-associated diseases.

G3BP1-linked mRNA partitioning supports selective protein synthesis in response to oxidative stress.

Cells limit energy-consuming mRNA translation during stress to maintain metabolic homeostasis. Sequestration of mRNAs by RNA binding proteins (RBPs) into RNA granules reduces their translation, but it remains unclear whether RBPs also function in partitioning of specific transcripts to polysomes (PSs) to guide selective translation and stress adaptation in cancer. To study transcript partitioning under cell stress, we catalogued mRNAs enriched in prostate carcinoma PC-3 cell PSs, as defined by polysome fractionation and RNA sequencing (RNAseq), and compared them to mRNAs complexed with the known SG-nucleator protein, G3BP1, as defined by spatially-restricted enzymatic tagging and RNAseq. By comparing these compartments before and after short-term arsenite-induced oxidative stress, we identified three major categories of transcripts, namely those that were G3BP1-associated and PS-depleted, G3BP1-dissociated and PS-enriched, and G3BP1-associated but also PS-enriched. Oxidative stress profoundly altered the partitioning of transcripts between these compartments. Under arsenite stress, G3BP1-associated and PS-depleted transcripts correlated with reduced expression of encoded mitochondrial proteins, PS-enriched transcripts that disassociated from G3BP1 encoded cell cycle and cytoprotective proteins whose expression increased, while transcripts that were both G3BP1-associated and PS-enriched encoded proteins involved in diverse stress response pathways. Therefore, G3BP1 guides transcript partitioning to reprogram mRNA translation and support stress adaptation.

Discovery of Eicosapentaenoic Acid Esters of Hydroxy Fatty Acids as Potent Nrf2 Activators.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids with anti-inflammatory and anti-diabetic properties. Despite the possible link between endogenous FAHFA levels and nuclear factor erythroid 2-related factor 2 (Nrf2), their possible function as antioxidants and the mechanisms involved in this are unknown. Here, we investigate FAHFAs’ plausible antioxidant potential with reference to their effect on the Nrf2 levels, oxidative stress, and lipid droplet oxidation in human hepatocytes (C3A). Six authentic FAHFAs were chemically synthesized and performed activity-based screening by reporter gene assay. Among them, eicosapentaenoic acid (EPA) esterified 12-hydroxy stearic acid (12-HSA) and 12-hydroxy oleic acid (12-HOA) FAHFAs showed less cytotoxicity compared to their free fatty acids and potent activators of Nrf2. To define their mode of action, relative levels of nuclear Nrf2 were determined, which found a higher amount of Nrf2 in nucleus of cells treated with 12-EPAHSA compared to the control. Furthermore, 12-EPAHSA increased the expression of Nrf2-dependent antioxidant enzyme genes (NQO1, GCLM, GCLC, SOD-1, and HO-1). Fluorescence imaging analysis of linoleic-acid-induced lipid droplets (LDs) in C3A cells treated with 12-EPAHSA revealed the strong inhibition of small-size LD oxidation. These results suggest that EPA-derived FAHFAs as a new class of lipids with less cytotoxicity, and strong Nrf2 activators with plausible antioxidant effects via the induction of cytoprotective proteins against oxidative stress, induced cellular damage.

Anthocyanins As Modulators of Cell Redox-Dependent Pathways in Non-Communicable Diseases.

Chronic Noncommunicable Diseases (NCDs), mostly represented by cardiovascular diseases, diabetes, chronic pulmonary diseases, cancers, and several chronic pathologies, are one of the main causes of morbidity and mortality, and are mainly related to the occurrence of metabolic risk factors. Anthocyanins (ACNs) possess a wide spectrum of biological activities, such as anti-inflammatory, antioxidant, cardioprotective and chemopreventive properties, which are able to promote human health. Although ACNs present an apparent low bioavailability, their metabolites may play an important role in the in vivo protective effects observed. This article directly addresses the scientific evidences supporting that ACNs could be useful to protect human population against several NCDs not only acting as antioxidant but through their capability to modulate cell redox-dependent signaling. In particular, ACNs interact with the NF-κB and AP-1 signal transduction pathways, which respond to oxidative signals and mediate a proinflammatory effect, and the Nrf2/ARE pathway and its regulated cytoprotective proteins (GST, NQO, HO-1, etc.), involved in both cellular antioxidant defenses and elimination/inactivation of toxic compounds, so countering the alterations caused by conditions of chemical/oxidative stress. In addition, supposed crosstalks could contribute to explain the protective effects of ACNs in different pathological conditions characterized by an altered balance among these pathways. Thus, this review underlines the importance of specific nutritional molecules for human health and focuses on the molecular targets and the underlying mechanisms of ACNs against various diseases.

Nrf2 activation through the inhibition of Keap1-Nrf2 protein-protein interaction.

Activation of the transcription factor Nrf2 via the Keap1-Nrf2-ARE signaling system regulates the transcription and subsequent expression of cellular cytoprotective proteins and plays a crucial role in preventing pathological conditions exacerbated by the overproduction of oxidative stress. In addition to electrophilic modulators, direct non-covalent inhibitors that interrupt the Keap1-Nrf2 protein-protein interaction (PPI) leading to Nrf2 activation have attracted a great deal of attention as potential preventive and therapeutic agents for oxidative stress-related diseases. Structural studies of Keap1-binding ligands, development of biochemical and cellular assays, and new structure-based design approaches have facilitated the discovery of small molecule PPI inhibitors. This perspective reviews the Keap1-Nrf2-ARE system, its physiological functions, and the recent progress in the discovery and the potential applications of direct inhibitors of Keap1-Nrf2 PPI.

Protective effects and mechanisms of bilirubin nanomedicine against acute pancreatitis

Acute pancreatitis (AP) is a sudden inflammatory reaction, caused by the activation of pancreatic enzymes in the pancreas, and in severe cases can lead to systemic inflammation and multiple organ failure. Oxidative stress contributed to the further deterioration of inflammation and played an important role in AP development. Bilirubin has been found to exert antioxidative, anti-inflammatory, and anti-apoptotic effects in a series of diseases accompanied by a high level of oxidative stress. However, the therapeutic effects of bilirubin for AP management have not yet been demonstrated. Additionally, the poor solubility and potential toxicity of bilirubin also limit its application. Thus, we developed bilirubin encapsulated silk fibrin nanoparticles (BRSNPs) to study the protective effects and mechanisms of bilirubin nanomedicine for the treatment of AP. BRSNPs could selectively delivery to the inflammatory lesion of the pancreas and release bilirubin in an enzyme-responsive manner. In the model of AP caused by L-Arginine hyperstimulation, BRSNPs exerted strong therapeutic effects against AP by the reduction of oxidative stress, decreased expression of pro-inflammatory cytokines, and impaired recruitment of macrophages and neutrophils. The mechanism study indicated that BRSNPs protected acinar cells against extensive oxidative damage and inflammation through inhibiting NF-κB pathway and activating the Nrf2/HO-1 pathway. Collectively, for the first time, this study demonstrated that bilirubin nanomedicine, BRSNPs, are effective in alleviating experimental acute pancreatitis, and the mechanisms are associated with its inhibition of NF-κB regulated pro-inflammatory signaling and activation of Nrf2-regulated cytoprotective protein expression.

<p>Miconazole Contributes to NRF2 Activation by Noncanonical P62-KEAP1 Pathway in Bladder Cancer Cells</p>

PurposeNuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2, a transcription factor capable of upregulating antioxidant response element (ARE)-mediated expression and cytoprotective proteins, plays critical roles in chemoprevention, inflammation and aging. NRF2 has recently been proposed as a novel target for cancer chemoprevention. The fungicide miconazole has shown promising antiproliferative effects in cancer cells.Materials and MethodsAfter miconazole treatment, the p62-KEAP1-NRF2 activation was analyzed by qPCR and Western blot. The nuclear translocation indicating NRF2 activation was further confirmed by immunofluorescence. Finally, the ROS production was detected by CM-H2DCFDA staining.ResultsWe demonstrate in this study that miconazole dramatically increases NRF2 activation in bladder cancer cells, in a dose- and time-dependent manner. Interestingly, levels of expression of p62, a noncanonical pathway that mediates NRF2 activation, appeared to increase in accordance with NRF2. We also investigated levels of the negative regulator kelch-like ECH-associated protein 1 (KEAP1), which is involved in NRF2 activation. As expected, a decrease in KEAP1 expression was found after miconazole exposure. Confirmation of NRF2 nuclear translocation was monitored by immunofluorescence. Miconazole-induced generation of reactive oxygen species (ROS) promoted NRF2 activation. Pretreatment of bladder cancer cells with ROS scavengers abolished NRF2 expression and nuclear translocation, indicating that miconazole activates the noncanonical p62-KEAP1-NRF2 pathway, which is regulated by ROS production.ConclusionOur study elucidates the mechanisms through which miconazole stimulates NRF2 which may contribute to cancer chemopreventive effects.

Upregulation of heme oxygenase-1 by Brahma-related gene 1 through Nrf2 signaling confers protective effect against high glucose-induced oxidative damage of retinal ganglion cells

High glucose (HG)-induced oxidative damage of retinal ganglion cells (RGCs) contributes to the pathogenesis of diabetic retinopathy, a severe complication of diabetes mellitus. Brahma-related gene 1 (Brg1) has currently emerged as a cytoprotective protein that alleviates oxidative damage induced by various stress. However, whether Brg1 is involved in the regulation of HG-induced oxidative damage of RGCs remains unknown. In this study, we aimed to investigate the potential role and underlying mechanism of Brg1 in regulating HG-induced damage of RGCs. We found that Brg1 expression was significantly downregulated in RGCs in response to HG treatment. Functional experiments showed that Brg1 knockdown enhanced HG-induced apoptosis and production of reactive oxygen species, while Brg1 overexpression suppressed HG-induced apoptosis and reactive oxygen species production, showing a protective effect. Moreover, Brg1 overexpression resulted in an increase in nuclear expression of nuclear factor-erythroid-2-related factor-2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) in RGCs. Notably, inhibition of Nrf2 or HO-1 significantly blocked Brg1-mediated protection against HG-induced damage. Overall, these findings demonstrate that Brg1 protects RGCs from HG-induced oxidative damage through promotion of Nrf2/HO-1 signaling, indicating a potential role of Brg1 in the pathogenesis of diabetic retinopathy.

Role of DJ-1 in Modulating Glycative Stress in Heart Failure.

Background DJ‐1 is a ubiquitously expressed protein typically associated with the development of early onset Parkinson disease. Recent data suggest that it also plays a role in the cellular response to stress. Here, we sought to determine the role DJ‐1 plays in the development of heart failure.Methods and ResultsInitial studies found that DJ‐1 deficient mice (DJ‐1 knockout; male; 8–10 weeks of age) exhibited more severe left ventricular cavity dilatation, cardiac dysfunction, hypertrophy, and fibrosis in the setting of ischemia‐reperfusion–induced heart failure when compared with wild‐type littermates. In contrast, the overexpression of the active form of DJ‐1 using a viral vector approach resulted in significant improvements in the severity of heart failure when compared with mice treated with a control virus. Subsequent studies aimed at evaluating the underlying protective mechanisms found that cardiac DJ‐1 reduces the accumulation of advanced glycation end products and activation of the receptor for advanced glycation end products—thus, reducing glycative stress.ConclusionsThese results indicate that DJ‐1 is an endogenous cytoprotective protein that protects against the development of ischemia‐reperfusion–induced heart failure by reducing glycative stress. Our findings also demonstrate the feasibility of using a gene therapy approach to deliver the active form of DJ‐1 to the heart as a therapeutic strategy to protect against the consequences of ischemic injury, which is a major cause of death in western populations.

Pro-death signaling of cytoprotective heat shock factor 1: upregulation of NOXA leading to apoptosis in heat-sensitive cells

Heat shock can induce either cytoprotective mechanisms or cell death. We found that in certain human and mouse cells, including spermatocytes, activated heat shock factor 1 (HSF1) binds to sequences located in the intron(s) of the PMAIP1 (NOXA) gene and upregulates its expression which induces apoptosis. Such a mode of PMAIP1 activation is not dependent on p53. Therefore, HSF1 not only can activate the expression of genes encoding cytoprotective heat shock proteins, which prevents apoptosis, but it can also positively regulate the proapoptotic PMAIP1 gene, which facilitates cell death. This could be the primary cause of hyperthermia-induced elimination of heat-sensitive cells, yet other pro-death mechanisms might also be involved.

Deletion of Nrf2 shortens lifespan in C57BL6/J male mice but does not alter the health and survival benefits of caloric restriction

Caloric restriction (CR) is the leading non-pharmaceutical dietary intervention to improve health- and lifespan in most model organisms. A wide array of cellular pathways is induced in response to CR and CR-mimetics, including the transcriptional activator Nuclear factor erythroid-2-related factor 2 (Nrf2), which is essential in the upregulation of multiple stress-responsive and mitochondrial enzymes. Nrf2 is necessary in tumor protection but is not essential for the lifespan extending properties of CR in outbred mice. Here, we sought to study Nrf2-knockout (KO) mice and littermate controls in male C57BL6/J, an inbred mouse strain. Deletion of Nrf2 resulted in shortened lifespan compared to littermate controls only under ad libitum conditions. CR-mediated lifespan extension and physical performance improvements did not require Nrf2. Metabolic and protein homeostasis and activation of tissue-specific cytoprotective proteins were dependent on Nrf2 expression. These results highlight an important contribution of Nrf2 for normal lifespan and stress response.

A cell-based assay system for activators of the environmental cell stress response

Improved health span and lifespan extension in a wide phylogenetic range of species is associated with the induction of the environmental cell stress response through a signalling pathway regulated by the transcription factor Nrf2. Phytochemicals which stimulate this response may form part of therapeutic interventions which stimulate endogenous cytoprotective mechanisms, thereby delaying the onset of age-related diseases and promoting healthy ageing in humans. In order to identify compounds that activate the Nrf2 pathway, a cell-based reporter system was established in HepG2 cells using a luciferase reporter gene under the control of the Nqo1 promoter. Sulforaphane, an isothiocyanate derived from cruciferous vegetables and a known activator of the Nrf2 pathway, was used to validate the reporter system. The transfected cell line HepG2 C1 was subsequently used to screen natural product libraries. Five compounds were identified as activating the bioluminescent reporter by greater than 5-fold. The two most potent compounds, MBC20 and MBC37, were further characterised and shown to stimulate endogenous cytoprotective gene and protein expression. The bioluminescent reporter system will allow rapid, in vitro identification of novel compounds that have the potential to improve health span through activation of the environmental stress response.

The tripartite interaction of phosphate, autophagy, and αKlotho in health maintenance.

Aging-related organ degeneration is driven by multiple factors including the cell maintenance mechanisms of autophagy, the cytoprotective protein αKlotho, and the lesser known effects of excess phosphate (Pi), or phosphotoxicity. To examine the interplay between Pi, autophagy, and αKlotho, we used the BK/BK mouse (homozygous for mutant Becn1F121A ) with increased autophagic flux, and αKlotho-hypomorphic mouse (kl/kl) with impaired urinary Pi excretion, low autophagy, and premature organ dysfunction. BK/BK mice live longer than WT littermates, and have heightened phosphaturia from downregulation of two key NaPi cotransporters in the kidney. The multi-organ failure in kl/kl mice was rescued in the double-mutant BK/BK;kl/kl mice exhibiting lower plasma Pi, improved weight gain, restored plasma and renal αKlotho levels, decreased pathology of multiple organs, and improved fertility compared to kl/kl mice. The beneficial effects of heightened autophagy from Becn1F121A was abolished by chronic high-Pi diet which also shortened life span in the BK/BK;kl/kl mice. Pi promoted beclin 1 binding to its negative regulator BCL2, which impairs autophagy flux. Pi downregulated αKlotho, which also independently impaired autophagy. In conclusion, Pi, αKlotho, and autophagy interact intricately to affect each other. Both autophagy and αKlotho antagonizes phosphotoxicity. In concert, this tripartite system jointly determines longevity and life span.