Redox-dependent Manner Research Articles

Desaminotyrosine is a redox-active microbial metabolite that bolsters macrophage antimicrobial functions while attenuating IL-6 production.

Intestinal microbiota contributes to host defense against pathogens while avoiding the induction of inflammation in homeostatic conditions, but the mechanism is not fully understood. To investigate the potential role of the bacterial metabolite desaminotyrosine (DAT) in regulating host defense and inflammation, we pretreated mouse bone marrow-derived macrophages (BMDMs) with DAT for 12 hours and then challenged with bacterial lipopolysaccharide (LPS). We found that DAT priming-enhanced type I interferon response while selectively inhibiting proinflammatory interleukin (IL)-6 production after exposure to LPS. This is related to the fact that DAT is a natural antioxidant determined by radical scavenging assay in a cell-free system. DAT-primed cells had increased levels of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) upon LPS stimulation. Countering the increased NADPH by supplementing extra oxidized NADP+ to cells reversed DAT's effect on LPS-induced Il-6 and interferon-stimulated gene expressions. DAT-primed cells also were more resistant to oxidative stress-induced generation of reactive oxygen species and cell death. DAT promoted the production of antimicrobial effector nitric oxide in a cellular redox-dependent manner, leading to enhanced macrophage antimicrobial activity during Salmonella enterica infection. Our data suggest that DAT acts as a host-microbiota crosstalk signal in shaping host immune defense and inflammatory response.

Blocking the utilization of carbon sources via two pathways to induce tumor starvation for cancer treatment

Glucose oxidase (GOx) is often used to starvation therapy. However, only consuming glucose cannot completely block the energy metabolism of tumor cells. Lactate can support tumor cell survival in the absence of glucose. Here, we constructed a nanoplatform (Met@HMnO2-GOx/HA) that can deplete glucose while inhibiting the compensatory use of lactate by cells to enhance the effect of tumor starvation therapy. GOx can catalyze glucose into gluconic acid and H2O2, and then HMnO2 catalyzes H2O2 into O2 to compensate for the oxygen consumed by GOx, allowing the reaction to proceed sustainably. Furthermore, metformin (Met) can inhibit the conversion of lactate to pyruvate in a redox-dependent manner and reduce the utilization of lactate by tumor cells. Met@HMnO2-GOx/HA nanoparticles maximize the efficacy of tumor starvation therapy by simultaneously inhibiting cellular utilization of two carbon sources. Therefore, this platform is expected to provide new strategies for tumor treatment.

Structural and thermodynamic framework for PIEZO1 modulation by small molecules.

Mechanosensitive PIEZO channels constitute potential pharmacological targets for multiple clinical conditions, spurring the search for potent chemical PIEZO modulators. Among them is Yoda1, a widely used synthetic small molecule PIEZO1 activator discovered through cell-based high-throughput screening. Yoda1 is thought to bind to PIEZO1's mechanosensory arm domain, sandwiched between two transmembrane regions near the channel pore. However, how the binding of Yoda1 to this region promotes channel activation remains elusive. Here, we first demonstrate that cross-linking PIEZO1 repeats A and B with disulfide bridges reduces the effects of Yoda1 in a redox-dependent manner, suggesting that Yoda1 acts by perturbing the contact between these repeats. Using molecular dynamics-based absolute binding free energy simulations, we next show that Yoda1 preferentially occupies a deeper, amphipathic binding site with higher affinity in PIEZO1 open state. Using Yoda1's binding poses in open and closed states, relative binding free energy simulations were conducted in the membrane environment, recapitulating structure-activity relationships of known Yoda1 analogs. Through virtual screening of an 8 million-compound library using computed fragment maps of the Yoda1 binding site, we subsequently identified two chemical scaffolds with agonist activity toward PIEZO1. This study supports a pharmacological model in which Yoda1 activates PIEZO1 by wedging repeats A and B, providing a structural and thermodynamic framework for the rational design of PIEZO1 modulators. Beyond PIEZO channels, the three orthogonal computational approaches employed here represent a promising path toward drug discovery in highly heterogeneous membrane protein systems.

BZIP transcription factor FabR: Redox-dependent mechanism controlling docosahexaenoic acid biosynthesis and H2O2 stress response in Schizochytrium sp.

Schizochytrium sp. is an important industrial strain for commercial production of docosahexaenoic acid (DHA), which plays essential physiological roles in infant development and human health. The regulatory network for DHA biosynthesis and lipid accumulation in Schizochytrium remains poorly understood. FabR (fatty acid biosynthesis repressor), a basic leucine zipper (bZIP) transcription factor, was transcriptionally downregulated under low-nitrogen condition. Deletion of fabR gene (mutant ΔfabR) increased production of total lipids and DHA by 30.1% and 46.5%, respectively. ΔfabR displayed H2O2 stress resistance higher than that of parental strain or complementation strain CfabR. FabR bound specifically to 7-bp pseudo-palindromic sequence 5′-ATTSAAT-3′ in upstream regions and repressed transcription of fatty acid biosynthesis genes (acl, fas, pfa) and antioxidant defense genes (cat, sod1, sod2, gpx). DNA binding activity of FabR was regulated in a redox-dependent manner. Under oxidative condition, FabR forms intermolecular disulfide bonds between two Cys46 residues of dimers; its DNA binding activity is thereby lost, and the transcription of its target genes is enhanced through derepression. Our findings clarify the redox-dependent mechanism that modulates FabR activity governing lipid and DHA biosynthesis and H2O2 stress response in Schizochytrium.

Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from Gluconacetobacter diazotrophicus.

NifA is a σ54 activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in NifA is poorly understood. In α- and β-proteobacteria, redox sensing involves two pairs of Cys residues within and immediately following the protein's central AAA+ domain. In this work, we examine if an additional Cys pair that is part of a C(X)5C motif and located immediately upstream of the DNA binding domain of NifA from the α-proteobacterium Gluconacetobacter diazotrophicus (Gd) is involved in redox sensing. We hypothesize that the Cys residues' redox state may directly influence the DNA binding domain's DNA binding affinity and/or alter the protein's oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. The Kd of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. The Kd of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 µM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner.

Molecular insights into RmcA-mediated c-di-GMP consumption: Linking redox potential to biofilm morphogenesis in Pseudomonas aeruginosa

The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3′,5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints.

Peroxiredoxins Play an Important Role in the Regulation of Immunity and Aging in Drosophila.

Aberrant immune responses and chronic inflammation can impose significant health risks and promote premature aging. Pro-inflammatory responses are largely mediated via reactive oxygen species (ROS) and reduction-oxidation reactions. A pivotal role in maintaining cellular redox homeostasis and the proper control of redox-sensitive signaling belongs to a family of antioxidant and redox-regulating thiol-related peroxidases designated as peroxiredoxins (Prx). Our recent studies in Drosophila have shown that Prxs play a critical role in aging and immunity. We identified two important 'hubs', the endoplasmic reticulum (ER) and mitochondria, where extracellular and intracellular stress signals are transformed into pro-inflammatory responses that are modulated by the activity of the Prxs residing in these cellular organelles. Here, we found that mitochondrial Prx activity in the intestinal epithelium is required to prevent the development of intestinal barrier dysfunction, which can drive systemic inflammation and premature aging. Using a redox-negative mutant, we demonstrated that Prx acts in a redox-dependent manner in regulating the age-related immune response. The hyperactive immune response observed in flies under-expressing mitochondrial Prxs is due to a response to abiotic signals but not to changes in the bacterial content. This hyperactive response, but not reduced lifespan phenotype, can be rescued by the ER-localized Prx.

NTRC regulates CP12 to activate Calvin–Benson cycle during cold acclimation

NADPH-dependent thioredoxin reductase C (NTRC) is a chloroplast redox regulator in algae and plants. Here, we used site-specific mutation analyses of the thioredoxin domain active site of NTRC in the green alga Chlamydomonas reinhardtii to show that NTRC mediates cold tolerance in a redox-dependent manner. By means of coimmunoprecipitation and mass spectrometry, a redox- and cold-dependent binding of the Calvin-Benson Cycle Protein 12 (CP12) to NTRC was identified. NTRC was subsequently demonstrated to directly reduce CP12 of C. reinhardtii as well as that of the vascular plant Arabidopsis thaliana in vitro. As a scaffold protein, CP12 joins the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to form an autoinhibitory supracomplex. Using size-exclusion chromatography, NTRC from both organisms was shown to control the integrity of this complex in vitro and thereby PRK and GAPDH activities in the cold. Thus, NTRC apparently reduces CP12, hence triggering the dissociation of the PRK/CP12/GAPDH complex in the cold. Like the ntrc::aphVIII mutant, CRISPR-based cp12::emx1 mutants also exhibited a redox-dependent cold phenotype. In addition, CP12 deletion resulted in robust decreases in both PRK and GAPDH protein levels implying a protein protection effect of CP12. Both CP12 functions are critical for preparing a repertoire of enzymes for rapid activation in response to environmental changes. This provides a crucial mechanism for cold acclimation.

Light-Dependent Regulatory Interactions between the Redox System and miRNAs and Their Biochemical and Physiological Effects in Plants.

Light intensity and spectrum play a major role in the regulation of the growth, development, and stress response of plants. Changes in the light conditions affect the formation of reactive oxygen species, the activity of the antioxidants, and, consequently, the redox environment in the plant tissues. Many metabolic processes, thus the biogenesis and function of miRNAs, are redox-responsive. The miRNAs, in turn, can modulate various components of the redox system, and this process is also associated with the alteration in the intensity and spectrum of the light. In this review, we would like to summarise the possible regulatory mechanisms by which the alterations in the light conditions can influence miRNAs in a redox-dependent manner. Daily and seasonal fluctuations in the intensity and spectral composition of the light can affect the expression of miRNAs, which can fine-tune the various physiological and biochemical processes due to their effect on their target genes. The interactions between the redox system and miRNAs may be modulated by light conditions, and the proposed function of this regulatory network and its effect on the various biochemical and physiological processes will be introduced in plants.

Exercise-stimulated extracellular vesicles promote cardiomyocyte pro-survival programming in a redox-dependent manner

Exercise-stimulated extracellular vesicles promote cardiomyocyte pro-survival programming in a redox-dependent manner

Interaction and association analysis of malting related traits in barley.

Barley is considered as a foundation of the brewing and malting industry. Varieties with superior malt quality traits are required for efficient brewing and distillation processes. Among these, the Diastatic Power (DP), wort-Viscosity (VIS), β-glucan content (BG), Malt Extract (ME) and Alpha-Amylase (AA) are controlled by several genes linked to numerous quantitative trait loci (QTL), identified for barley malting quality. One of the well-known QTL, QTL2, associated with barley malting trait present on chromosome 4H harbours a key gene, called as HvTLP8 that has been identified for influencing the barley malting quality through its interaction with β-glucan in a redox-dependent manner. In this study, we examined to develop a functional molecular marker for HvTLP8 in the selection of superior malting cultivars. We first examined the expression of HvTLP8 and HvTLP17 containing carbohydrate binding domains in barley malt and feed varieties. The higher expression of HvTLP8 prompted us to further investigate its role as a marker for malting trait. By exploring the 1000 bp downstream 3' UTR region of HvTLP8, we found single nucleotide polymorphism (SNP) in between Steptoe (feed variety) and Morex (malt variety), which was further validated by Cleaved Amplified Polymorphic Sequence (CAPS) marker assay. Analysis of 91 individuals from the Steptoe x Morex doubled haploid (DH) mapping population revealed CAPS polymorphism in HvTLP8. Highly significant (p<0.001) correlations among ME, AA and DP malting traits were observed. The correlation coefficient (r) between these traits ranged from 0.53 to 0.65. However, the polymorphism in HvTLP8 did not correlate effectively with ME, AA, and DP. Altogether, these findings will help us to further design the experiment regarding the HvTLP8 variation and its association with other desirable traits.

Chloroplast translation factor EF-Tu of Arabidopsis thaliana can be inactivated via oxidation of a specific cysteine residue.

Translational elongation factor EF-Tu, which delivers aminoacyl-tRNA to the ribosome, is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803. However, the sensitivity to ROS of chloroplast-localized EF-Tu (cpEF-Tu) of plants remains to be elucidated. In the present study, we generated a recombinant cpEF-Tu protein of Arabidopsis thaliana and examined its sensitivity to ROS in vitro. In cpEF-Tu that lacked a bound nucleotide, one of the two cysteine residues, Cys149 and Cys451, in the mature protein was sensitive to oxidation by H2O2, with the resultant formation of sulfenic acid. The translational activity of cpEF-Tu, as determined with an in vitro translation system, derived from Escherichia coli, that had been reconstituted without EF-Tu, decreased with the oxidation of a cysteine residue. Replacement of Cys149 with an alanine residue rendered cpEF-Tu insensitive to inactivation by H2O2, indicating that Cys149 might be the target of oxidation. In contrast, cpEF-Tu that had bound either GDP or GTP was less sensitive to oxidation by H2O2 than nucleotide-free cpEF-Tu. The addition of thioredoxin f1, a major thioredoxin in the Arabidopsis chloroplast, to oxidized cpEF-Tu allowed the reduction of Cys149 and the reactivation of cpEF-Tu, suggesting that the oxidation of cpEF-Tu might be a reversible regulatory mechanism that suppresses the chloroplast translation system in a redox-dependent manner.

Thioredoxin h2 inhibits the MPKK5-MPK3 cascade to regulate the CBF-COR signaling pathway in Citrullus lanatus suffering chilling stress.

Thioredoxins (TRXs) are ubiquitous oxidoreductases and present as a multigenic family. TRXs determine the thiol redox balance, which is crucial for plants in the response to cold stress. However, limited knowledge is available about the role of TRXs in watermelon (Citrullus lanatus), which is highly sensitive to chilling stress in agricultural practice. Here, we identified 18 genes encoding 14 typical and 4 atypical TRXs from the watermelon genome, and found that ClTRXh2 localized at the plasma membrane was largely induced by chilling. Virus-induced gene silencing of ClTRXh2 resulted in watermelon plants that were more sensitive to chilling stress. We further found that ClTRXh2 physically interacted with mitogen-activated protein kinase kinase 5 (ClMPKK5), which was confirmed to phosphorylate and activate ClMPK3 in vitro, and the activation of ClMPK3 by ClMPKK5 was blocked by a point mutation of the Cys-229 residue to Ser in ClMPKK5. Additionally, ClTRXh2 inhibited the chilling-induced activation of ClMPK3, suggesting that the ClMPKK5-ClMPK3 cascade is regulated in a redox-dependent manner. We showed that ClMPK3-silenced plants had increased tolerance to chilling, as well as enhanced transcript abundances of the C-repeat/DREB binding factor (ClCBF) and cold-responsive (ClCOR) genes. Taken together, our results indicate that redox status mediated by ClTRXh2 inhibits ClMPK3 phosphorylation through the interaction between ClTRXh2 and ClMPKK5, which subsequently regulates the CBF-COR signaling pathway when submitted to chilling stress. Hence, our results provide a link between thiol redox balance and MAPK cascade signaling, revealing a conceptual framework to understand how TRX regulates chilling stress tolerance in watermelon.

Mitotic spindle disassembly in human cells relies on CRIPT having hierarchical redox signals.

Swift and complete spindle disassembly in late mitosis is essential for cell survival, yet how it happens is largely unknown in mammalian cells. Here we used real-time live cell microscopy and biochemical assays to show that the primordial dwarfism (PD)-related cysteine-rich protein CRIPT dictates the spindle disassembly in a redox-dependent manner in human cells. This previously reported cytoplasmic protein was found to have a confined nuclear localization with a nucleolar concentration during interphase but was distributed to spindles and underwent redox modifications to form disulfide bonds in CXXC pairs during mitosis. Then, it directly interacted with, and might transfer a redox response to, tubulin subunits via a putative redox exchange among cysteine residues to induce microtubule depolymerization. Expression of CRIPT proteins with mutations of these cysteine residues blocked spindle disassembly, generating two cell types with long-lasting metaphase spindles or spindle remnants. Live-cell recordings of a disease-relevant mutant (CRIPTC3Y) revealed that microtubule depolymerization at spindle ends during anaphase and the entire spindle dissolution during telophase might share a common CRIPT-bearing redox-controlled mechanism.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

Redox-dependent structure and dynamics of macrophage migration inhibitory factor reveal sites of latent allostery

Macrophage migration inhibitory factor (MIF) is a multifunctional immunoregulatory protein that is a key player in the innate immune response. Given its overexpression at sites of inflammation and in diseases marked by increasingly oxidative environments, a comprehensive understanding of how cellular redox conditions impact the structure and function of MIF is necessary. We used NMR spectroscopy and mass spectrometry to investigate biophysical signatures of MIF under varied solution redox conditions. Our results indicate that the MIF structure is modified and becomes increasingly dynamic in an oxidative environment, which may be a means to alter the MIF conformation and functional response in a redox-dependent manner. We identified latent allosteric sites within MIF through mutational analysis of redox-sensitive residues, revealing that a loss of redox-responsive residues attenuates CD74 receptor activation. Leveraging sites of redox sensitivity as targets for structure-based drug design therefore reveals an avenue to modulate MIF function in its "disease state."

Is Nucleoredoxin a Master Regulator of Cellular Redox Homeostasis? Its Implication in Different Pathologies.

Nucleoredoxin (NXN), an oxidoreductase enzyme, contributes to cellular redox homeostasis by regulating different signaling pathways in a redox-dependent manner. By interacting with seven proteins so far, namely disheveled (DVL), protein phosphatase 2A (PP2A), phosphofructokinase-1 (PFK1), translocation protein SEC63 homolog (SEC63), myeloid differentiation primary response gene-88 (MYD88), flightless-I (FLII), and calcium/calmodulin-dependent protein kinase II type alpha (CAMK2A), NXN is involved in the regulation of several key cellular processes, including proliferation, organogenesis, cell cycle progression, glycolysis, innate immunity and inflammation, motility, contraction, protein transport into the endoplasmic reticulum, neuronal plasticity, among others; as a result, NXN has been implicated in different pathologies, such as cancer, alcoholic and polycystic liver disease, liver fibrogenesis, obesity, Robinow syndrome, diabetes mellitus, Alzheimer’s disease, and retinitis pigmentosa. Together, this evidence places NXN as a strong candidate to be a master redox regulator of cell physiology and as the hub of different redox-sensitive signaling pathways and associated pathologies. This review summarizes and discusses the current insights on NXN-dependent redox regulation and its implication in different pathologies.

Long-range retrograde mechanical coupling in the PIEZO1 channel

Mechanosensitive PIEZO channels possess a unique bowl-like structure spanning approximately 25 nm in diameter, making them some of the largest membrane proteins known to date. We had previously identified gating-associated conformational changes in two discrete regions of the N-terminal mechanosensory domain of PIEZO1 by temporally correlating fluorescence signals from genetically-inserted probes (cyclic permuted GFPs) with fluid shear stress-induced channel activation, measured using calcium imaging. Here, we show that signals from both probes are largely unchanged by mutations known to accelerate or slow down PIEZO1 inactivation, suggesting our probes capture gating transitions from resting to open/inactivated states. Likewise, application of the mechanosensitive channel inhibitor GsMtx4 does not inhibit these signals, despite the ability of the toxin to inhibit shear stress-induced PIEZO1-dependent calcium responses. Remarkably, shear stress-induced signals emitted by the most distal probe, near the N-terminus, are strongly inhibited in a redox-dependent manner by introducing a previously reported inhibitory disulfide bridge in the C-terminal central pore region, located more than 10 nm away from the probe. Whereas anterograde mechanical coupling from sensory domains to pore domains forms a prevailing paradigm for stimulus-mediated ion channel activation, our results suggest that this coupling also exists in the retrograde direction in PIEZO1, from the pore to the mechanosensory domain.

Perturbation of Cellular Redox Homeostasis Dictates Divergent Effects of Polybutyl Cyanoacrylate (PBCA) Nanoparticles on Autophagy.

Nanoparticles (NPs) are used in our everyday life, including as drug delivery vehicles. However, the effects of NPs at the cellular level and their impacts on autophagy are poorly understood. Here, we demonstrate that the NP drug delivery vehicle poly(butyl cyanoacrylate) (PBCA) perturbs redox homeostasis in human epithelial cells, and that the degree of redox perturbation dictates divergent effects of PBCA on autophagy. Specifically, PBCA promoted functional autophagy at low concentrations, whereas it inhibited autophagy at high concentrations. Both effects were completely abolished by the antioxidant N-acetyl cysteine (NAC). High concentrations of PBCA inhibited MAP1LC3B/GABARAP lipidation and LC3 flux, and blocked bulk autophagic cargo flux induced by mTOR inhibition. These effects were mimicked by the redox regulator H2O2. In contrast, low concentrations of PBCA enhanced bulk autophagic cargo flux in a Vps34-, ULK1/2- and ATG13-dependent manner, yet interestingly, without an accompanying increase in LC3 lipidation or flux. PBCA activated MAP kinase signaling cascades in a redox-dependent manner, and interference with individual signaling components revealed that the autophagy-stimulating effect of PBCA required the action of the JNK and p38–MK2 pathways, whose activities converged on the pro-autophagic protein Beclin-1. Collectively, our results reveal that PBCA exerts a dual effect on autophagy depending on the severity of the NP insult and the resulting perturbation of redox homeostasis. Such a dual autophagy-modifying effect may be of general relevance for redox-perturbing NPs and have important implications in nanomedicine.

Abstract 11086: MG53 Suppresses NfκB Activation to Mitigate Age-Related Heart Failure

Introduction: Aging is associated with chronic oxidative stress and inflammation that affect the tissue repair and regeneration capacity. MG53 is a TRIM family protein that facilitates repair of cell membrane injury in a redox-dependent manner. However, it is not clear how MG53 regulates cardiac inflammation during aging. Hypothesis: We hypothesize that the expression of MG53 is associated with age-related heart failure, and treatment with recombinant human MG53 (rhMG53) protein in aged mice might have beneficial effect on the age-related decline of heart function. Methods: qPCR and Western blots were conducted for targeted genes/proteins in non-failing and failing human left ventricular tissues, and mouse heart tissues. A total of sixty mice (24 months, 30 male and 30 female) were enrolled for treatment with rhMG53 (6 mg/kg, subcutaneous, daily for 6 weeks) or saline as control, and subjected to cardiac functional measurement with echocardiography and pressure-volume loop, and cardiac apoptotic and ROS assays. Blood were collected to quantify the serum levels of biomarkers for liver and cardiovascular injury. Results: We demonstrate that the expression of MG53 is reduced in failing human heart and aging mouse heart, concomitant with elevated NFκB activation. We evaluate the safety and efficacy of longitudinal, systemic administration of recombinant human MG53 (rhMG53) protein in aged mice. Echocardiography and pressure-volume loop measurements reveal beneficial effects of rhMG53 treatment in improving heart function of aging mice. Biochemical and histological studies demonstrate the cardioprotective effects of rhMG53 are linked to suppression of NFκB-mediated inflammation, reducing apoptotic cell death and oxidative stress in the aged heart. Repetitive administration of rhMG53 in aged mice does not have adverse effects on immune response and major vital organ functions. Conclusions: In this study, we showed decreased expression of MG53 and increased NFκB activation in both failing human hearts and aging mouse hearts. Moreover, longitudinal administration of rhMG53 mitigated the heart dysfunction in the aged mice. These findings support the therapeutic value of rhMG53 in treating age-related decline in cardiac function.