Cytoprotective Proteins Research Articles

Inflammation, coagulation, and cellular injury in heat-induced shock.

The number of heatstroke victims hit record numbers in 2022 as global warming continues. In heat-induced injuries, circulatory shock is the most severe and deadly complication. This review aims to examine the mechanisms and potential approaches to heat-induced shock and the life-threatening complications of heatstroke. A computer-based online search was performed using the PubMed database and Web of Science database for published articles concerning heatstroke, shock, inflammation, coagulopathy, endothelial cell, cell death, and heat shock proteins. Dehydration and heat-induced cardiomyopathy were reported as the major causes of heat-induced shock, although other heat-induced injuries are also involved in the pathogenesis of circulatory shock. In addition to dehydration, the blood volume decreases considerably due to the increased vascular permeability as a consequence of endothelial damage. Systemic inflammation is induced by factors that include elevated cytokine and chemokine levels, dysregulated coagulation/fibrinolytic responses, and the release of damage-associated molecular patterns (DAMPs) from necrotic cell death that cause distributive shock. The cytoprotective heat shock proteins can also facilitate circulatory disturbance under excess heat stress. Multiple mechanisms are involved in the pathogenesis of heat-induced shock. In addition to dehydration, heat stress-induced cardiomyopathy due to the thermal damage of mitochondria, upregulated inflammation via damage-associated molecular patterns released from oncotic cells, unbalanced coagulation/fibrinolysis, and endothelial damage are the major factors that are related to circulatory shock.

Quercetin 3-O-rutinoside prevents radiation induced oxidative damage and inflammation by coordinated regulation of Nrf2/ NF-κB/ NLRP3- inflammasome signaling in gastrointestine

BackgroundIrradiation causes extensive alteration in the cellular homeostasis leading to severe organ damage and dysfunction. The lack of understanding of the mechanism conferring radioprotection is often considered a major limitation in the development of agents that suppress the damaging effects of radiation. PurposeIn this study, we demonstrate that Quercetin 3-O-rutinoside (Q-3-R), a flavonoid, can significantly protect the gastrointestine against gamma radiation induced damage. MethodsQ-3-R (10 mg/kg body weight) treated C57BL/6 mice were investigated for anti-oxidant status, regulations of critical targets of Nrf2, NF-κB signaling and the inflammasome complex in the irradiated intestine. ResultsProphylactic administration of Q-3-R stabilized radiation induced reactive oxygen species, maintained total antioxidant and glutathione levels in the intestine. Q-3-R pretreatment activated Nrf2, supported its nuclear translocation and upregulated cytoprotective proteins, HO-1, thioredoxin1, GST and GCLC. The key inflammatory mediator, NF-kB p65 was found down-regulated in Q-3-R treated jejunum. NF-kB p65 downregulation caused suppression of genes of the inflammasome complex, pro-inflammatory cytokine (IL-6 and TNF-α) expression and myeloperoxidase activity. ML385, a specific Nrf2 inhibitor that, when used at a concentration of 30 mg/kg prevented Q-3-R mediated activation of Nrf2 and upregulated NF-kB, suggesting that Q-3-R mediates protection against radiation primarily via Nrf2 activation. Q-3-R also prevented intestinal apoptosis by inhibiting mitochondrial translocation of cytochrome c into the cytosol and subsequent activation of caspase-3. Molecular docking studies confirmed the interactions of Q-3-R with key antioxidant and anti-inflammatory proteins. ConclusionThe findings suggest that Q-3-R effectively coordinated the regulation of cellular anti-oxidant and inflammatory response, thereby minimizing apoptotic event in the irradiated intestine.

Quantitating Nrf2-regulated cytoprotective proteins using an LC-MS/MS data-independent acquisition method

Quantitating Nrf2-regulated cytoprotective proteins using an LC-MS/MS data-independent acquisition method

The Nrf2 in Obesity: A Friend or Foe?

Obesity and its complications have become serious global health concerns recently and increasing work has been carried out to explicate the underlying mechanism of the disease development. The recognized correlations suggest oxidative stress and inflammation in expanding adipose tissue with excessive fat accumulation play important roles in the pathogenesis of obesity, as well as its associated metabolic syndromes. In adipose tissue, obesity-mediated insulin resistance strongly correlates with increased oxidative stress and inflammation. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been described as a key modulator of antioxidant signaling, which regulates the transcription of various genes coding antioxidant enzymes and cytoprotective proteins. Furthermore, an increasing number of studies have demonstrated that Nrf2 is a pivotal target of obesity and its related metabolic disorders. However, its effects are controversial and even contradictory. This review aims to clarify the complicated interplay among Nrf2, oxidative stress, lipid metabolism, insulin signaling and chronic inflammation in obesity. Elucidating the implications of Nrf2 modulation on obesity would provide novel insights for potential therapeutic approaches in obesity and its comorbidities.

Characterization of the mitochondrial unfolded protein response in human coronary artery endothelial cells

Abstract Background The unfolded protein response of the endoplasmic reticulum (UPRER) is highly activated in cardiovascular diseases and aims to restore homeostasis by improving protein folding. An analogue cytoprotective mitochondrial unfolded protein response (UPRMito) regulated by an orthologue of the activating transcription factor 5 (ATF5) was described in model organism c. elegans. However, in humans it is not clear if there is a distinct UPRMito, and if there is an activation of an UPRMito during stress induced by cardiovascular diseases. The endothelium is subjected to high levels of mitochondrial stress, e.g. in atherosclerosis. Purpose To provide a systematic characterization of the UPRMito in human coronary artery endothelial cells (HCAEC). Methods and results HCAEC were treated with Nicotinamide Riboside (NR), a NAD+ precursor that activates UPRMito without inducing cellular stress. NR treatment did not induce toxicity and improved cell viability at a concentration of 100μM NR. Quantitative PCR confirmed a dose-dependent upregulation of UPRMito markers. Additionally, we measured expression of ATF4, which is discussed to regulate an integrated stress response in mammals by activating both the UPRMito and UPRER. ATF4 upregulation upon NR treatment was more pronounced than ATF5 upregulation. Analysis of GTEX-Data showed that ATF5 is almost exclusively expressed in liver tissue, while ATF4 shows ubiquitous expression. Among 54 tissues, aortic tissue and coronary arterial tissue are among the five tissues with the highest ATF4 expression. Analysis of RNA-seq-data of human carotid atherosclerosis revealed that the two transcription factors are differentially regulated. ATF4 is downregulated in unstable plaque, while ATF5 is upregulated. Moreover, ATF4 is in both conditions highly more abundant than ATF5. Accordingly, endothelial cells incubated under atherosclerotic conditions (oxLDL + high glucose) exhibited a ATF4 down- and ATF5 upregulation. Genes previously described to be involved in the UPRMito exhibited a similar expression pattern as ATF4. To further elucidate these findings, we will perform transcriptomic analyses of HCAEC treated with atherosclerotic stimuli (IL-1β, oxLDL), UPRER inductors (Thapsigargin, Tunicamycin) and stressors of mitochondrial proteostasis (Oligomycin, MitoBloCK-6), to analyze, if ATF5- or ATF4-related pathways are dysregulated. Specific inhibitors and UPRMito activators will be used to investigate the significance of ATF4 and ATF5 on endothelial cell function (proliferation, migration, apoptosis, monocyte adhesion). Conclusion The UPRMito is dysregulated during the pathogenesis of atherosclerosis in HCAEC. Further findings are required to elucidate, if the UPRMito is activated separately and specifically by ATF5 or as part of an integrated cellular stress response by ATF4. A deeper understanding of these stress responses is crucial for the identification of novel therapeutic targets in atherosclerosis. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): University hospital Bonn

Macrophage malfunction in Triptolide-induced indirect hepatotoxicity.

Background and Objective: Indirect hepatotoxicity is a new type of drug-induced hepatotoxicity in which the character of a drug that may induce its occurrence and the underlying mechanism remains elusive. Previously, we proved that Triptolide (TP) induced indirect hepatotoxicity upon LPS stimulation resulting from the deficiency of cytoprotective protein of hepatocyte. However, whether immune cells participated in TP-induced indirect hepatotoxicity and the way immune cells change the liver hypersensitivity to LPS still need to be deeply investigated. In this study, we tried to explore whether and how macrophages are involved in TP-induced indirect hepatotoxicity. Method: Firstly, TP (500 μg/kg) and LPS (0.1 mg/kg) were administrated into female C57BL/6 mice as previously reported. Serum biochemical indicators, morphological changes, hepatic macrophage markers, as well as macrophage M1/M2 markers were detected. Secondly, macrophage scavenger clodronate liposomes were injected to prove whether macrophages participated in TP-induced indirect hepatotoxicity. Also, the ability of macrophages to secrete inflammatory factors and macrophage phagocytosis were detected. Lastly, reverse docking was used to find the target of TP on macrophage and the possible target was verified in vivo and in RAW264.7 cells. Results: TP pretreatment increased the liver hypersensitization to LPS accompanied by the recruitment of macrophages to the liver and promoted the transformation of macrophages to M1 type. Depletion of hepatic macrophages almost completely alleviated the liver injury induced by TP/LPS. TP pretreatment increased the secretion of pro-inflammatory factors and weakened the phagocytic function of macrophages upon LPS exposure. Reverse docking results revealed that MerTK might be the real target of TP. Conclusion: TP disrupts inflammatory cytokines profile and phagocytic function of hepatic macrophages, resulting in the production of massive inflammatory factors and the accumulation of endotoxin in the liver, ultimately leading to the indirect hepatotoxicity of TP. MerTK might be the target of TP on the macrophage, while the binding of TP to MerTK should be investigated in vivo and in vitro.

Short-term microplastic exposure triggers cellular damage through oxidative stress in the soft coral Coelogorgia palmosa

ABSTRACT Microplastics are a persistent and ubiquitous source of pollution in the marine environment, representing a severe threat to tropical coral reefs. The effects of microplastics on reef-building (hard) corals have been documented (interference with normal digestion process, polyp retraction, oxidative stress, impairment of the photosynthetic machinery, bleaching). However, the impact of microplastics on soft corals, the second most abundant benthos of tropical reefs, remains to be thoroughly studied. In this work, we analysed the effects of a short-term microplastic exposure on the cellular physiology of the soft coral Coelogorgia palmosa. We found that samples exposed to >50 mg l−1 of microplastic showed significant increase in the activities of the antioxidant enzymes glutathione reductase, catalase, and superoxide dismutase, suggesting a rise in oxidative stress. Furthermore, exposure to microplastics increased lipid peroxidation, indicating oxidative damage. Overall, our results show that similar to hard corals, microplastic ingestion causes oxidative stress and cellular damage in soft corals. Our study provides a first assessment of physiological effects of microplastic exposure on the soft coral, Coelogorgia palmosa, highlighting the need for further investigations about these contaminants and their influence on marine benthic fauna. Such information is crucial to understand how different reef organisms respond to microplastic pollution and who the ecological winners or losers will be in an increasingly polluted marine environment. Key policy highlights The effect of microplastic contamination at the cellular and molecular level has never been investigated before in alcyonacean corals. Concentrations of 50–70 mg l−1 of polyethylene caused significant rise in oxidative stress in Coelogorgia palmosa after 48 h. After 48 h exposure to 50 mg l−1 significant oxidative damage was recorded. No significant modulation in the expression of the cytoprotective protein Hsp60 was observed after exposure to microplastics.

The Antigastric Cancer Effect of Triptolide is Associated With H19/NF-κB/FLIP Axis.

Background and Objective: Triptolide (TP), one of the fat-soluble components extracted from the Chinese medicinal herb Tripterygium wilfordii Hook F. (TWHF), possesses strong antitumor bioactivities, but its dose-dependent side effects restrict its wide application. This study was designed to investigate whether inflammatory factors increased the antitumor effects of the nontoxic dose of TP on gastric cancer cells and tried to explore the possible molecular mechanisms. Method: AGS and MKN45 cells were treated with different doses of TP and TNF-α. Cell viability and apoptosis were detected in vitro. In addition, NF-κB mediated prosurvival signals and cytoprotective proteins, especially FLICE-inhibitory protein (FLIP), were detected to determine their effects on TP/TNF-α–induced apoptosis. Moreover, the function of lncRNA H19/miR-204-5p/NF-κB/FLIP axis was investigated in vitro, and the antigastric cancer effect of TP plus TNF-α was proved in the mice xenograft model. Result: In vitro experimental results showed that TP pretreatment promoted apoptosis in AGS and MKN45 cells upon TNF-α exposure. TP/TNF-α–mediated apoptosis was partly mediated by the inhibitory effect of NF-κB–mediated FLIP expression. Oncogene H19 lying in the upstream pathway of NF-κB played a vital role upon TNF-α exposure, and bioinformatics analysis proved that H19 participated in TP/TNF-α–induced apoptosis via binding of miR-204-5p. Lastly, a low dose of TP and TNF-α inhibited the tumor weight and tumor volume of AGS and MKN45 cells in vivo. Conclusion: TP pretreatment increased apoptosis in TNF-α–stimulated gastric cancer cells, which are dependent on the disruption of the H19/miR-204-5p/NF-κB/FLIP axis. Cotreatment of TP and TNF-α is a better option for enhancing the anticancer effect and lowering the side effect of TP.

In Situ Upregulating Heat Shock Protein 70 via Gastric Nano-Heaters for the Interference of Helicobacter pylori Infection.

Taking inspiration from the mechanism of Helicobacter pylori infection can lead to innovative antibacterial ways to fight antibiotics resistance. Herein, a gastric nano-heater iron-cobalt alloy shielded with graphitic shells (FeCo@G) is developed to interfere with H. pylori infection under an alternating magnetic field. FeCo@G shows a high and stable specific loss power (SLP = 534.1 W g-1) in the acidic environment and provides efficient magnetothermal stimulation in the stomach. Such stimulation upregulates the cytoprotective heat shock protein 70 (HSP70) in gastric epithelial cells, which antagonizes the infection of H. pylori. This finding is further supported by the transcriptomic analysis verifying the upregulation of HSP70 in the stomach. Moreover, the nano-heater shows a high inhibition rate of H. pylori in vivo with good biocompatibility; 95% of FeCo@G is excreted from the mouse's gastrointestinal tract within 12 h. In summary, FeCo@G allows magnetothermal therapy to be used in harsh gastric environments, providing an approach for the therapy against H. pylori.

Nanodrugs Manipulating Endoplasmic Reticulum Stress for Highly Effective Antitumor Therapy.

Cancer is one of the leading causes of death worldwide due to high morbidity and mortality. Many attempts and efforts have been devoted to fighting cancer. Owing to the significant role of the endoplasmic reticulum (ER) in cell function, inducing ER stress can be promising for cancer treatment. However, the sustained activation of cytoprotective unfolded protein response (UPR) presents a tremendous obstacle for drugs in inducing unsolved ER stress in tumor cells, especially small-molecule drugs with poor bioavailability. Therefore, many emerging nanodrugs inducing and amplifying ER stress have been developed for efficient cancer treatment. More importantly, the novel discovery of ER stress in immunogenic cell death (ICD) makes it possible to repurpose antitumor drugs for immunotherapy through nanodrug-based strategies amplifying ER stress. Therefore, this mini-review aims to provide a comprehensive summary of the latest developments of the strategies underlying nanodrugs in the treatment of cancer via manipulating ER stress. Meanwhile, the prospects of ER stress–inducing nanodrugs for cancer treatment are systematically discussed, which provide a sound platform for novel therapeutic insights and inspiration for the design of nanodrugs in treating cancer.

DJ-1 is not a deglycase and makes a modest contribution to cellular defense against methylglyoxal damage in neurons.

Human DJ‐1 is a cytoprotective protein whose absence causes Parkinson's disease and is also associated with other diseases. DJ‐1 has an established role as a redox‐regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ‐1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α‐keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ‐1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ‐1 protects cells against insults that can cause disease. We find that DJ‐1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady‐state kinetics of DJ‐1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ‐1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ‐1. Sensitive and quantitative isotope‐dilution mass spectrometry shows that DJ‐1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ‐1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ‐1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.

Redox-sensitive DJ-1 protein: an insight into physiological roles, secretion, and therapeutic target

Graphical abstract Abstract The highly conserved DJ-1 protein fundamentally acts as a redox sensor conferring antioxidative cytoprotection under oxidative insults. DJ-1 preferentially undergoes oxidation at 106 cysteine residue (C106) under oxidative stress. Although initially identified as an oncogene, emerging evidence suggests the essential roles of DJ-1 in modulating numerous physiological processes involved in cellular growth, development, survival, and death. Compromised DJ-1 expression and function directly or indirectly trigger signaling cascades leading to pathophysiological conditions including neurodegeneration, cancer, stroke, and inflammatory diseases. Besides the intracellular functions, enhanced DJ-1 secretion into the extracellular fluid, including cerebrospinal fluid and blood, is related to Parkinson’s disease (PD) and cancer pathogenesis. Here, we review the current knowledge regarding DJ-1’s roles as a ubiquitous cytoprotective protein controlling numerous signaling pathways, secretion, and therapeutic potentials.

N-Glycosylation Deficiency Reduces the Activation of Protein C and Disrupts Endothelial Barrier Integrity.

Phosphomannomutase 2 (PMM2) deficiency is the most prevalent congenital disorder of glycosylation. It is associated with coagulopathy, including protein C deficiency. Since all components of the anticoagulant and cytoprotective protein C system are glycosylated, we sought to investigate the impact of an N-glycosylation deficiency on this system as a whole. To this end, we developed a PMM2 knockdown model in the brain endothelial cell line hCMEC/D3. The resulting PMM2low cells were less able to generate activated protein C (APC), due to lower surface expression of thrombomodulin and endothelial protein C receptor. The low protein levels were due to downregulated transcription of the corresponding genes (THBD and PROCR, respectively), which itself was related to downregulation of transcription regulators Krüppel-like factors 2 and 4 and forkhead box C2. PMM2 knockdown was also associated with impaired integrity of the endothelial cell monolayer-partly due to an alteration in the structure of VE-cadherin in adherens junctions. The expression of protease-activated receptor 1 (involved in the cytoprotective effects of APC on the endothelium) was not affected by PMM2 knockdown. Thrombin stimulation induced hyperpermeability in PMM2low cells. However, pretreatment of cells with APC before thrombin simulation was still associated with a barrier-protecting effect. Taken as a whole, our results show that the partial loss of PMM2 in hCMEC/D3 cells is associated with impaired activation of protein C and a relative increase in barrier permeability.

Combined transcriptomic, proteomic and synthesis profiling reveal distinct proteostatic signatures for skeletal muscle of adult and old mice

Muscle mass is reduced with age and numerous studies have examined the potential role of global changes in proteostasis, through protein synthesis and/or degradation in the overall loss of muscle mass with age, although no consensus has been reached.We hypothesise that changes in expression, synthesis and degradation may be very different for individual proteins and such findings may be overshadowed by examining global changes in protein turnover. In addition, we hypothesize that alterations in the synthesis rates of specific individual proteins in the muscles of old mice when compared with that of adult are likely to impact on the age‐related loss of muscle mass. We used deuterated water labelling to determine the synthesis rates of individual muscle proteins and have integrated these data with RNA Sequencing data and protein abundances to provide a powerful analysis of individual proteins in muscles of adult (6‐8months) and old (26months) C57BL/6J mice by which time ~15% of gastrocnemius muscle had been lost.For protein synthesis analysis, all animals received an initial loading dose of 2H2O via two IP injections of 99% 2H2O in PBS, 4hrs apart. Mice were then given 8% 2H2O in normal drinking water to maintain plasma enrichment of 2H2O at around 4.5% and gastrocnemius muscles were harvested for up to 60 days when mice were culled. Muscle RNA was isolated, RNA libraries generated using poly‐A selection, sequenced using Illumina HiSeq 4000 platform and analysed with DESEQ2 and gene ontology and pathway tools. To determine the effect of age on differentially expressed genes, protein abundance and turnover, we analysed the common list of 185 proteins and cognate transcripts for which we had data on transcript and turnover parameters.Functional annotation of proteins in the unlabelled dataset demonstrated an upregulation of protein degradation pathways with aging, including ubiquitin protein ligase binding and the unfolded protein response suggesting a clear role of protein degradation as a major contributor to the loss of muscle mass with age in mice. The top four KEGG disease pathways enriched when comparing protein abundance in the muscles of adult and old mice were all degenerative diseases caused or exacerbated by protein misfolding and accumulation. For most proteins, synthesis rates were unchanged, and thus, any change in abundance has to be reconciled with a commensurate change in degradation rate. These global data demonstrate that the net negative protein balance within the muscles of old mice is driven by elevated protein degradation rather than reduced translation. However, there were some notable outliers from the analysis of protein synthesis data showing a decrease in the synthesis of several structural proteins and an increase in the synthesis of metabolic or cytoprotective proteins in muscles of old compared with adult mice. The rate of synthesis of a protein was not always associated with transcript level suggesting caution in extrapolating the suggestion that static mRNA levels reflect the rate of protein synthesis or the protein levels in skeletal muscle.

Laminar Shear Stress Enhances Cytosolic PINK1 Expression: Increased Mitophagic Sensitivity Toward Mitochondrial Dysfunction

PURPOSEPhosphatase and tensin homolog (PTEN)‐induced putative kinase 1 (PINK1) is an essential molecule in mitophagy process and known to act as a cytoprotective protein in mammalian cells. Mutation or deficiency of PINK1 has been closely related to several disease conditions. The purpose of this study was to determine PINK1 expression levels and subcellular localization under exercise‐mimic laminar shear stress (LSS) condition in human aortic endothelial cells (HAECs) or in exercising mice, and its implication on endothelial homeostasis and cardiovascular disease (CVD) prevention.METHODS & RESULTSFirst, we measured full‐length PINK1 (FL‐PINK1) mRNA and protein expression levels in HAECs under unidirectional laminar shear stress (LSS) at 15 dyne/cm2 for 48h. LSS significantly elevated both FL‐PINK1 mRNA and protein expressions in HAECs compared to static control. Mitochondrial fractionation assays showed a decrease in FL‐PINK1 accumulation in the mitochondria with a compensatory increase in the cytosolic FL‐PINK1 level under LSS. Confocal microscopic analysis confirmed these subcellular localization patterns suggesting downregulation of mitophagy induction. Also, mitophagy flux was decreased under LSS, determined by a mtKeima probe. Mitochondrial morphometric analysis and mitochondrial membrane potential (JC‐1) showed mitochondrial elongation and increased mitochondrial membrane potential, respectively, suggesting that an elevation of cytosolic PINK1 is not related to an immediate induction of mitophagy. Based on this observation, we further hypothesized that the elevation of the cytosolic pool of FL‐PINK1 would increase the sensitivity to mitophagic signals in pathological conditions. Preconditioned HAECs with LSS showed lower mtDNA lesions under angiotensin II stimulation (100 nM, 6h). Moreover, LSS‐preconditioned ECs showed rapid Parkin recruitment and mitophagy induction in response to mitochondrial toxin (i.e. CCCP) treatment compared to the control. We measured PINK1 expression at ECs of the thoracic aorta in exercised mice, a physiological LSS‐enhanced model, which was significantly elevated compared to sedentary animals. In addition, exercise‐preconditioned mice were more protective to angiotensin II‐induced mtDNA lesion formation in the mouse abdominal aorta than sedentary mice, suggesting a potential protective mechanism of exercise in a PINK1‐dependent manner.CONCLUSIONLSS increases a cytosolic pool of FL‐PINK1, which may elevate the mitophagic sensitivity toward dysfunctional mitochondria or activate other cytoprotective mechanisms in endothelial cells. Our data suggest that exercise may support mitochondrial homeostasis in vascular endothelial cells by enhancing PINK1‐dependent cell protection mechanisms.

Surface Mapping and Functional Interactions of GPx4, a Cytoprotective Peripheral Membrane Protein

The study of peripheral membrane proteins (PMPs) has long been fraught with difficulty due to the need for reliable and robust membrane models. Many PMPs are soluble in an inactive state and recruited to the membrane to perform their respective function. This is the source of difficulty: the small size of the proteins and their inherent dynamic interactions with membranes and specific lipids make them incredibly challenging to characterize using X‐ray crystallography or cryo‐EM. Using protein NMR and better optimized membrane mimics, a robust study of PMPs may proceed.Glutathione peroxidase 4 (GPx4) is a PMP with the unique function of protection of membranes from lipid hydroperoxidation. This enzyme prevents cell death from ferroptosis by engaging the membrane and reducing lipid hydroperoxides. GPx4 has emerged as a high‐profile therapeutic target due to the ability of inhibitors to leverage ferroptosis against certain types of cancer cells.We utilize a wide variety of membrane models and native substrates to map the binding interaction of the GPx4 with the membrane and substrate lipids. Electrostatic interactions of the cationic patch are also found to be important in the moonlighting function of GPx4 as a cross‐linker of DNA‐bound protamines. Finally, our experimentally determined membrane interface using DPC micelles to formulate and confirm a novel DLPC:DPC reverse micelle system that can act as a membrane model for future PMP studies. The novel DLPC:DPC formulation is the first step in developing reverse micelles as more biologically accurate membrane models that can be tuned depending on membrane chemistry.

Oxidative Stress Suppresses Nrf2 Protein Synthesis through Global Protein Synthesis Inhibition

Oxidative and electrophilic stresses are common aggressors in chronic diseases ranging from arthritis to cancer to neurodegenerative disease. The transcription factor Nrf2 is an essential mediator of the cell’s cytoprotective response to these stressors. Nrf2 is constantly synthesized, ubiquitinated, and degraded with a short half‐life of 20 minutes that maintains low basal Nrf2 protein levels. Electrophiles and oxidative stress cause Nrf2 to accumulate and subsequently to activate cytoprotective genes by modifying cysteines of the Keap1 protein, the repressor that targets Nrf2 for ubiquitination. We previously found that paradoxically, reactive oxygen species (ROS) stimulate the production of cytoprotective proteins, but inhibit Nrf2 protein synthesis, revealing a “Dr. Jekyll and Mr. Hyde” effect of ROS on Nrf2 accumulation. We tested the hypothesis that oxidative conditions that activate the Nrf2 pathway simultaneously inhibit global protein synthesis, thereby suppressing Nrf2 protein synthesis. We investigated both ROS generated by redox cycling of the oxidizable phenol, di‐tert‐butylhydroquinone (dtBHQ), and direct addition of H2O2 to HaCaT keratinocyte cells. We found that 100 μM to 400 μM H2O2 and 50 μM dtBHQ inhibit both Nrf2 protein synthesis and global protein synthesis. Addition of antioxidants, such as the manganese porphyrin MnTMPyP, rescued both Nrf2‐specific and global protein synthesis. Therefore, antioxidants such as MnTMPyP that mitigate the suppressive effects of ROS on global protein synthesis during Nrf2 activation could enhance the Nrf2/cytoprotective response under conditions of electrophilic and oxidative stress.

Endothelial HSP72 is not reduced in type 2 diabetes nor is it a key determinant of endothelial insulin sensitivity.

Impaired endothelial insulin signaling and consequent blunting of insulin-induced vasodilation is a feature of type 2 diabetes (T2D) that contributes to vascular disease and glycemic dysregulation. However, the molecular mechanisms underlying endothelial insulin resistance remain poorly known. Herein, we tested the hypothesis that endothelial insulin resistance in T2D is attributed to reduced expression of heat shock protein 72 (HSP72). HSP72 is a cytoprotective chaperone protein that can be upregulated with heating and is reported to promote insulin sensitivity in metabolically active tissues, in part via inhibition of JNK activity. Accordingly, we further hypothesized that, in individuals with T2D, 7 days of passive heat treatment via hot water immersion to waist level would improve leg blood flow responses to an oral glucose load (i.e., endogenous insulin stimulation) via induction of endothelial HSP72. In contrast, we found that: 1) endothelial insulin resistance in T2D mice and humans was not associated with reduced HSP72 in aortas and venous endothelial cells, respectively; 2) after passive heat treatment, improved leg blood flow responses to an oral glucose load did not parallel with increased endothelial HSP72; and 3) downregulation of HSP72 (via small-interfering RNA) or upregulation of HSP72 (via heating) in cultured endothelial cells did not impair or enhance insulin signaling, respectively, nor was JNK activity altered. Collectively, these findings do not support the hypothesis that reduced HSP72 is a key driver of endothelial insulin resistance in T2D but provide novel evidence that lower-body heating may be an effective strategy for improving leg blood flow responses to glucose ingestion-induced hyperinsulinemia.

Coronary Sinus Integrated Lactate Decreases After Ischemia and Reperfusion with GGA Induced hsp70

<h3>Purpose: Background</h3> Ischemia reperfusion(IR) increases lactate. No one has examined if cardiac-specific coronary sinus lactate(CSL) can be reduced with prior cytoprotective heat shock protein 70(hsp70i) induction. We previous demonstrated improved IR in vivo with inducted hsp70i. Geranylgeranylacetone(GGA), an hsp70i inducer, has never been administered IV preischemically. Interventions to decrease CSL may improve clinical parameters. <h3>Methods</h3> Rabbit hearts underwent 30 cold cardioplegic ischemia then 60 min reperfusion. One group received IVGGA 24 hours prior(GGA+) and the other vehicle(GGA-). CSlactate was collected prior to ischemia and throughout reperfusion. We aimed to determine IVGGA effects on myocardial hsp70i and lactate. Hsp70 western blot was performed. <h3>Results</h3> Baseline CSlactate was similar between GGA+ and GGA-(Figure 1). Both peaked CSlactate at 1 minute reperfusion. However GGA+ peak was less. At every time point GGA+ was less. GGA+ CSlactate continued to decrease throughout reperfusion however in GGA- CSlactate increased later. Integrated CS lactate area was less for GGA+(Figure 1). <h3>Conclusion</h3> In summary, protective IVGGA resulted in five lactate benefits: lactate was less at 1 minute reperfusion peak,decreased faster in early reperfusion, was reduced at all time points, does not have a second rise and lastly results in overall less integrated lactate production in GGA+. GGA induced hsp70. IVGGA may have clinical applications in endothelial protection in IR and COVID.

Epithelial Heat Shock Proteins Mediate the Protective Effects of Limosilactobacillus reuteri in Dextran Sulfate Sodium-Induced Colitis.

Defects in gut barrier function are implicated in gastrointestinal (GI) disorders like inflammatory bowel disease (IBD), as well as in systemic inflammation. With the increasing incidence of IBD worldwide, more attention should be paid to dietary interventions and therapeutics with the potential to boost the natural defense mechanisms of gut epithelial cells. The current study aimed to investigate the protective effects of Limosilactobacillus reuteri ATCC PTA 4659 in a colitis mouse model and delineate the mechanisms behind it. Wild-type mice were allocated to the control group; or given 3% dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis; or administered L. reuteri for 7 days as pretreatment; or for 14 days starting 7 days before subjecting to the DSS. Peroral treatment with L. reuteri improved colitis severity clinically and morphologically and reduced the colonic levels of Tumor necrosis factor-α (TNF-α) (Tnf), Interleukin 1-β (Il1β), and nterferon-γ (Ifng), the crucial pro-inflammatory cytokines in colitis onset. It also prevented the CD11b+Ly6G+ neutrophil recruitment and the skewed immune responses in mesenteric lymph nodes (MLNs) of CD11b+CD11c+ dendritic cell (DC) expansion and Foxp3+CD4+ T-cell reduction. Using 16S rRNA gene amplicon sequencing and RT-qPCR, we demonstrated a colitis-driven bacterial translocation to MLNs and gut microbiota dysbiosis that were in part counterbalanced by L. reuteri treatment. Moreover, the expression of barrier-preserving tight junction (TJ) proteins and cytoprotective heat shock protein (HSP) 70 and HSP25 was reduced by colitis but boosted by L. reuteri treatment. A shift in expression pattern was also observed with HSP70 in response to the pretreatment and with HSP25 in response to L. reuteri-DSS. In addition, the changes of HSPs were found to be correlated to bacterial load and epithelial cell proliferation. In conclusion, our results demonstrate that the human-derived L. reuteri strain 4659 confers protection in experimental colitis in young mice, while intestinal HSPs may mediate the probiotic effects by providing a supportive protein–protein network for the epithelium in health and colitis.