DNA-controlled protein fluorescence: Design of aptamer-split peptide hetero-modulator for GFP to respond to intracellular ATP levels.

Enabling the precise control of protein functions with artificially programmed reaction patterns is beneficial for investigating biological processes. Although several strategies have been established that employ the programmability of nucleic acid, they have been limited to DNA hybridization without external stimuli or target binding. Here, we report an approach for the DNA-mediated control of the tripartite split-GFP assembly via aptamers with responsiveness to intracellular small molecules as stimuli. We designed a novel structure-switching aptamer-peptide conjugate as a hetero modulator for split GFP in response to ATP. By conjugating two peptides (S10/11) derived from the tripartite split-GFP to ATP aptamer, we achieved GFP reassembly using only ATP as a trigger molecule. The response to ATP at ≥4 mM concentrations indicated that it can be applied to respond to intracellular ATP in live cells. Furthermore, our hetero-modulator exhibited high and long-term stability, with a half-life of approximately four days in a serum stability assay, demonstrating resistance to nuclease degradation. We validated that our aptamer-modulator split GFP was successfully reconstituted in the cell in response to intracellular ATP levels. Our aptamer-modulated split GFP platform can be utilized to monitor a wide range of intracellular metabolites by replacing the aptamer sequence.

Sensitization of GSH synthesis by curcumin curtails acrolein-induced alveolar epithelial apoptosis via Keap1 cysteine conjugation: A randomized controlled trial and experimental animal model of pneumonitis.

INTRODUCTION: Epidemiological studies have reported an association between exposures to ambient air pollution and respiratory diseases, including chronic obstructive pulmonary disease (COPD). Pneumonitis is a critical driving factor of COPD and exposure to air pollutants (e.g., acrolein) is associated with increased incidence of pneumonitis. OBJECTIVES: Currently available anti-inflammatory therapies provide little benefit against respiratory diseases. To this end, we investigated the preventive role of curcumin against air pollutant-associated pneumonitis and its underlying mechanism. METHODS: A total of 40 subjects was recruited from Chengdu, China which is among the top three cities in terms of respiratory mortality related to air pollution. The participants were randomly provided either placebo or curcumin supplements for 2 weeks and blood samples were collected at the baseline and at the end of the intervention to monitor systemic markers. In our follow up mechanistic study, C57BL/6 mice (n = 40) were randomly allocated into 4 groups: Control group (saline + no acrolein), Curcumin only group (curcumin + no acrolein), Acrolein only group (saline + acrolein), and Acrolein + Curcumin group (curcumin + acrolein). Curcumin was orally administered at 100 mg/kg body weight once a day for 10 days, and then the mice were subjected to nasal instillation of acrolein (5 mg/kg body weight). Twelve hours after single acrolein exposure, all mice were euthanized. RESULTS: Curcumin supplementation, with no noticeable adverse responses, reduced circulating pro-inflammatory cytokines in association with clinical pneumonitis as positive predictive while improving those of anti-inflammatory cytokines. In the pre-clinical study, curcumin reduced pneumonitis manifestations by suppression of intrinsic and extrinsic apoptotic signaling, which is attributed to enhanced redox sensing of Nrf2 and thus sensitized synthesis and restoration of GSH, at least in part, through curcumin-Keap1 conjugation. CONCLUSIONS: Our study collectively suggests that curcumin could provide an effective preventive measure against air pollutant-enhanced pneumonitis and thus COPD.

The role of microRNA-33 as a key regulator in hepatic lipogenesis signaling and a potential serological biomarker for NAFLD with excessive dietary fructose consumption in C57BL/6N mice.

Limited studies reported mechanisms by which microRNAs (miRNA) are interlinked in the etiology of fructose-induced non-alcoholic fatty liver disease (NAFLD). Here, we aimed to investigate the significance of miRNAs in fructose-induced NAFLD pathogenesis through unbiased approaches. In experiment I, C57BL/6N mice were fed either water or 34% fructose for six weeks ad libitum. In experiment II, time course effects of fructose intervention were monitored using the same conditions; mice were killed at the baseline, fourth, and sixth weeks. Bioinformatic analyses for hepatic proteomics revealed that SREBP1 is the most significant upstream regulator influenced by fructose; miR-33-5p (miR-33) was identified as the key miRNA responsible for SREBP1 regulation upon fructose intake, which was validated by in vitro transfection assay. In experiment II, we confirmed that the longer mice consumed fructose, the more severe liver injury markers (e.g., serum AST) appeared. Moreover, hepatic Srebp1 mRNA expression was increased depending upon the duration of fructose consumption. Hepatic miR-33 was time-dependently decreased by fructose while serum miR-33 expression was increased; these observations indicated that miR-33 from the liver might be released upon cell damage. Finally we observed that fructose-induced ferroptosis might be a cause of liver toxicity, resulting from oxidative damage. Collectively, our findings suggest that fructose-induced oxidative damage induces ferroptosis, and miR-33 could be used as a serological biomarker of fructose-induced NAFLD.

In Vitro and In Vivo Inhibitory Effect of Citrus Junos Tanaka Peel Extract against Oxidative Stress-Induced Apoptotic Death of Lung Cells.

Various stresses derived from both internal and external oxidative environments lead to the excessive production of reactive oxygen species (ROS) causing progressive intracellular oxidative damage and ultimately cell death. The objective of this study was to evaluate the protective effects of Citrus junos Tanaka peel extract (CE) against oxidative-stress induced the apoptosis of lung cells and the associated mechanisms of action using in vitro and in vivo models. The protective effect of CE was evaluated in vitro in NCI-H460 human lung cells exposed to pro-oxidant H2O2. The preventive effect of CE (200 mg/kg/day, 10 days) against pulmonary injuries following acrolein inhalation (10 ppm for 12 h) was investigated using an in vivo mouse model. Herein, we demonstrated the inhibitory effect of CE against the oxidative stress-induced apoptosis of lung cells under a highly oxidative environment. The function of CE is linked with its ability to suppress ROS-dependent, p53-mediated apoptotic signaling. Furthermore, we evaluated the protective role of CE against apoptotic pulmonary injuries associated with the inhalation of acrolein, a ubiquitous and highly oxidizing environmental respiratory pollutant, through the attenuation of oxidative stress. The results indicated that CE exhibits a protective effect against the oxidative stress-induced apoptosis of lung cells in both in vitro and in vivo models.

Protective effects of p-coumaric acid against acetaminophen-induced hepatotoxicity in mice.

Acetaminophen (N-acetyl-p-aminophenol, AAP) is an effective analgesic and antipyretic drug with minimal toxicity when used at therapeutic doses. However, AAP overdose is the most common cause of drug-induced acute liver failure and one of the main causes of morbidity and mortality. p-Coumaric acid (PCA) is the most abundant isomer of hydroxycinnamic acid in nature, and it can be widely found in fruits, vegetables, and plants products. PCA has strong antioxidant activity and exhibits protective effects in numerous disease models associated with reactive oxygen species (ROS) generation. In this study, we investigated the protective effects of PCA on AAP-induced hepatotoxicity and the underlying mechanisms using an in vivo model. We found that PCA ameliorates AAP-induced hepatotoxicity as well as the reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity. Furthermore, we observed that PCA suppressed hepatic apoptosis via ROS-mediated DNA damage responses and inflammation by modulating the mitogen-activated protein kinase (MAPK) signaling axis in an ROS-dependent manner. These findings indicate that the administration of PCA protects against AAP-induced hepatotoxicity, suggesting it could be a novel therapeutic strategy for AAP-induced liver injury.

Idh2 deficiency accelerates renal dysfunction in aged mice.

The free radical or oxidative stress theory of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species (ROS) that are produced as by-products of normal metabolic processes in mitochondria. The oxidative stress may arise as a result of either increased ROS production or decreased ability to detoxify ROS. The availability of the mitochondrial NADPH pool is critical for the maintenance of the mitochondrial antioxidant system. The major enzyme responsible for generating mitochondrial NADPH is mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2). Depletion of IDH2 in mice (idh2-/-) shortens life span and accelerates the degeneration of multiple age-sensitive traits, such as hair grayness, skin pathology, and eye pathology. Among the various internal organs tested in this study, IDH2 depletion-induced acceleration of senescence was uniquely observed in the kidney. Renal function and structure were greatly deteriorated in 24-month-old idh2-/- mice compared with wild-type. In addition, disruption of redox status, which promotes oxidative damage and apoptosis, was more pronounced in idh2-/- mice. These data support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice, and thus support the oxidative stress theory of aging.

Increased susceptibility of IDH2-deficient mice to dextran sodium sulfate-induced colitis.

Inflammatory bowel disease (IBD) is a group of chronic, relapsing, immunological, inflammatory disorders of the gastrointestinal tract including ulcerative colitis (UC) and Crohn's disease (CD). It has been reported that UC, which is studied using a dextran sodium sulfate (DSS)-induced colitis model, is associated with the production of reactive oxygen species (ROS) and the apoptosis of intestine epithelial cells (IEC). Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) has been reported as an essential enzyme in the mitochondrial antioxidant system via generation of NADPH. Therefore, we evaluated the role of IDH2 in DSS-induced colitis using IDH2-deficient (IDH2-/-) mice. We observed that DSS-induced colitis in IDH2-/- mice was more severe than that in wild-type IDH2+/+ mice. Our results also suggest that IDH2 deficiency exacerbates PUMA-mediated apoptosis, resulting from NF-κB activation regulated by histone deacetylase (HDAC) activity. In addition, DSS-induced colitis is ameliorated by an antioxidant N-acetylcysteine (NAC) through attenuation of oxidative stress, resulting from deficiency of the IDH2 gene. In conclusion, deficiency of IDH2 leads to increased mitochondrial ROS levels, which inhibits HDAC activity, and the activation of NF-κB via acetylation is enhanced by attenuated HDAC activity, which causes PUMA-mediated apoptosis of IEC in DSS-induced colitis. The present study supported the rationale for targeting IDH2 as an important cancer chemoprevention strategy, particularly in the prevention of colorectal cancer.

Amelioration of late-onset hepatic steatosis in IDH2-deficient mice.

Nonalcoholic fatty liver disease (NAFLD) has a high prevalence in the general population and can evolve into nonalcoholic steatohepatosis (NASH), cirrhosis, and complications such as liver failure and hepatocellular carcinoma. Recently, we reported that mitochondrial NADP+-dependent isocitrate dehydrogenase, encoded by the IDH2, plays an important role in the regulation of redox balance and oxidative stress levels, which are tightly associated with intermediary metabolism and energy production. In the present study, we showed that in mice targeted disruption of IDH2 attenuates age-associated hepatic steatosis by the activation of p38/cJun NH2-terminal kinase (JNK) and p53, presumably induced by the elevation of mitochondrial reactive oxygen species (ROS), which in turn resulted in the suppression of hepatic lipogenesis and inflammation via the upregulation of fibroblast growth factor 21 (FGF21) and the inhibition of NFκB signaling pathways. Our finding uncovers a new mechanism involved in hepatocellular steatosis and IDH2 may be a valuable therapeutic target for the management of NAFLD.

Oxalomalate reduces expression and secretion of vascular endothelial growth factor in the retinal pigment epithelium and inhibits angiogenesis: Implications for age-related macular degeneration.

Clinical and experimental observations indicate a critical role for vascular endothelial growth factor (VEGF), secreted by the retinal pigment epithelium (RPE), in pathological angiogenesis and the development of choroidal neovascularization (CNV) in age-related macular degeneration (AMD). RPE-mediated VEGF expression, leading to angiogenesis, is a major signaling mechanism underlying ocular neovascular disease. Inhibiting this signaling pathway with a therapeutic molecule is a promising anti-angiogenic strategy to treat this disease with potentially fewer side effects. Oxalomalate (OMA) is a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase (IDH), which plays an important role in cellular signaling pathways regulated by reactive oxygen species (ROS). Here, we have investigated the inhibitory effect of OMA on the expression of VEGF, and the associated underlying mechanism of action, using in vitro and in vivo RPE cell models of AMD. We found that OMA reduced the expression and secretion of VEGF in RPE cells, and consequently inhibited CNV formation. This function of OMA was linked to its capacity to activate the pVHL-mediated HIF-1α degradation in these cells, partly via a ROS-dependent ATM signaling axis, through inhibition of IDH enzymes. These findings reveal a novel role for OMA in inhibiting RPE-derived VEGF expression and angiogenesis, and suggest unique therapeutic strategies for treating pathological angiogenesis and AMD development.

IDH2 deficiency promotes mitochondrial dysfunction and dopaminergic neurotoxicity: implications for Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and its pathogenesis is under intense investigation. Substantial evidence indicates that mitochondrial dysfunction and oxidative stress play central roles in the pathophysiology of PD, through activation of mitochondria-dependent apoptotic molecular pathways. Several mitochondrial internal regulating factors act to maintain mitochondrial function. However, the mechanism by which these internal regulating factors contribute to mitochondrial dysfunction in PD remains elusive. One of these factors, mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2), has been implicated in the regulation of mitochondrial redox balance and reduction of oxidative stress-induced cell injury. Here we report that IDH2 regulates mitochondrial dysfunction and cell death in MPP(+)/MPTP-induced DA neuronal cells, and in a mouse model of PD. Down-regulation of IDH2 increased DA neuron sensitivity to MPP(+); lowered IDH2 levels facilitated induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Deficient IDH2 also promoted loss of DA SNpc neurons in an MPTP mouse model of PD. Interestingly, Mito-TEMPO, a mitochondrial ROS-specific scavenger, protected degeneration of SNpc DA neurons in the MPTP model of PD. These findings demonstrate that IDH2 contributes to degeneration of the DA neuron in the neurotoxin model of PD and establish IDH2 as a molecular target of potential therapeutic significance for this disabling neurological illness.