Chronological and reproductive aging-associated changes in resistance to oxidative stress in post-reproductive female mice.

Effort toward reproduction is often thought to negatively influence health and survival. Reproduction has been shown to influence metabolism, but the pathways and mechanisms have yet to be thoroughly elucidated. In the current experiments, our aim was to dissect the role of young and old ovarian tissues in the response to oxidative stress, through changes in liver oxidative stress response proteins. Liver proteins were analyzed in control mice at 4, 13, and 27 months of age and compared to 23-month-old mice which received young ovarian tissue transplants (intact or follicle-depleted) at 13 months of age. In control mice, of the 29 oxidative stress response proteins measured, 31% of the proteins decreased, 52% increased, and 17% were unchanged from 13 to 27 months. The greatest changes were seen during the period of reproductive failure, from 4 to 13 months of age. In transplanted mice, far more proteins were decreased from 13 to 23 months (93% in follicle-containing young ovary recipients; 62% in follicle-depleted young ovary recipients). Neither transplant group reflected changes seen in control mice between 13 and 27 months. Estradiol levels in transplant recipient mice were not increased compared with age-matched control mice. The current results suggest the presence of a germ cell- and estradiol-independent ovarian influence on aging-associated changes in the response to oxidative stress, which is manifest differently in reproductive-aged adults and post-reproductive-aged mice. The results presented here separate chronological and ovarian aging and the influence of estradiol in the response to aging-associated oxidative stress and support a novel, estradiol-independent role for the ovary in female health and survival.

Increased expression of BRD4 isoforms long (BRD4-L) and short (BRD4-S) promotes chemotherapy resistance in high-grade serous ovarian carcinoma.

Chemoresistance in ovarian carcinoma is a puzzling issue that urges understanding of strategies used by cancer cells to survive DNA damage and to escape cell death. Expanding efforts to understand mechanisms driving chemoresistance and to develop alternative therapies targeting chemoresistant tumors are critical. Amplification of BRD4 is frequently associated with chemoresistant ovarian carcinoma, but little is known about the biological effects of the overexpression of BRD4 isoforms in this malignancy. Here, we described the consequences of BRD4-L and BRD4-S overexpression in ovarian carcinoma shedding a light on a complex regulation of BRD4 isoforms. We demonstrated that the BRD4-L transcript expression is required to generate both isoforms, BRD4-L and BRD4-S. We showed that the BRD4-S mRNA expression positively correlated with BRD4-S protein levels, while BRD4-L isoform showed negative correlation between mRNA and protein levels. Moreover, we demonstrated that an overexpression of BRD4 isoforms is associated with chemoresistance in ovarian cancer.

Impaired proteostatic mechanisms other than decreased protein synthesis limit old skeletal muscle recovery after disuse atrophy.

BACKGROUND: Skeletal muscle mass and strength diminish during periods of disuse but recover upon return to weight bearing in healthy adults but are incomplete in old muscle. Efforts to improve muscle recovery in older individuals commonly aim at increasing myofibrillar protein synthesis via mammalian target of rapamycin (mTOR) stimulation despite evidence demonstrating that old muscle has chronically elevated levels of mammalian target of rapamycin complex 1 (mTORC1) activity. We hypothesized that protein synthesis is higher in old muscle than adult muscle, which contributes to a proteostatic stress that impairs recovery. METHODS: We unloaded hindlimbs of adult (10-month) and old (28-month) F344BN rats for 14 days to induce atrophy, followed by reloading up to 60 days with deuterium oxide (D2 O) labelling to study muscle regrowth and proteostasis. RESULTS: We found that old muscle has limited recovery of muscle mass during reloading despite having higher translational capacity and myofibrillar protein synthesis (0.029 k/day ± 0.002 vs. 0.039 k/day ± 0.002, P < 0.0001) than adult muscle. We showed that collagen protein synthesis was not different (0.005 k (1/day) ± 0.0005 vs. 0.004 k (1/day) ± 0.0005, P = 0.15) in old compared to adult, but old muscle had higher collagen concentration (4.5 µg/mg ± 1.2 vs. 9.8 µg/mg ± 0.96, P < 0.01), implying that collagen breakdown was slower in old muscle than adult muscle. This finding was supported by old muscle having more insoluble collagen (4.0 ± 1.1 vs. 9.2 ± 0.9, P < 0.01) and an accumulation of advanced glycation end products (1.0 ± 0.06 vs. 1.5 ± 0.08, P < 0.001) than adult muscle during reloading. Limited recovery of muscle mass during reloading is in part due to higher protein degradation (0.017 1/t ± 0.002 vs. 0.028 1/t ± 0.004, P < 0.05) and/or compromised proteostasis as evidenced by accumulation of ubiquitinated insoluble proteins (1.02 ± 0.06 vs. 1.22 ± 0.06, P < 0.05). Last, we showed that synthesis of individual proteins related to protein folding/refolding, protein degradation and neural-related biological processes was higher in old muscle during reloading than adult muscle. CONCLUSIONS: Our data suggest that the failure of old muscle to recover after disuse is not due to limitations in the ability to synthesize myofibrillar proteins but because of other impaired proteostatic mechanisms (e.g., protein folding and degradation). These data provide novel information on individual proteins that accumulate in protein aggregates after disuse and certain biological processes such as protein folding and degradation that likely play a role in impaired recovery. Therefore, interventions to enhance regrowth of old muscle after disuse should be directed towards the identified impaired proteostatic mechanisms and not aimed at increasing protein synthesis.

Metabolic derangement in polycystic kidney disease mouse models is ameliorated by mitochondrial-targeted antioxidants.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressively enlarging cysts. Here we elucidate the interplay between oxidative stress, mitochondrial dysfunction, and metabolic derangement using two mouse models of PKD1 mutation, PKD1RC/null and PKD1RC/RC. Mouse kidneys with PKD1 mutation have decreased mitochondrial complexes activity. Targeted proteomics analysis shows a significant decrease in proteins involved in the TCA cycle, fatty acid oxidation (FAO), respiratory complexes, and endogenous antioxidants. Overexpressing mitochondrial-targeted catalase (mCAT) using adeno-associated virus reduces mitochondrial ROS, oxidative damage, ameliorates the progression of PKD and partially restores expression of proteins involved in FAO and the TCA cycle. In human ADPKD cells, inducing mitochondrial ROS increased ERK1/2 phosphorylation and decreased AMPK phosphorylation, whereas the converse was observed with increased scavenging of ROS in the mitochondria. Treatment with the mitochondrial protective peptide, SS31, recapitulates the beneficial effects of mCAT, supporting its potential application as a novel therapeutic for ADPKD.

Myocardial Hypertrophy and Compensatory Increase in Systolic Function in a Mouse Model of Oxidative Stress.

Free radicals, or reactive oxygen species, have been implicated as one of the primary causes of myocardial pathologies elicited by chronic diseases and age. The imbalance between pro-oxidants and antioxidants, termed "oxidative stress", involves several pathological changes in mouse hearts, including hypertrophy and cardiac dysfunction. However, the molecular mechanisms and adaptations of the hearts in mice lacking cytoplasmic superoxide dismutase (Sod1KO) have not been investigated. We used echocardiography to characterize cardiac function and morphology in vivo. Protein expression and enzyme activity of Sod1KO were confirmed by targeted mass spectrometry and activity gel. The heart weights of the Sod1KO mice were significantly increased compared with their wildtype peers. The increase in heart weights was accompanied by concentric hypertrophy, posterior wall thickness of the left ventricles (LV), and reduced LV volume. Activated downstream pathways in Sod1KO hearts included serine-threonine kinase and ribosomal protein synthesis. Notably, the reduction in LV volume was compensated by enhanced systolic function, measured by increased ejection fraction and fractional shortening. A regulatory sarcomeric protein, troponin I, was hyper-phosphorylated in Sod1KO, while the vinculin protein was upregulated. In summary, mice lacking cytoplasmic superoxide dismutase were associated with an increase in heart weights and concentric hypertrophy, exhibiting a pathological adaptation of the hearts to oxidative stress.

Role of necroptosis in chronic hepatic inflammation and fibrosis in a mouse model of increased oxidative stress.

Mice deficient in the antioxidant enzyme Cu/Zn-superoxide dismutase (Sod1-/- or Sod1KO mice) have increased oxidative stress, show accelerated aging and develop spontaneous hepatocellular carcinoma (HCC) with age. Similar to humans, HCC development in Sod1KO mice progresses from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH) with fibrosis, which eventually progresses to HCC. Oxidative stress plays a role in NAFLD to NASH progression, and liver inflammation is the main mechanism that drives the disease progression from NASH to fibrosis. Because necroptosis is a major source of inflammation, we tested the hypothesis that increased necroptosis in the liver plays a role in increased inflammation and fibrosis in Sod1KO mice. Phosphorylation of MLKL (P-MLKL), a well-accepted marker of necroptosis, and expression of MLKL protein were significantly increased in the livers of Sod1KO mice compared to wild type (WT) mice indicating increased necroptosis. Similarly, phosphorylation of RIPK3 and RIPK3 protein levels were also significantly increased. Markers of pro-inflammatory M1 macrophages, NLRP3 inflammasome, and transcript levels of pro-inflammatory cytokines and chemokines, e.g., TNFα, IL-6, IL-1ß, and Ccl2 that are associated with human NASH, were significantly increased. Expression of antioxidant enzymes and heat shock proteins, and markers of fibrosis and oncogenic transcription factor STAT3 were also upregulated and autophagy was downregulated in the livers of Sod1KO mice. Short term treatment of Sod1KO mice with necrostatin-1s (Nec-1s), a necroptosis inhibitor, reversed these conditions. Our data show for the first time that necroptosis-mediated inflammation contributes to fibrosis in a mouse model of increased oxidative stress and accelerated aging, that also exhibits progressive HCC development.

Cancer cachexia in a mouse model of oxidative stress.

BACKGROUND: Cancer is associated with muscle atrophy (cancer cachexia) that is linked to up to 40% of cancer-related deaths. Oxidative stress is a critical player in the induction and progression of age-related loss of muscle mass and weakness (sarcopenia); however, the role of oxidative stress in cancer cachexia has not been defined. The purpose of this study was to examine if elevated oxidative stress exacerbates cancer cachexia. METHODS: Cu/Zn superoxide dismutase knockout (Sod1KO) mice were used as an established mouse model of elevated oxidative stress. Cancer cachexia was induced by injection of one million Lewis lung carcinoma (LLC) cells or phosphate-buffered saline (saline) into the hind flank of female wild-type mice or Sod1KO mice at approximately 4 months of age. The tumour developed for 3 weeks. Muscle mass, contractile function, neuromuscular junction (NMJ) fragmentation, metabolic proteins, mitochondrial function, and motor neuron function were measured in wild-type and Sod1KO saline and tumour-bearing mice. Data were analysed by two-way ANOVA with Tukey-Kramer post hoc test when significant F ratios were determined and α was set at 0.05. Unless otherwise noted, results in abstract are mean ±SEM. RESULTS: Muscle mass and cross-sectional area were significantly reduced, in tumour-bearing mice. Metabolic enzymes were dysregulated in Sod1KO mice and cancer exacerbated this phenotype. NMJ fragmentation was exacerbated in tumour-bearing Sod1KO mice. Myofibrillar protein degradation increased in tumour-bearing wild-type mice (wild-type saline, 0.00847 ± 0.00205; wildtype LLC, 0.0211 ± 0.00184) and tumour-bearing Sod1KO mice (Sod1KO saline, 0.0180 ± 0.00118; Sod1KO LLC, 0.0490 ± 0.00132). Muscle mitochondrial oxygen consumption was reduced in tumour-bearing mice compared with saline-injected wild-type mice. Mitochondrial protein degradation increased in tumour-bearing wild-type mice (wild-type saline, 0.0204 ± 0.00159; wild-type LLC, 0.167 ± 0.00157) and tumour-bearing Sod1KO mice (Sod1KO saline, 0.0231 ± 0.00108; Sod1 KO LLC, 0.0645 ± 0.000631). Sciatic nerve conduction velocity was decreased in tumour-bearing wild-type mice (wild-type saline, 38.2 ± 0.861; wild-type LLC, 28.8 ± 0.772). Three out of eleven of the tumour-bearing Sod1KO mice did not survive the 3-week period following tumour implantation. CONCLUSIONS: Oxidative stress does not exacerbate cancer-induced muscle loss; however, cancer cachexia may accelerate NMJ disruption.

Short-term Calorie Restriction and 17α-Estradiol Administration Elicit Divergent Effects on Proteostatic Processes and Protein Content in Metabolically Active Tissues.

17α-Estradiol (17α-E2) is a "non-feminizing" estrogen that extends life span in male, but not female, mice. We recently reported that 17α-E2 had robust beneficial effects on metabolic and inflammatory parameters in aged male mice. However, it remains unclear if 17α-E2 also delays other "hallmarks" of aging, particularly maintaining proteostasis. Here, we used isotope labeling methods in older mice to examine proteostatic mechanisms. We compared weight-matched mild calorie restricted (CR) and 17α-E2 treated male mice with the hypothesis that 17α-E2 would increase protein synthesis for somatic maintenance. 17α-E2 had no effect on protein synthesis or DNA synthesis in multiple tissues, including white adipose tissue. Conversely, mild short-term CR decreased DNA synthesis and increased the protein to DNA synthesis ratio in multiple tissues. Examination of individual protein synthesis and content did not differentiate treatments, although it provided insight into the regulation of protein content between tissues. Contrary to our hypothesis, we did not see the predicted differences in protein to DNA synthesis following 17α-E2 treatment. However, mild short-term CR elicited differences consistent with both lifelong CR and other treatments that curtail aging processes. These data indicated that despite similar maintenance of body mass, 17α-E2 and CR treatments elicit distinctly different proteostatic outcomes.

Disparate Central and Peripheral Effects of Circulating IGF-1 Deficiency on Tissue Mitochondrial Function.

Age-related decline in circulating levels of insulin-like growth factor (IGF)-1 is associated with reduced cognitive function, neuronal aging, and neurodegeneration. Decreased mitochondrial function along with increased reactive oxygen species (ROS) and accumulation of damaged macromolecules are hallmarks of cellular aging. Based on numerous studies indicating pleiotropic effects of IGF-1 during aging, we compared the central and peripheral effects of circulating IGF-1 deficiency on tissue mitochondrial function using an inducible liver IGF-1 knockout (LID). Circulating levels of IGF-1 (~ 75%) were depleted in adult male Igf1f/f mice via AAV-mediated knockdown of hepatic IGF-1 at 5 months of age. Cognitive function was evaluated at 18 months using the radial arm water maze and glucose and insulin tolerance assessed. Mitochondrial function was analyzed in hippocampus, muscle, and visceral fat tissues using high-resolution respirometry O2K as well as redox status and oxidative stress in the cortex. Peripherally, IGF-1 deficiency did not significantly impact muscle mass or mitochondrial function. Aged LID mice were insulin resistant and exhibited ~ 60% less adipose tissue but increased fat mitochondrial respiration (20%). The effects on fat metabolism were attributed to increases in growth hormone. Centrally, IGF-1 deficiency impaired hippocampal-dependent spatial acquisition as well as reversal learning in male mice. Hippocampal mitochondrial OXPHOS coupling efficiency and cortex ATP levels (~ 50%) were decreased and hippocampal oxidative stress (protein carbonylation and F2-isoprostanes) was increased. These data suggest that IGF-1 is critical for regulating mitochondrial function, redox status, and spatial learning in the central nervous system but has limited impact on peripheral (liver and muscle) metabolism with age. Therefore, IGF-1 deficiency with age may increase sensitivity to damage in the brain and propensity for cognitive deficits. Targeting mitochondrial function in the brain may be an avenue for therapy of age-related impairment of cognitive function. Regulation of mitochondrial function and redox status by IGF-1 is essential to maintain brain function and coordinate hippocampal-dependent spatial learning. While a decline in IGF-1 in the periphery may be beneficial to avert cancer progression, diminished central IGF-1 signaling may mediate, in part, age-related cognitive dysfunction and cognitive pathologies potentially by decreasing mitochondrial function.

Mitochondrial oxidative stress impairs contractile function but paradoxically increases muscle mass via fibre branching.

BACKGROUND: Excess reactive oxygen species (ROS) and muscle weakness occur in parallel in multiple pathological conditions. However, the causative role of skeletal muscle mitochondrial ROS (mtROS) on neuromuscular junction (NMJ) morphology and function and muscle weakness has not been directly investigated. METHODS: We generated mice lacking skeletal muscle-specific manganese-superoxide dismutase (mSod2KO) to increase mtROS using a cre-Lox approach driven by human skeletal actin. We determined primary functional parameters of skeletal muscle mitochondrial function (respiration, ROS, and calcium retention capacity) using permeabilized muscle fibres and isolated muscle mitochondria. We assessed contractile properties of isolated skeletal muscle using in situ and in vitro preparations and whole lumbrical muscles to elucidate the mechanisms of contractile dysfunction. RESULTS: The mSod2KO mice, contrary to our prediction, exhibit a 10-15% increase in muscle mass associated with an ~50% increase in central nuclei and ~35% increase in branched fibres (P < 0.05). Despite the increase in muscle mass of gastrocnemius and quadriceps, in situ sciatic nerve-stimulated isometric maximum-specific force (N/cm2 ), force per cross-sectional area, is impaired by ~60% and associated with increased NMJ fragmentation and size by ~40% (P < 0.05). Intrinsic alterations of components of the contractile machinery show elevated markers of oxidative stress, for example, lipid peroxidation is increased by ~100%, oxidized glutathione is elevated by ~50%, and oxidative modifications of myofibrillar proteins are increased by ~30% (P < 0.05). We also find an approximate 20% decrease in the intracellular calcium transient that is associated with specific force deficit. Excess superoxide generation from the mitochondrial complexes causes a deficiency of succinate dehydrogenase and reduced complex-II-mediated respiration and adenosine triphosphate generation rates leading to severe exercise intolerance (~10 min vs. ~2 h in wild type, P < 0.05). CONCLUSIONS: Increased skeletal muscle mtROS is sufficient to elicit NMJ disruption and contractile abnormalities, but not muscle atrophy, suggesting new roles for mitochondrial oxidative stress in maintenance of muscle mass through increased fibre branching.

Effect of biomechanical stress on endogenous antioxidant networks in bovine articular cartilage.

Mechanosensitve pathways in chondrocytes are essential for maintaining articular cartilage homeostasis. Traumatic loading increases cartilage oxidation and causes cell death and osteoarthritis. However, sub-lethal doses of the pro-oxidant molecule tert-Butyl hydroperoxide (tBHP) protects against loading-induced chondrocyte death. We hypothesized that compressive cyclic loading at moderate strains (<20%) causes sub-lethal cartilage oxidation that induces an adaptive increase in the endogenous antioxidant defense network. We tested this hypothesis by subjecting healthy bovine articular cartilage explants to in vitro static or cyclic (1 Hz) compressive loading at 50 kPa (15% strain, "physiologic") versus 300 kPa (40% strain, "hyper-physiologic") for 12 h per day for 2 days. We also treated unloaded explants with 100 µM tBHP for 12 h per day for 2 days to differentiate between biomechanical and chemical pro-oxidant stimulation. All loading conditions induced glutathione oxidation relative to unloaded controls, but only the 50 kPa cyclic loading condition increased total glutathione content (twofold). This increase was associated with a greater expression of glutamate-cysteine ligase, the rate-limiting step in glutathione synthesis, compared to 300 kPa cyclic loading. 50 kPa cyclic loading also increased the expression of superoxide dismutase-1 and peroxiredoxin-3. Like 50 kPa loading, tBHP treatment also increased total glutathione content. However, tBHP treatment and 50 kPa cyclic loading differed in their effect on the expression of genes regulating antioxidant defense and cartilage matrix synthesis and degradation. These findings suggest that glutathione metabolism is a mechanosensitive antioxidant defense pathway in chondrocytes and that intermittent pro-oxidant treatment alone is insufficient to account for all changes in mediators of cartilage homeostasis associated with cyclic loading. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:760-769, 2018.

A fish oil diet induces mitochondrial uncoupling and mitochondrial unfolded protein response in epididymal white adipose tissue of mice.

White adipose tissue (WAT) mitochondrial dysfunction is linked to the pathogenesis of obesity driven insulin resistance. Dietary conditions that alter fat mass are known to affect white adipocyte mitochondrial function, however, the impact of high calorie diets on white adipocyte mitochondria is not fully understood. The aim of this study is to assess the effect of a diet rich in saturated or polyunsaturated fat on mitochondrial unfolded protein response (UPRmt), a retrograde signaling response that maintains mitochondrial homeostasis, in epididymal WAT (eWAT). Mice were fed a low fat diet (LFD), saturated fat diet (SFD) or fish oil (unsaturated fat diet, UFD) and assessed changes in eWAT mitochondria. Compared to mice fed a LFD, SFD-fed mice have reduced mitochondrial biogenesis markers, mitochondrial fatty acid oxidation enzymes and TCA cycle enzymes, suggesting an impaired mitochondrial function that could contribute to increased fat mass. In contrast, isocaloric UFD-fed mice have increased expression of mitochondrial uncoupling protein 1 (UCP1) and peroxisomal fatty acid oxidation enzymes suggesting that elevated mitochondrial uncoupling and peroxisomal fatty acid oxidation could contribute to the reduction in fat mass. Interestingly, expression of UPRmt-associated proteins caseinolytic peptidase (ClpP) and heat shock protein 60 (Hsp60) are induced by UFD, whereas SFD reduced the expression of ClpP. Based on our data, we propose that induction of UPRmt helps to preserve a functional mitochondria and efficient utilization of fat by UFD whereas a dampened UPRmt response might impair mitochondrial function and promote fat accumulation by SFD. Thus, our findings suggest a potential role of UPRmt in mediating the beneficial effects of fish oil.

Catalase-dependent H2O2 consumption by cardiac mitochondria and redox-mediated loss in insulin signaling.

We have recently demonstrated that catalase content in mouse cardiac mitochondria is selectively elevated in response to high dietary fat, a nutritional state associated with oxidative stress and loss in insulin signaling. Catalase and various isoforms of glutathione peroxidase and peroxiredoxin each catalyze the consumption of H2O2 Catalase, located primarily within peroxisomes and to a lesser extent mitochondria, has a low binding affinity for H2O2 relative to glutathione peroxidase and peroxiredoxin. As such, the contribution of catalase to mitochondrial H2O2 consumption is not well understood. In the current study, using highly purified cardiac mitochondria challenged with micromolar concentrations of H2O2, we found that catalase contributes significantly to mitochondrial H2O2 consumption. In addition, catalase is solely responsible for removal of H2O2 in nonrespiring or structurally disrupted mitochondria. Finally, in mice fed a high-fat diet, mitochondrial-derived H2O2 is responsible for diminished insulin signaling in the heart as evidenced by reduced insulin-stimulated Akt phosphorylation. While elevated mitochondrial catalase content (∼50%) enhanced the capacity of mitochondria to consume H2O2 in response to high dietary fat, the selective increase in catalase did not prevent H2O2-induced loss in cardiac insulin signaling. Taken together, our results indicate that mitochondrial catalase likely functions to preclude the formation of high levels of H2O2 without perturbing redox-dependent signaling.

Conformation-Dependent Human p52Shc Phosphorylation by Human c-Src.

Phosphorylation of the human p52Shc adaptor protein is a key determinant in modulating signaling complex assembly in response to tyrosine kinase signaling cascade activation. The underlying mechanisms that govern p52Shc phosphorylation status are unknown. In this study, p52Shc phosphorylation by human c-Src was investigated using purified proteins to define mechanisms that affect the p52Shc phosphorylation state. We conducted biophysical characterizations of both human p52Shc and human c-Src in solution as well as membrane-mimetic environments using the acidic lipid phosphatidylinositol 4-phosphate or a novel amphipathic detergent (2,2-dihexylpropane-1,3-bis-ß-D-glucopyranoside). We then identified p52Shc phosphorylation sites under various solution conditions, and the amount of phosphorylation at each identified site was quantified using mass spectrometry. These data demonstrate that the p52Shc phosphorylation level is altered by the solution environment without affecting the fraction of active c-Src. Mass spectrometry analysis of phosphorylated p52Shc implies functional linkage among phosphorylation sites. This linkage may drive preferential coupling to protein binding partners during signaling complex formation, such as during initial binding interactions with the Grb2 adaptor protein leading to activation of the Ras/MAPK signaling cascade. Remarkably, tyrosine residues involved in Grb2 binding were heavily phosphorylated in a membrane-mimetic environment. The increased phosphorylation level in Grb2 binding residues was also correlated with a decrease in the thermal stability of purified human p52Shc. A schematic for the phosphorylation-dependent interaction between p52Shc and Grb2 is proposed. The results of this study suggest another possible therapeutic strategy for altering protein phosphorylation to regulate signaling cascade activation.

The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response.

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

An enhancer element harboring variants associated with systemic lupus erythematosus engages the TNFAIP3 promoter to influence A20 expression.

Functional characterization of causal variants present on risk haplotypes identified through genome-wide association studies (GWAS) is a primary objective of human genetics. In this report, we evaluate the function of a pair of tandem polymorphic dinucleotides, 42 kb downstream of the promoter of TNFAIP3, (rs148314165, rs200820567, collectively referred to as TT>A) recently nominated as causal variants responsible for genetic association of systemic lupus erythematosus (SLE) with tumor necrosis factor alpha inducible protein 3 (TNFAIP3). TNFAIP3 encodes the ubiquitin-editing enzyme, A20, a key negative regulator of NF-κB signaling. A20 expression is reduced in subjects carrying the TT>A risk alleles; however, the underlying functional mechanism by which this occurs is unclear. We used a combination of electrophoretic mobility shift assays (EMSA), mass spectrometry (MS), reporter assays, chromatin immunoprecipitation-PCR (ChIP-PCR) and chromosome conformation capture (3C) EBV transformed lymphoblastoid cell lines (LCL) from individuals carrying risk and non-risk TNFAIP3 haplotypes to characterize the effect of TT>A on A20 expression. Our results demonstrate that the TT>A variants reside in an enhancer element that binds NF-κB and SATB1 enabling physical interaction of the enhancer with the TNFAIP3 promoter through long-range DNA looping. Impaired binding of NF-κB to the TT>A risk alleles or knockdown of SATB1 expression by shRNA, inhibits the looping interaction resulting in reduced A20 expression. Together, these data reveal a novel mechanism of TNFAIP3 transcriptional regulation and establish the functional basis by which the TT>A risk variants attenuate A20 expression through inefficient delivery of NF-κB to the TNFAIP3 promoter. These results provide critical functional evidence supporting a direct causal role for TT>A in the genetic predisposition to SLE.

Glutathionylation of α-ketoglutarate dehydrogenase: the chemical nature and relative susceptibility of the cofactor lipoic acid to modification.

α-Ketoglutarate dehydrogenase (KGDH) is reversibly inhibited when rat heart mitochondria are exposed to hydrogen peroxide (H2O2). H2O2-induced inhibition occurs through the formation of a mixed disulfide between a protein sulfhydryl and glutathione. Upon consumption of H2O2, glutaredoxin can rapidly remove glutathione, resulting in regeneration of enzyme activity. KGDH is a key regulatory site within the Krebs cycle. Glutathionylation of the enzyme may therefore represent an important means to control mitochondrial function in response to oxidative stress. We have previously provided indirect evidence that glutathionylation occurs on lipoic acid, a cofactor covalently bound to the E2 subunit of KGDH. However, lipoic acid contains two vicinal sulfhydryls and rapid disulfide exchange might be predicted to preclude stable glutathionylation. The current study sought conclusive identification of the site and chemistry of KGDH glutathionylation and factors that control the degree and rate of enzyme inhibition. We present evidence that, upon reaction of free lipoic acid with oxidized glutathione in solution, disulfide exchange occurs rapidly, producing oxidized lipoic acid and reduced glutathione. This prevents the stable formation of a glutathione-lipoic acid adduct. Nevertheless, 1:1 lipoic acid-glutathione adducts are formed on KGDH because the second sulfhydryl on lipoic acid is unable to participate in disulfide exchange in the enzyme's native conformation. The maximum degree of KGDH inhibition that can be achieved by treatment of mitochondria with H2O2 is 50%. Results indicate that this is not due to glutathionylation of a subpopulation of the enzyme but, rather, the unique susceptibility of lipoic acid on a subset of E2 subunits within each enzyme complex. Calcium enhances the rate of glutathionylation by increasing the half-life of reduced lipoic acid during enzyme catalysis. This does not, however, alter the maximal level of inhibition, providing further evidence that specific lipoic acid residues within the E2 complex are susceptible to glutathionylation. These findings offer chemical information necessary for the identification of mechanisms and physiological implications of KGDH glutathionylation.

Disulfide bond as a switch for copper-zinc superoxide dismutase activity in asthma.

AIM: Loss of superoxide dismutase (SOD) activity is a defining biochemical feature of asthma. However, mechanisms for the reduced activity are unknown. We hypothesized that loss of asthmatic SOD activity is due to greater susceptibility to oxidative inactivation. RESULT: Activity assays of blood samples from asthmatics and healthy controls revealed impaired dismutase activity of copper-zinc SOD (CuZnSOD) in asthma. CuZnSOD purified from erythrocytes or airway epithelial cells from asthmatic was highly susceptible to oxidative inactivation. Proteomic analyses identified that inactivation was related to oxidation of cysteine 146 (C146), which is usually disulfide bonded to C57. The susceptibility of cysteines pointed to an alteration in protein structure, which is likely related to the loss of disulfide bond. We speculated that a shift to greater intracellular reducing potential might account for the change. Strikingly, measures of reduced and oxidized glutathione confirmed greater reducing intracellular state in asthma, compared with controls. Similarly, greater free thiol in CuZnSOD was confirmed by ~2-fold greater N-ethylmaleimide binding to C146 in asthma as compared with controls. INNOVATION: Greater reducing potential under a chronic inflammatory state of asthma, thus, leads to susceptibility of CuZnSOD to oxidative inactivation due to cleavage of C57-C146 disulfide bond and exposure of usually unavailable cysteines. CONCLUSION: Vulnerability of CuZnSOD influenced by redox likely amplifies injury and inflammation during acute asthma attacks when reactive oxygen species are explosively generated. Overall, this study identifies a new paradigm for understanding the chemical basis of inflammation, in which redox regulation of thiol availability dictates protein susceptibility to environmental and endogenously generated reactive species.

A quantitative proteomic profile of the Nrf2-mediated antioxidant response of macrophages to oxidized LDL determined by multiplexed selected reaction monitoring.

The loading of macrophages with oxidized low density lipoprotein (LDL) is a key part of the initiation and progression of atherosclerosis. Oxidized LDL contains a wide ranging set of toxic species, yet the molecular events that allow macrophages to withstand loading with these toxic species are not completely characterized. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the cellular stress response. However, the specific parts of the Nrf2-dependent stress response are diverse, with both tissue- and treatment-dependent components. The goal of these experiments was to develop and use a quantitative proteomic approach to characterize the Nrf2-dependent response in macrophages to oxidized LDL. Cultured mouse macrophages, the J774 macrophage-like cell line, were treated with a combination of oxidized LDL, the Nrf2-stabilizing reagent tert- butylhydroquinone (tBHQ), and/or Nrf2 siRNA. Protein expression was determined using a quantitative proteomics assay based on selected reaction monitoring. The assay was multiplexed to monitor a set of 28 antioxidant and stress response proteins, 6 housekeeping proteins, and 1 non-endogenous standard protein. The results have two components. The first component is the validation of the multiplexed, quantitative proteomics assay. The assay is shown to be fundamentally quantitative, precise, and accurate. The second component is the characterization of the Nrf2-mediated stress response. Treatment with tBHQ and/or Nrf2 siRNA gave statistically significant changes in the expression of a subset of 11 proteins. Treatment with oxidized LDL gave statistically significant increases in the expression of 7 of those 11 proteins plus one additional protein. All of the oxLDL-mediated increases were attenuated by Nrf2 siRNA. These results reveal a specific, multifaceted response of the foam cells to the incoming toxic oxidized LDL.

The nutrigenetics of hyperhomocysteinemia: quantitative proteomics reveals differences in the methionine cycle enzymes of gene-induced versus diet-induced hyperhomocysteinemia.

Hyperhomocysteinemia has long been associated with atherosclerosis and thrombosis and is an independent risk factor for cardiovascular disease. Its causes include both genetic and environmental factors. Although homocysteine is produced in every cell as an intermediate of the methionine cycle, the liver contributes the major portion found in circulation, and fatty liver is a common finding in homocystinuric patients. To understand the spectrum of proteins and associated pathways affected by hyperhomocysteinemia, we analyzed the mouse liver proteome of gene-induced (cystathionine beta-synthase (CBS)) and diet-induced (high methionine) hyperhomocysteinemic mice using two-dimensional difference gel electrophoresis and Ingenuity Pathway Analysis. Nine proteins were identified whose expression was significantly changed by 2-fold (p < or = 0.05) as a result of genotype, 27 proteins were changed as a result of diet, and 14 proteins were changed in response to genotype and diet. Importantly, three enzymes of the methionine cycle were up-regulated. S-Adenosylhomocysteine hydrolase increased in response to genotype and/or diet, whereas glycine N-methyltransferase and betaine-homocysteine methyltransferase only increased in response to diet. The antioxidant proteins peroxiredoxins 1 and 2 increased in wild-type mice fed the high methionine diet but not in the CBS mutants, suggesting a dysregulation in the antioxidant capacity of those animals. Furthermore, thioredoxin 1 decreased in both wild-type and CBS mutants on the diet but not in the mutants fed a control diet. Several urea cycle proteins increased in both diet groups; however, arginase 1 decreased in the CBS(+/-) mice fed the control diet. Pathway analysis identified the retinoid X receptor signaling pathway as the top ranked network associated with the CBS(+/-) genotype, whereas xenobiotic metabolism and the NRF2-mediated oxidative stress response were associated with the high methionine diet. Our results show that hyperhomocysteinemia, whether caused by a genetic mutation or diet, alters the abundance of several liver proteins involved in homocysteine/methionine metabolism, the urea cycle, and antioxidant defense.