Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.     

Characterization of Mitochondrial YME1L Protease Oxidative Stress-Induced Conformational State

Oxidative stress is a common challenge to mitochondrial function where reactive oxygen species are capable of significant organelle damage. The generation of mitochondrial reactive oxygen species occurs in the inner membrane and matrix compartments as a consequence of subunit function in the electron transport chain and citric acid cycle, respectively. Maintenance of mitochondrial proteostasis and stress response is facilitated by compartmentalized proteases that couple the energy of ATP hydrolysis to unfolding and the regulated removal of damaged, misfolded, or aggregated proteins. The mitochondrial protease YME1L functions in the maintenance of proteostasis in the intermembrane space. YME1L is an inner membrane-anchored hexameric protease with distinct N-terminal, transmembrane, AAA + (ATPases associated with various cellular activities), and C-terminal M41 zinc-dependent protease domains. The effect of oxidative stress on enzymes such as YME1L tasked with maintaining proteostasis is currently unclear. We report here that recombinant YME1L undergoes a reversible conformational change in response to oxidative stress that involves the interaction of one hydrogen peroxide molecule per YME1L monomer with affinities equal to 31 ± 2 and 26 ± 1 mM for conditions lacking or including nucleotide, respectively. Our data also reveal that oxidative stress does not significantly impact nucleotide binding equilibria, but does stimulate a 2-fold increase in the rate constant for high-affinity ATP binding from (8.9 ± 0.2) × 10⁵ M⁻¹ s⁻¹ to (1.5 ± 0.1) × 10⁶ M⁻¹ s⁻¹. Taken together, these data may suggest a mechanism for the regulated processing of YME1L by other inner membrane proteases such as OMA1.     

Nitrosative/Oxidative Stress Conditions Regulate Thioredoxin-Interacting Protein (TXNIP) Expression and Thioredoxin-1 (TRX-1) Nuclear Localization

Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H₂O₂, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.     

Oxidative and Nitrosative Modifications of Tropoelastin Prevent Elastic Fiber Assembly in Vitro

Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. Tropoelastin (TE), the precursor of elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO⁻) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO⁻ and HOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.     

Nuclear-translocated Glyceraldehyde-3-phosphate Dehydrogenase Promotes Poly(ADP-ribose) Polymerase-1 Activation during Oxidative/Nitrosative Stress in Stroke

In addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke.     

Nano-Boehmite Induced Oxidative and Nitrosative Stress Responses in Vigna radiata L.

Journal of Plant Growth Regulation - Wide spectrum and increasing use of nano-sized aluminum oxyhydroxide (boehmite, nBhm) particles have left a risk of their environmental exposure and...     

Duloxetine Protects Human Neuroblastoma Cells from Oxidative Stress-Induced Cell Death Through Akt/Nrf-2/HO-1 Pathway

The contribution of oxidative stress to the pathophysiology of depression has been described in numerous studies. Particularly, an increased production of reactive oxygen species (ROS) caused by mitochondrial dysfunction can lead to neuronal cell death. Human neuroblastoma SH-SY5Y cells were used to investigate the neuroprotective effect of the antidepressant duloxetine against rotenone-induced oxidative stress. SH-SY5Y cells were pretreated with duloxetine (1–5 µM) for 24 h followed by a 24-h rotenone exposure (10 µM). The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) inhibitor LY294002 (10 µM) and the heme oxygenase 1 (HO-1) inhibitor zinc protoporphyrin IX-ZnPP (5 µM) were added to cultures 1 h prior duloxetine treatments. After treatments cell viability and ROS generation were assessed. NF-E2-related factor-2 (Nrf2) nuclear translocation was assessed by immunofluorescent staining after 4 and 8 h of duloxetine incubation. Furthermore, the Nrf2 and HO-1 mRNA expression was carried out after 4–48 h of duloxetine treatment by qRT-PCR. Duloxetine pretreatment antagonized rotenone-induced overproduction of ROS and cell death in SH-SY5Y cells. In addition, a 1-h pretreatment with LY294002 abolished duloxetine’s protective effect. Duloxetine also induced nuclear translocation of the Nrf2 and the expression of its target gene, HO-1. Finally, the HO-1 inhibitor, ZnPP, suppressed the duloxetine protective effect. Overall, these results indicate that the mechanism of duloxetine neuroprotective action against oxidative stress and cell death might rely on the Akt/Nrf2/HO-1 pathways.     

斑马鱼Gfi-1 基因结构和功能分析

目的:研究斑马鱼Gfi-1基因在物种间进化的保守性和功能分析。方法:运用生物信息学方法分析斑马鱼Gfi-1基因结构特征和保守性等。结果:斑马鱼Gfi-1基因在蛋白水平与小鼠、人高度保守;分析斑马鱼和人的Gfi-1基因外显子、内含子和ATG起始、终止密码子也具有高度相似性;从进化树分析斑马鱼Gfi-1基因与人、小鼠、犬、猴等在进化上高度保守;分析斑马鱼、人、小鼠Gfi-1基因在染色体上的位置和相邻基因,显示出惊人的相似性。结论:斑马鱼Gfi-1基因在进化上高度保守,为脊椎动物保守基因,为其后续在造血系统和造血微环境方面的研究提供了理论支持和铺垫。     

Divalent Metal Ions in Plant Mitochondria and Their Role in Interactions with Proteins and Oxidative Stress-Induced Damage to Respiratory Function

Understanding the metal ion content of plant mitochondria and metal ion interactions with the proteome are vital for insights into both normal respiratory function and the process of protein damage during oxidative stress. We have analyzed the metal content of isolated Arabidopsis (Arabidopsis thaliana) mitochondria, revealing a 26:8:6:1 molar ratio for iron:zinc:copper:manganese and trace amounts of cobalt and molybdenum. We show that selective changes occur in mitochondrial copper and iron content following in vivo and in vitro oxidative stresses. Immobilized metal affinity chromatography charged with Cu²⁺, Zn²⁺, and Co²⁺ was used to identify over 100 mitochondrial proteins with metal-binding properties. There were strong correlations between the sets of immobilized metal affinity chromatography-interacting proteins, proteins predicted to contain metal-binding motifs, and protein sets known to be oxidized or degraded during abiotic stress. Mitochondrial respiratory chain pathways and matrix enzymes varied widely in their susceptibility to metal-induced loss of function, showing the selectivity of the process. A detailed study of oxidized residues and predicted metal interaction sites in the tricarboxylic acid cycle enzyme aconitase identified selective oxidation of residues in the active site and showed an approach for broader screening of functionally significant oxidation events in the mitochondrial proteome.Transition metal ions are essential in myriad biochemical functions by being incorporated into or associating with proteins to elicit functions in living cells. In plant mitochondria, key functions of metal cofactors include metabolism, electron transport, ATP synthesis, and the detoxification of reactive oxygen species (ROS). For example, copper (Cu) and iron (Fe) ions facilitate the transfer of electrons in the electron transport chain (ETC; Bligny and Douce, 1977; Pascal and Douce, 1993), proteins of the tricarboxylic acid (TCA) cycle utilize metal ion cofactors to catalyze primary metabolic reactions (Miernyk and Randall, 1987; Jordanov et al., 1992), manganese (Mn) and Fe are required for antioxidant defense enzymes (Alscher et al., 2002), and zinc (Zn) is required for the protein import apparatus in both carrier protein transport to the inner membrane (Lister et al., 2002) and presequence degradation (Moberg et al., 2003). Cobalt (Co) is known to substitute for other metal ions in the activation of NAD-malic enzyme and succinyl-CoA ligase from plant mitochondrial extracts (Palmer and Wedding, 1966; Macrae, 1971), but it is not known whether there is an in vivo requirement for trace amounts of Co for plant respiratory metabolism.Metal ions, however, can also be highly toxic to cells and cell organelle functions. The redox-inactive heavy metal cadmium exhibits strong affinity for oxygen, nitrogen, and sulfur atoms (Nieboer and Richardson, 1980) and can inhibit enzyme activity by direct blocking of protein function or displacement of natural metal centers. There are numerous reports of heavy metals depleting cellular glutathione pools, leading to diminished antioxidant protection in the cell and resulting in ROS accumulation (Schutzendubel and Polle, 2002). Cadmium has been reported to both directly and indirectly inhibit plant mitochondrial function (Kesseler and Brand, 1994; Smiri et al., 2009), as have Co complexes (Guzhova et al., 1979). Redox-active metal catalysts such as Cu and Fe cations can also be cytotoxic, as they react with ROS via the Haber-Weiss reaction or Fenton-type reactions to produce the hydroxyl anion (Stohs and Bagchi, 1995). Inhibitory effects of exogenously added Cu and Fe on plant respiratory function have been reported (Kampfenkel et al., 1995; Padua et al., 1996, 1999). Therefore, the presence of free metal cations, redox active or inactive, in mitochondria may significantly contribute to the initiation and perpetuation of oxidative stress.One of the best described mechanisms for metal-linked damage is metal-catalyzed oxidation (MCO) of proteins, which involves the oxidation of susceptible amino acids such as Arg, Lys, Pro, and His, among a plethora of other poorly characterized consequences (Stadtman, 1990). It has been proposed that MCO of proteins can be a highly specific event where proteins are more susceptible to damage if they bind metal ions and when the site of protein oxidation can be defined on the protein surface that binds to the metal ions (Stadtman, 1990). One of the major consequences of MCO is the irreversible formation of reactive carbonyls on amino acid side chains (Stadtman, 1990). Such carbonyls are known to accumulate in the wheat (Triticum aestivum) mitochondrial proteome during environmental stress, even more so than in other ROS-producing subcellular organelles of plants (Bartoli et al., 2004). The selectivity of protein susceptibility to MCO was also demonstrated in rice (Oryza sativa), where distinct subpopulations of the mitochondrial matrix proteome were carbonylated following Cu²⁺ and hydrogen peroxide (H₂O₂) treatment (Kristensen et al., 2004). The targeted damage of select sets of plant mitochondrial proteins has also been observed in other studies, but without clear linkage to the role of metal ions. For example, altered protein abundance has been observed in Arabidopsis (Arabidopsis thaliana; Sweetlove et al., 2002) and pea (Pisum sativum; Taylor et al., 2005) mitochondria after the initiation of oxidative or environmental stress. Additionally, inhibition of respiratory metabolism by the lipid peroxidation by-product 4-hydroxy-2-nonenal has been shown to operate through modification of a specific subset of proteins (Taylor et al., 2002; Winger et al., 2005, 2007). However, the mechanisms of targeted oxidative modification, the role of metals, and the consequences for mitochondrial metabolic function are not very well understood. Furthermore, whether or not selectivity of protein damage in mitochondria is based on relative metal ion affinity and if the sites of damage can be predicted by the sites of metal ion binding are not known.In this study, we investigated metal homeostasis in the Arabidopsis mitochondrion during oxidative stress. The interactions between metal ions and proteins were also investigated using immobilized metal affinity chromatography (IMAC). Functional assays were used to determine the targets and consequences of metal ion interaction in the mitochondrion and to explore the linkages to the redox nature of the metal and the loss of mitochondrial functions. Finally, a detailed study of the oxidized peptides of aconitase was undertaken to probe the linkage between metal-binding sites, the oxidation of amino acids, and the inactivation of this critical TCA cycle enzyme.     

The Role of PARP-1 and PARP-2 Enzymes in Metabolic Regulation and Disease

While originally described as DNA damage repair agents, recent data suggest a role for poly(ADP-ribose) polymerase (PARP) enzymes in metabolic regulation by influencing mitochondrial function and oxidative metabolism. Here we review how PARP activity has a major metabolic impact and the role of PARP-1 and PARP-2 in diverse metabolic complications.     

Neurokinin 1 Receptor Mediates Membrane Blebbing and Sheer Stress-Induced Microparticle Formation in HEK293 Cells

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.     

Neuroprotective Effects of Theaflavins Against Oxidative Stress-Induced Apoptosis in PC12 Cells

Oxidative stress can induce neuronal apoptosis via the production of superoxide and hydroxyl radicals. This process is as a major pathogenic mechanism in neurodegenerative disorders. In this study, we aimed to clarify whether theaflavins protect PC12 cells from oxidative stress damage induced by H₂O₂. A cell model of PC12 cells undergoing oxidative stress was created by exposing cells to 200 μM H₂O₂ in the presence or absence of varying concentrations of theaflavins (5, 10, and 20 μM). Cell viability was monitored using the MTT assay and Hoechst 33258 staining, showing that 10 μM theaflavins enhanced cell survival following 200 μM H₂O₂ induced toxicity and increased cell viability by approximately 40?%. Additionally, we measured levels of intracellular reactive oxygen species (ROS) and antioxidant enzyme activity. This suggested that the neuroprotective effect of theaflavins against oxidative stress in PC12 cells is derived from suppression of oxidant enzyme activity. Furthermore, Western blot analyses indicated that theaflavins downregulated the ratio of pro-apoptosis/anti-apoptosis proteins Bax/Bcl-2. Theaflavins also downregulated the expression of caspase-3 compared with a H₂O₂-treated group that had not been treated with theaflavins. Interestingly, this is the first study to report that the four main components of theaflavins found in black tea can protect neural cells (PC12) from apoptosis induced by H₂O₂. These findings provide the foundations for a new field of using theaflavins or its source, black tea, in the treatment of neurodegenerative diseases caused by oxidative stress.