Increased mitochondrial prooxidant activity mediates up-regulation of Complex I S-glutathionylation via protein thiyl radical in the murine heart of eNOS−/−

In response to oxidative stress, mitochondrial Complex I is reversibly S-glutathionylated. We hypothesized that protein S-glutathionylation (PrSSG) of Complex I is mediated by a kinetic mechanism involving reactive protein thiyl radical (PrS^•) and GSH in vivo. Previous studies have shown that in vitro S-glutathionylation of isolated Complex I at the 51 and 75-kDa subunits was detected under the conditions of ^•O₂⁻ production, and mass spectrometry confirmed that formation of Complex I PrS^• mediates PrSSG. Exposure of myocytes to menadione resulted in enhanced Complex I PrSSG and PrS^• (Kang et al., Free Radical Biol. Med. 52:962–973; 2012). In this investigation, we tested our hypothesis in the murine heart of eNOS^−/−. The eNOS^−/− mouse is known to be hypertensive and develops the pathological phenotype of progressive cardiac hypertrophy. The mitochondria isolated from the eNOS^−/− myocardium exhibited a marked dysfunction with impaired state 3 respiration, a declining respiratory control index, and decreasing enzymatic activities of ETC components. Further biochemical analysis and EPR measurement indicated defective aconitase activity, a marked increase in ^•O₂⁻ generation activity, and a more oxidized physiological setting. These results suggest increasing prooxidant activity and subsequent oxidative stress in the mitochondria of the eNOS^−/− murine heart. When Complex I from the mitochondria of the eNOS^−/− murine heart was analyzed by immunospin trapping and probed with anti-GSH antibody, both PrS^• and PrSSG of Complex I were significantly enhanced. Overexpression of SOD2 in the murine heart dramatically diminished the detected PrS^•, supporting the conclusion that mediation of Complex I PrSSG by oxidative stress-induced PrS^• is a unique pathway for the redox regulation of mitochondrial function in vivo.     

Angiotensin IV upregulates the activity of protein phosphatase 1α in Neura-2A cells

The peptide angiotensin IV (Ang IV) is a derivative of angiotensin II. While insulin regulated amino peptidase (IRAP) has been proposed as a potential receptor for Ang IV, the signalling pathways of Ang IV through IRAP remain elusive. We applied high-resolution mass spectrometry to perform a systemic quantitative phosphoproteome of Neura-2A (N2A) cells treated with and without Ang IV using sta ble-isotope labeling by amino acids in cell culture (SILAC), and identified a reduction in the phosphorylation of a major Ser/Thr protein phosphorylase 1 (PP1) upon Ang IV treatment. In addition, spinophilin (spn), a PP1 regulatory protein that plays important functions in the neural system, was expressed at higher levels. Immunoblotting revealed decreased phosphorylation of p70S6 kinase (p70^S6K) and the major cell cycle modulator retinoblastoma protein (pRB). These changes are consistent with an observed decrease in cell proliferation. Taken together, our study suggests that Ang IV functions via regulating the activity of PP1.     

Regulation of HIF-1α activity by overexpression of thioredoxin is independent of thioredoxin reductase status

Berberine (BBR) is one of the major alkaloids and has been reported to have a variety of pharmacologic effects, including inhibition of cell cycle progression. Here, we investigated the mechanisms of BBR protection of neuronal cells from cell death induced by the Parkinson’s disease-related neurotoxin 6-hydroxydopamine (6-OHDA). Pretreatment of SH-SY5Y cells with BBR significantly reduced 6-OHDAinduced generation of reactive oxygen species (ROS), caspase-3 activation, and subsequent cell death. BBR also upregulated heme oxygenase-1 (HO-1) expression, which conferred protection against 6-OHDA-induced dopaminergic neuron injury and besides, effect of BBR on HO-1 was reversed by siRNA-Nrf2. Furthermore, BBR induced PI3K/Akt and p38 activation, which are involved in the induction of Nrf2 expression and neuroprotection. These results suggest that BBR may be useful as a therapeutic agent for the treatment of dopaminergic neuronal diseases.     

PCBP2 enhances the antiviral activity of IFN-α against HCV by stabilizing the mRNA of STAT1 and STAT2

Interferon-α (IFN-α) is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV) during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b). However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3'Untranslated Region (UTR) of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3'UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.     

Modulation of host HIF-1α activity and the tryptophan pathway contributes to the anti-Toxoplasma gondii potential of nanoparticles

BackgroundToxoplasmosis constitutes a large global burden that is further exacerbated by the shortcomings of available therapeutic options, thus underscoring the urgent need for better anti-Toxoplasma gondii therapy or strategies. Recently, we showed that the anti-parasitic action of inorganic nanoparticles (NPs) could, in part, be due to changes in redox status as well as in the parasite mitochondrial membrane potential.MethodsIn the present study, we explored the in vitro mode of action of the anti-T. gondii effect of NPs by evaluating the contributions of host cellular processes, including the tryptophan pathway and hypoxia-inducing factor activity. NPs, at concentrations ranging from 0.01 to 200 µg/ml were screened for anti-parasitic activity. Sulfadiazine and/or pyrimethamine served as positive controls.ResultsWe found that interplay among multiple host cellular processes, including HIF-1α activity, indoleamine 2,3-dioxygenase activity, and to a larger extent the tryptophan pathway, contribute to the anti-parasitic action of NPs.ConclusionTo our knowledge, this is the first study to demonstrate an effect of NPs on the tryptophan and/or kynurenine pathway.General significanceOur findings deepen our understanding of the mechanism of action of NPs and suggest that modulation of the host nutrient pool may represent a viable approach to the development of new and effective anti-parasitic agents.     

Regulation of IκB kinase by GβL through recruitment of the protein phosphatases

G protein β-like (GβL) is a member of WD repeat-containing family which are involved in various intracellular signaling events. In our previous report, we demonstrated that GβL regulates TNFα-stimulated NF-κB signaling by interacting with and inhibiting phosphorylation of IκB kinase. However, GβL itself does not seem to regulate IKK directly, because it contains no functional domains except WD domains. Here, using immunoprecipitation and proteomic analyses, we identified protein phosphatase 4 as a new binding partner of GβL. We also found that GβL interacts with PP2A and PP6, other members of the same phosphatase family. By interacting with protein phosphatases, which do not directly bind to IKKβ, GβL mediates the association of phosphatases with IKKβ. Overexpression of protein phosphatases inhibited TNFκ-induced activation of NF-κB signaling, which is an effect similar to that of GβL overexpression. Down-regulation of GβL by small interfering RNA diminished the inhibitory effect of phosphatases, resulting in restoration of NF-κB signaling. Thus, we propose that GβL functions as a negative regulator of NF-κB signaling by recruiting protein phosphatases to the IKK complex.     

BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response

Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.