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Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation.

ABSTRACT: Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection.

SUBMITTER: Yang CS

PROVIDER: S-EPMC3616771 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation.

Yang Chul-Su CS Lee Jong-Soo JS Rodgers Mary M Min Chan-Ki CK Lee June-Yong JY Kim Hee Jin HJ Lee Kwang-Hoon KH Kim Chul-Joong CJ Oh Byungha B Zandi Ebrahim E Yue Zhenyu Z Kramnik Igor I Liang Chengyu C Jung Jae U JU

Cell host & microbe 20120301 3

Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating ...[more]

PMID: 22423966

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Project description:The phagocytic NADPH oxidase generates superoxide by transferring electrons from cytosolic NADPH to extracellular O2. The activity of the oxidase at the plasma membrane can be measured as electron current (I(e)), and the voltage dependence of I(e) was recently reported to exhibit a strong rectification in human eosinophils, with the currents being nearly voltage independent at negative potentials. To investigate the underlying mechanism, we performed voltage-clamp experiments on inside-out patches from human eosinophils activated with PMA. Electron current was evoked by bath application of different concentrations of NADPH, whereas slow voltage ramps (0.8 mV/ms), ranging from -120 to 200 mV, were applied to obtain 'steady-state' current-voltage relationships (I-V). The amplitude of I(e) recorded at -40 mV was minimal at 8 microM NADPH and saturated above 1 mM, with half-maximal activity (K(m)) observed at approx. 110 microM NADPH. Comparison of I-V values obtained at different NADPH concentrations revealed that the voltage-dependence of I(e) is strongly influenced by the substrate concentration. Above 0.1 mM NADPH, I(e) was markedly voltage-dependent and steeply decreased with depolarization within the physiological membrane potential range (-60 to 60 mV), the I-V curve strongly rectifying only below -100 mV. At lower NADPH concentrations the I-V curve was progressively shifted to more positive potentials and I(e) became voltage-independent also within the physiological range. Consequently, the K(m) of the oxidase decreased by approx. 40% (from 100 to 60 microM) when the membrane potential increased from -60 to 60 mV. We concluded that the oxidase activity depends on both membrane potential and [NADPH], and that the shape of the I(e)-V curve is influenced by the concentration of NADPH in the submillimolar range. The surprising voltage-independence of I(e) reported in whole-cell perforated patch recordings was most likely due to substrate limitation and is not an intrinsic property of the oxidase.

Project description:Superoxide radical plays an important role in redox cell signaling and physiological processes; however, overproduction of superoxide or insufficient activity of antioxidants leads to oxidative stress and contributes to the development of pathological conditions such as endothelial dysfunction and hypertension. Meanwhile, the studies of superoxide in biological systems represent unique challenges associated with short lifetime of superoxide, insufficient reactivity of the superoxide probes, and lack of site-specific detection of superoxide. In this work we have developed 15N-and deuterium-enriched spin probe 15N-CAT1H for high sensitivity and site-specific detection of extracellular superoxide. We have tested simultaneous tracking of extracellular superoxide by 15N-CAT1H and intramitochondrial superoxide by conventional 14N-containing spin probe mitoTEMPO-H in immune cells isolated from spleen, splenocytes, under basal conditions or stimulated with inflammatory cytokines IL-17A and TNFα, NADPH oxidase activator PMA, or treated with inhibitors of mitochondrial complex I rotenone or complex III antimycin A. 15N-CAT1H provides two-fold increase in sensitivity and improves detection since EPR spectrum of 15N-CAT1 nitroxide does not overlap with biological radicals. Furthermore, concurrent use of cell impermeable 15N-CAT1H and mitochondria-targeted 14N-mitoTEMPO-H allows simultaneous detection of extracellular and mitochondrial superoxide. Analysis of IL-17A- and TNFα-induced superoxide showed parallel increase in 15N-CAT1 and 14N-mitoTEMPO signals suggesting coupling between phagocytic NADPH oxidase and mitochondria. The interplay between mitochondrial superoxide production and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II infusion in wild-type mice increased the extracellular basal splenocyte superoxide which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase activator PMA. Nox2 depletion attenuated angiotensin II mediated stimulation and inhibited both extracellular and mitochondrial PMA-induced superoxide production. These data indicate that splenocytes isolated from hypertensive angiotensin II-infused mice are "primed" for enhanced superoxide production from both phagocytic NADPH oxidase and mitochondria. Our data demonstrate that novel 15N-CAT1H provides high sensitivity superoxide measurements and combination with mitoTEMPO-H allows independent and simultaneous detection of extracellular and mitochondrial superoxide. We suggest that this new approach can be used to study the site-specific superoxide production and analysis of important sources of oxidative stress in cardiovascular conditions.

Project description:Background and aimsResistin has been implicated in cardiovascular disease and poor interventional cardiovascular outcomes. Previous studies by our group demonstrated resistin promoted vascular smooth muscle cell (VSMC) migration through protein kinase C epsilon (PKCε) pathways, while few others showed that resistin induced reactive oxygen species (ROS) generation in various cell types. In this study, we aim to systemically examine the functional role of resistin at the cellular and tissue levels as well as the potential mechanistic relationship between resistin-induced PKCε activation and ROS production.MethodsPlasma collected from patients undergoing carotid interventions was analyzed for resistin level and ROS. VSMCs were treated with resistin in the presence or absence of PKCε and NADPH oxidase (Nox)-specific inhibitors. Intracellular ROS production was analyzed using confocal microscopy and Nox activity with chemiluminescence. In vivo studies were performed in apolipoprotein E knock out (ApoE-/-) mice to determine therapeutic effects of PKCε-specific inhibitor, using the guide-wire injury model.ResultsWe observed significant correlation between plasma resistin and circulating levels of oxidative stress in patients with severe atherosclerotic disease. We also demonstrated that resistin induced ROS production via PKCε-mediated Nox activation. Resistin-induced ROS production was time-dependent, and Nox4 was the primary isoform involved. Inhibition of Nox completely abolished resistin-exaggerated VSMC proliferation, migration and dedifferentiation, as well as pro-inflammatory cytokine release. Upstream modulation of PKCε significantly reduced resistin-mediated cytosolic ROS, Nox activity and VSMC dysfunction. Moreover, PKCε-specific inhibitor mitigated resistin-induced Nox activation and intimal hyperplasia in ApoE-/- mice.ConclusionsResistin-associated VSMC dysfunction and intimal hyperplasia are related to PKCε-dependent Nox activation and ROS generation. Targeting the PKCε-Nox pathway may represent a novel strategy in managing resistin-associated atherosclerotic complications.

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Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation.

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Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation.

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