Lipid antioxidants: free radical scavenging versus regulation of enzymatic lipid peroxidation

The essentiality of polyunsaturated lipids makes membranes susceptible to peroxidative modifications. One of the most contemporary examples includes selective peroxidation of cardiolipin in mitochondria of cells undergoing apoptosis. Cardiolipin peroxidation products are required for the mitochondrial membrane permeabilization, release of pro-apoptotic factors and completion of the cell death program. Therefore, search for effective inhibitors of cardiolipin peroxidation is critical to discovery and development of anti-apoptotic antioxidants. Mitochondria contain significant amounts of α-tocopherol, a well known scavenger of reactive free radicals. In the present study, we used an oxidative lipidomics approach to evaluate the effect of α-tocopherol and its homologues with different lengths of the side-chain such as 2,5,7,8,-tetramethyl-2(4-methylpentyl)-6-chromanol and 2,2,5,7,8-pentamethyl-6-chromanol, on oxidation of tetralinoleoyl cardiolipin induced by cytochrome c in the presence of hydrogen peroxide. Our data indicate that vitamin E homologues inhibit not only accumulation of tetralinoleoyl cardiolipin hydroperoxides but also hydroxy-derivatives of tetralinoleoyl cardiolipin formed in the enzymatic peroxidase half-reaction catalyzed by cytochrome c. This suggests that protective effects of vitamin E homologues against tetralinoleoyl cardiolipin peroxidation catalyzed by cytochrome c/hydrogen peroxide are realized largely due to their effects on the peroxidase activity of cytochrome c towards tetralinoleoyl cardiolipin rather than via their scavenging activity

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis

Surface-Binding to Cardiolipin Nanodomains Triggers Cytochrome c Pro-apoptotic Peroxidase Activity via Localized Dynamics

The peroxidation of cardiolipins by reactive oxygen species, which is regulated and enhanced by cytochrome c (cyt c), is a critical signaling event in mitochondrial apoptosis. We probe the molecular underpinnings of this mitochondrial death signal through structural and functional studies of horse heart cyt c binding to mixed-lipid membranes containing cardiolipin with mono- and polyunsaturated acyl chains. Lipidomics reveal the selective oxidation of polyunsaturated fatty acid (PUFA) cardiolipin (CL), while multidimensional solid-state NMR probes the structure and dynamics of the membrane and the peripherally bound protein. The hydrophilic milieu at the membrane interface stabilizes a native-like fold, but also leads to localized flexibility at the membrane-interacting protein face. PUFA CL acts as both a preferred substrate and a dynamic regulator by affecting the dynamics of the cyt c N70-I85 Ω loop, which covers the heme cavity

Excessive phospholipid peroxidation distinguishes ferroptosis from other cell death modes including pyroptosis

Lipid peroxidation (LPO) drives ferroptosis execution. However, LPO has been shown to contribute also to other modes of regulated cell death (RCD). To clarify the role of LPO in different modes of RCD, we studied in a comprehensive approach the differential involvement of reactive oxygen species (ROS), phospholipid peroxidation products, and lipid ROS flux in the major prototype modes of RCD viz. apoptosis, necroptosis, ferroptosis, and pyroptosis. LC-MS oxidative lipidomics revealed robust peroxidation of three classes of phospholipids during ferroptosis with quantitative predominance of phosphatidylethanolamine species. Incomparably lower amounts of phospholipid peroxidation products were found in any of the other modes of RCD. Nonetheless, a strong increase in lipid ROS levels was detected in non-canonical pyroptosis, but only during cell membrane rupture. In contrast to ferroptosis, lipid ROS apparently was not involved in non-canonical pyroptosis execution nor in the release of IL-1 beta and IL-18, while clear dependency on CASP11 and GSDMD was observed. Our data demonstrate that ferroptosis is the only mode of RCD that depends on excessive phospholipid peroxidation for its cytotoxicity. In addition, our results also highlight the importance of performing kinetics and using different methods to monitor the occurrence of LPO. This should open the discussion on the implication of particular LPO events in relation to different modes of RCD

Lipid homeostasis and inflammatory activation are disturbed in classically activated macrophages with peroxisomal β-oxidation deficiency

Macrophage activation is characterized by pronounced metabolic adaptation. Classically activated macrophages show decreased rates of mitochondrial fatty acid oxidation and oxidative phosphorylation and acquire a glycolytic state together with their pro-inflammatory phenotype. In contrast, alternatively activated macrophages require oxidative phosphorylation and mitochondrial fatty acid oxidation for their anti-inflammatoryfunction. Although it is evident that mitochondrial metabolism is regulated during macrophage polarization and essential for macrophagefunction, little is known on the regulation and role of peroxisomal -oxidation during macrophage activation. In this study, we show that peroxisomal -oxidation is strongly decreased in classically activated bone-marrow-derived macrophages (BMDM) and mildly induced in alternatively activated BMDM. To examine the role of peroxisomal -oxidation in macrophages, we used Mfp2(-/-) BMDM lacking the key enzyme of this pathway. Impairment of peroxisomal -oxidation in Mfp2(-/-) BMDM did not cause lipid accumulation but rather an altered distribution of lipid species with very-long-chain fatty acids accumulating in the triglyceride and phospholipid fraction. These lipid alterations in Mfp2(-/-) macrophages led to decreased inflammatory activation of Mfp2(-/-) BMDM and peritoneal macrophages evidenced by impaired production of several inflammatory cytokines and chemokines, but did not affect anti-inflammatory polarization. The disturbed inflammatory responses of Mfp2(-/-) macrophages did not affect immune cell infiltration, as mice with selective elimination of MFP2 from myeloid cells showed normal monocyte and neutrophil influx upon challenge with zymosan. Together, these data demonstrate that peroxisomal -oxidation is involved in fine-tuning the phenotype of macrophages, probably by influencing the dynamic lipid profile during macrophage polarization

Factoring-in agglomeration of carbon nanotubes and nanofibers for better prediction of their toxicity versus asbestos

<p>Abstract</p> <p>Background</p> <p>Carbon nanotubes (CNT) and carbon nanofibers (CNF) are allotropes of carbon featuring fibrous morphology. The dimensions and high aspect ratio of CNT and CNF have prompted the comparison with naturally occurring asbestos fibers which are known to be extremely pathogenic. While the toxicity and hazardous outcomes elicited by airborne exposure to single-walled CNT or asbestos have been widely reported, very limited data are currently available describing adverse effects of respirable CNF.</p> <p>Results</p> <p>Here, we assessed pulmonary inflammation, fibrosis, oxidative stress markers and systemic immune responses to respirable CNF in comparison to single-walled CNT (SWCNT) and asbestos. Pulmonary inflammatory and fibrogenic responses to CNF, SWCNT and asbestos varied depending upon the agglomeration state of the particles/fibers. Foci of granulomatous lesions and collagen deposition were associated with dense particle-like SWCNT agglomerates, while no granuloma formation was found following exposure to fiber-like CNF or asbestos. The average thickness of the alveolar connective tissue - a marker of interstitial fibrosis - was increased 28 days post SWCNT, CNF or asbestos exposure. Exposure to SWCNT, CNF or asbestos resulted in oxidative stress evidenced by accumulations of 4-HNE and carbonylated proteins in the lung tissues. Additionally, local inflammatory and fibrogenic responses were accompanied by modified systemic immunity, as documented by decreased proliferation of splenic T cells <it>ex vivo </it>on day 28 post exposure. The accuracies of assessments of effective surface area for asbestos, SWCNT and CNF (based on geometrical analysis of their agglomeration) versus estimates of mass dose and number of particles were compared as predictors of toxicological outcomes.</p> <p>Conclusions</p> <p>We provide evidence that effective surface area along with mass dose rather than specific surface area or particle number are significantly correlated with toxicological responses to carbonaceous fibrous nanoparticles. Therefore, they could be useful dose metrics for risk assessment and management.</p

C-ferroptosis is an iron-dependent form of regulated cell death in cyanobacteria

Ferroptosis is an oxidative and iron-dependent form of regulated cell death (RCD) recently described in eukaryotic organisms like animals, plants, and parasites. Here, we report that a similar process takes place in the photosynthetic prokaryote Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis sp. PCC 6803 cells undergo a cell death pathway that can be suppressed by the canonical ferroptosis inhibitors, CPX, vitamin E, Fer-1, liproxstatin-1, glutathione (GSH), or ascorbic acid (AsA). Moreover, as described for eukaryotic ferroptosis, this pathway is characterized by an early depletion of the antioxidants GSH and AsA, and by lipid peroxidation. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program.Fil: Aguilera, Anabella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; ArgentinaFil: Berdun, Federico Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; ArgentinaFil: Bartoli, Carlos Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Steelheart Molina, Maria Charlotte. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Alegre, Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Bayir, Hülya. University of Pittsburgh; Estados UnidosFil: Tyurina, Yulia Y.. University of Pittsburgh; Estados UnidosFil: Kagan, Valerian E.. University of Pittsburgh; Estados UnidosFil: Salerno, Graciela Lidia. Fundación para Investigaciones Biológicas Aplicadas; ArgentinaFil: Pagnussat, Gabriela Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Martin, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; Argentin

Myeloperoxidase-dependent oxidation of etoposide in human myeloid progenitor CD34+ cells

ABSTRACT Etoposide is a widely used anticancer drug successfully utilized for treatment of many types of cancer in children and adults. Its use, however, is associated with an increased risk of development of secondary acute myelogenous leukemia (t-AML) involving MLL gene MOL #68718

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: Implications for AMP Kinase

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc iso- lated from kidneys is phosphorylated on Thr28, leading to a par- tial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing supe- rior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type un- phosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (����m), and ROS levels are reduced compared with wild type. As we show by high resolu- tion crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a cen- tral position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kid- ney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue- specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ����m hyperpolarization, a known cause of ROS and trigger of apoptosis

Phosphatidylserine Targets Single-Walled Carbon Nanotubes to Professional Phagocytes In Vitro and In Vivo

Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid “eat-me” signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells