Since the (re)discovery of cytochrome c (cyt c) in the early 1920s and subsequent detailed characterization of its structure and function in mitochondrial electron transport, it took over 70 years to realize that cyt c plays a different, not less universal role in programmed cell death, apoptosis, by interacting with several proteins and forming apoptosomes. Recently, two additional essential functions of cyt c in apoptosis have been discovered that are carried out via its interactions with anionic phospholipids: a mitochondria specific phospholipid, cardiolipin (CL), and plasma membrane phosphatidylserine (PS). Execution of apoptotic program in cells is accompanied by substantial and early mitochondrial production of reactive oxygen species (ROS). Because antioxidant enhancements protect cells against apoptosis, ROS production was viewed not as a meaningless side effect of mitochondrial disintegration but rather playing some - as yet unidentified - role in apoptosis. This conundrum has been resolved by establishing that mitochondria contain a pool of cyt c, which interacts with CL and acts as a CL oxygenase. The oxygenase is activated during apoptosis, utilizes generated ROS and causes selective oxidation of CL. The oxidized CL is required for the release of pro-apoptotic factors from mitochondria into the cytosol. This redox mechanism of cyt c is realized earlier than its other well-recognized functions in the formation of apoptosomes and caspase activation. In the cytosol, released cyt c interacts with another anionic phospholipid, PS, and catalyzes its oxidation in a similar oxygenase reaction. Peroxidized PS facilitates its externalization essential for the recognition and clearance of apoptotic cells by macrophages. Redox catalysis of plasma membrane PS oxidation constitutes an important redox-dependent function of cyt c in apoptosis and phagocytosis. Thus, cyt c acts as an anionic phospholipid specific oxygenase activated and required for the execution of essential stages of apoptosis. This review is focused on newly discovered redox mechanisms of complexes of cyt c with anionic phospholipids and their role in apoptotic pathways in health and disease.
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.
Palladium (Pd) nanoparticles are recognized as components of airborne automotive pollution produced by abrasion of catalyst materials in the car exhaust system. Here we produced dispersions of hydrophilic spherical Pd nanoparticles (Pd-NP) of uniform shape and size (10.4 ± 2.7 nm) in one step by Bradley's reaction (solvothermal decomposition in an alcohol or ketone solvent) as a model particle for experimental studies of the Pd particles in air pollution. The same approach provided mixtures of Pd-NP and nanoparticles of non-redox-active metal oxides, such as Al(2)O(3). Particle aggregation in applied media was studied by DLS and nanoparticle tracking analysis. The putative health effects of the produced Pd nanoparticles and nanocomposite mixtures were evaluated in vitro, using human primary bronchial epithelial cells (PBEC) and a human alveolar carcinoma cell line (A549). Viability of these cells was tracked by vital dye exclusion, and apoptosis was also assessed. In addition, we monitored the release of IL-8 and PGE(2) in response to noncytotoxic doses of the nanoparticles. Our studies demonstrate cellular uptake of Pd nanoparticles only in PBEC, as determined by TEM, with pronounced and dose-dependent effects on cellular secretion of soluble biomarkers in both cell types and a decreased responsiveness of human epithelial cells to the pro-inflammatory cytokine TNF-α. When cells were incubated with higher doses of the Pd nanoparticles, apoptosis induction and caspase activation were apparent in PBEC but not in A549 cells. These studies demonstrate the feasibility of using engineered Pd nanoparticles to assess the health effects of airborne automotive pollution.
MicroRNAs have been implicated as important mediators of cancer cell homeostasis, and accumulating data suggest compelling roles for them in the apoptosis pathway. X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor and an important barrier to apoptotic cell death, but the mechanisms which determine the diverse range of XIAP expression seen in cancer remains unclear. In this study, we present evidence that miR-24 directly targets the 3′UTR of the XIAP mRNA to exert translational repression. Using a heuristic algorithm of bioinformatics analysis and in vitro screening, we identified miR-24 as a candidate regulator of XIAP expression. Array CGH and SKY analysis reveal that genomic copy number loss at the miR-24 locus is concordant with loss of endogenous miR-24 in cancer cells. Using a luciferase construct of the XIAP 3′UTR, we showed that miR-24 specifically coordinates to the XIAP mRNA. And interference with miR-24’s binding of the critical seed region, resulting from site-directed mutagenesis of the 3′UTR, significantly abrogated miR-24’s effects on XIAP expression. Moreover, miR-24 over-expression can overcome apoptosis-resistance in cancer cells via down-regulation of XIAP expression, and the resulting cancer cell death induced by TRAIL is executed by the canonical caspase-mediated apoptosis pathway. In summary, our data suggest a novel mechanism by which miR-24 directly modulates XIAP expression level and consequently the apoptosis threshold in cancer cells.
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