Although the function of metallothionein (MT), a 6-to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca 2؉ act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT ؊͞؊ ) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed. Metallothioneins (MT) are 6-to 7-kDa intracellular cysteine-rich (30 mol%) metal binding proteins whose function remains elusive (1). A critical role for MT in protection against toxic non-essential metals such as cadmium is apparent (2), and MT appears to act as an antioxidant under a variety of conditions (3). More recently, in vitro data support the hypothesis that MT is a critical link between cellular redox state and metal ion homeostasis (4-6). In this regard, cysteines of metal thiolate clusters confer unique redox sensitivity to an otherwise redox inert metal ligand (e.g., zinc) and facilitate the potential for MT to participate in intracellular signal transduction pathways (7). In the current study, we examine this latter novel hypothesis in intact cells and tissue.We chose to study the interaction of MT and the free radical, nitric oxide (NO), because (i) the bioregulatory targets of NO usually contain cysteines and͞or metals at their active or allosteric site (8); (ii) NO (or a secondary product) reacts with MT in vitro, leading to the release of zinc (9) or cadmium (10); (iii) NO can form stable EPR-detectable complexes with MT in vitro (11); and (iv) MT can reduce the sensitivity of cells to potential toxic levels of NO (12). Application of a chimeric construct (called FRET-MT) based on a recently described cameleon for calmodulin (13) revealed an NO-induced conformational change in MT, indicative of metal release, thereby providing the first demonstrations of (i) changes in intramolecular FRET (fluorescence resonance energy transfer) of a native protein; and (ii) metal release from MT in response to physiologic stimuli in intact cells. Furthermore, the lack of myogen...

endothelium; S-nitrosylation; imaging; fluorescence resonance energy transfer; Zinquin NITRIC OXIDE (NO) IS A UBIQUITOUS signaling molecule that is known to have important biological roles in the cardiovascular, nervous, and immune systems. Although most bioregulatory targets of NO contain cysteine and/or iron at the allosteric or regulatory sites (39), other molecular reactions may contribute to the biology of NO. After iron, zinc is the most abundant intracellular metal. Virtually all intracellular zinc is associated with proteins (primarily via complex interactions with cysteines), where it is known to be an integral component of numerous metalloenzymes, structural proteins, and transcription factors. Unlike iron, zinc itself is redox inert and does not react with NO. In vitro data reveals, however, that S-nitrosylation of cysteines in zinc thiolate structures affects the activity of zinc-dependent transcription factors (3,21,22) and enzymes (5, 10). Furthermore, NO is capable of increasing the amount of labile Zn 2ϩ in living cells from the hippocampus (6) and the vascular endothelium (2,23,35). Accordingly, it is highly plausible that zinc may play a role in aspects of NO signaling.Metallothionein (MT), a cysteine-rich (30% of all amino acid residues), heavy-metal-binding protein, is critical to intracellular Zn 2ϩ homeostasis, as it has the ability to bind up to seven zinc atoms per MT molecule. Although the function of MT remains unclear (31), it was originally hypothesized to affect zinc in a manner homologous to that of calmodulin for calcium (38). More recently, it has been suggested that MT acts as a sensor of cellular redox such that a shift to moreoxidizing conditions leads to release of zinc, whereas a shift to a more-reducing environment leads to binding of zinc (26). In vitro, NO is capable of interacting with MT to form either dinitrosyl iron sulfur complexes (18,36) or S-nitrosylation to result in the NO-mediated release of cadmium (28), copper (17), or zinc (22). We recently used fluorescence resonance energy transfer (FRET) in cultured pulmonary artery endothelial cells to show that green fluorescent protein-modified MT (FRET-MT) undergoes conformational changes in the presence of NO that are consistent with the release of free metal (zinc and/or copper) from its thiolate clusters (34). Alterations in metal ion homeostasis may underlie the protective effect of MT against NO toxicity (36) or the participation of MT in NO-modified, complex physiological properties such as the myogenic reflex (34).In the current study, we used the zinc-specific fluorophore Zinquin (46) to image changes in intracellular

The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of hemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids which blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its heme-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 hr before through 24 hrs after the irradiation.

Reactive oxygen species have been shown to play a significant role in hyperoxia-induced acute lung injury, in part, by inducing apoptosis of pulmonary endothelium. However, the signaling roles of phospholipid oxidation products in pulmonary endothelial apoptosis have not been studied. Using an oxidative lipidomics approach, we identified individual molecular species of phospholipids involved in the apoptosis-associated peroxidation process in a hyperoxic lung. C57BL/6 mice were killed 72 h after exposure to hyperoxia (100% oxygen). We found that hyperoxia-induced apoptosis (documented by activation of caspase-3 and -7 and histochemical terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of pulmonary endothelium) was accompanied by nonrandom oxidation of pulmonary lipids. Two anionic phospholipids, mitochondria-specific cardiolipin (CL) and extramitochondrial phosphatidylserine (PS), were the two major oxidized phospholipids in hyperoxic lung. Using electrospray ionization mass spectrometry, we identified several oxygenation products in CL and PS. Quantitative assessments revealed a significant decrease of CL and PS molecular species containing C(18:2), C(20:4), C(22:5), and C(22:6) fatty acids. Similarly, exposure of mouse pulmonary endothelial cells (MLEC) to hyperoxia (95% oxygen; 72 h) resulted in activation of caspase-3 and -7 and significantly decreased the content of CL molecular species containing C(18:2) and C(20:4) as well as PS molecular species containing C(22:5) and C(22:6). Oxygenated molecular species were found in the same two anionic phospholipids, CL and PS, in MLEC exposed to hyperoxia. Treatment of MLEC with a mitochondria-targeted radical scavenger, a conjugate of hemi-gramicidin S with nitroxide, XJB-5-131, resulted in significantly lower oxidation of both CL and PS and a decrease in hyperoxia-induced changes in caspase-3 and -7 activation. We speculate that cytochrome c driven oxidation of CL and PS is associated with the signaling role of these oxygenated species participating in the execution of apoptosis and clearance of pulmonary endothelial cells, thus contributing to hyperoxic lung injury.