Many cell types contain metal-ion unsaturated metallothionein (MT). Considering the Zn 2+ binding affinity of metallothionein, the existence of this species in the intracellular environment constitutes a substantial "thermodynamic sink." Indeed, the mM concentration of glutathione may be thought of in the same way. In order to understand how apo-MT and the rest of the Zn-proteome manage to co-exist, experiments examined the in vitro reactivity of Zn-proteome with apo-MT, glutathione (GSH), and a series of common Zn 2+ chelating agents including N,N,N',N'-(2-pyridylethyl) ethylenediammine (TPEN), EDTA, and [(2,2'-oxyproplylene-dinitrilo]tetraacetic acid (EGTA). Less than 10% of Zn-proteome from U87 mg cells reacted with apo-MT or GSH. In contrast, each of the synthetic chelators was 2−3 times more reactive. TPEN, a cell permeant reagent, also reacted rapidly with both Zn-proteome and Zn-MT in LLC-PK 1 cells. Taking a specific zinc finger protein for further study, apo-MT, GSH, and TPEN inhibited the binding of Zn 3 -Sp1 with its cognate DNA site (GC-1) in the sodium-glucose co-transporter promoter of mouse kidney. In contrast, preformation of Zn 3 -Sp1-(GC-1) prevented reaction with apo-MT and GSH; TPEN remained active but at a higher concentration. Whereas, Zn 3 -Sp1 is active in cells containing apo-MT and GSH, exposure of LLC-PK 1 cells to TPEN for 24 h largely inactivated its DNA binding activity. The results help to rationalize the steady state presence of cellular apo-MT in the midst of the many, diverse members of the Znproteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn 2+ .

The localization of prenylated Ras at the plasma membrane promotes activation of Ras by receptor tyrosine kinases and stimulates oncogenic signaling by mutant Ras. The Nogo-B receptor (NgBR) is a transmembrane receptor that contains a conserved hydrophobic pocket. Here, we demonstrate that the NgBR promotes the membrane accumulation of Ras by directly binding prenylated Ras at the plasma membrane. We show that NgBR knockdown diminishes the membrane localization of Ras in multiple cell types. NgBR overexpression in NIH-3T3 fibroblasts increases membrane-associated Ras, induces the transformed phenotype in vitro, and promotes the formation of fibrosarcoma in nude mice. NgBR knockdown in human breast cancer cells reduces Ras membrane localization, inhibits EGF-stimulated Ras signaling, and diminishes tumorigenesis of xenografts in nude mice. Our data demonstrate that NgBR is a unique receptor that promotes accumulation of prenylated Ras at the plasma membrane and promotes EGF pathways.

is synthesized in the cytosol and imported into the mitochondrial matrix, where it is activated and functions as the primary antioxidant for cellular respiration. The specific mechanisms that target SOD-2 to the mitochondria remain unclear. We hypothesize that inducible heat shock protein 70 (iHSP70) targets SOD-2 to the mitochondria via a mechanism facilitated by ATP, and this process is impaired in persistent pulmonary hypertension of the newborn (PPHN). We observed that iHSP70 interacts with SOD-2 and targets SOD-2 to the mitochondria. Interruption of iHSP70-SOD-2 interaction with 2-phenylethylenesulfonamide-(PFT-, a specific inhibitor of substrate binding to iHSP70 COOH terminus) and siRNA-mediated knockdown of iHSP70 expression disrupted SOD-2 transport to mitochondria. Increasing intracellular ATP levels by stimulation of respiration with CaCl 2 facilitated the mitochondrial import of SOD-2, increased SOD-2 activity, and decreased the mitochondrial superoxide (O 2 ·Ϫ ) levels in PPHN pulmonary artery endothelial cells (PAEC) by promoting iHSP70-SOD-2 dissociation at the outer mitochondrial membrane. In contrast, oligomycin, an inhibitor of mitochondrial ATPase, decreased SOD-2 expression and activity and increased O 2 ·Ϫ levels in the mitochondria of control PAEC. The basal ATP levels and degree of iHSP70-SOD-2 dissociation were lower in PPHN PAEC and lead to increased SOD-2 degradation in cytosol. In normal pulmonary arteries (PA), PFT-impaired the relaxation response of PA rings in response to nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine. Pretreatment with Mito-Q, a mitochondrial targeted O 2 ·Ϫ scavenger, restored the relaxation response in PA rings pretreated with PFT-. Our observations suggest that iHSP70 chaperones SOD-2 to the mitochondria. Impaired SOD-2-iHSP70 dissociation decreases SOD-2 import and contributes to mitochondrial oxidative stress in PPHN.persistent pulmonary hypertension of the newborn; pulmonary artery endothelium; oxidative stress; vasodilation; nitric oxide OXIDATIVE STRESS PLAYS A KEY ROLE in the pathogenesis of pulmonary hypertension (14, 41a). Persistent pulmonary hypertension of the newborn (PPHN) is associated with increased generation of reactive oxygen species (ROS), including superoxide radical (O 2 ·Ϫ ) (10). ROS induces pulmonary vascular remodeling, which leads to increased vascular resistance and impaired pulmonary vasodilation in PPHN (46). Studies in a fetal lamb model of PPHN induced by prenatal constriction of the ductus arteriosus have previously identified increased NA-DPH oxidase activity and uncoupling of endothelial nitric oxide synthase (eNOS) as the major sources of O 2 ·Ϫ in the cytosol (10,33,36 ·Ϫ generation in the mitochondria, which in turn plays a major role in the toxicity of hyperoxia (44). Newborns are at risk of experiencing oxidative stress because O 2 concentrations increase by several times at birth compared with in utero environment. Adaptive increases in cellular antioxidant defenses, particularly superoxide dismutases (SODs), du...

Nogo-B receptor (NgBR) was identified as a specific receptor for binding Nogo-B, and is essential for the stability of Niemann-Pick type C2 protein (NPC2) and NPC2-dependent cholesterol trafficking. Here, we report that NgBR expression levels decrease in the fatty liver and that NgBR plays previously unrecognized roles in regulating hepatic lipogenesis via NPC2-independent pathways. To further elucidate the pathophysiological role of NgBR in mammals, we generated NgBR liver-specific knockout mice and investigated the roles of NgBR in hepatic lipid homeostasis. Our results showed that NgBR knockout in mouse liver did not decrease either NPC2 levels or increase NPC2-dependent intracellular cholesterol levels. However, NgBR deficiency still resulted in remarkable cellular lipid accumulation that was associated with increased free fatty acids (FFA) and triglycerides (TG) in hepatocytes in vitro and in mouse livers in vivo. Mechanistically, NgBR deficiency specifically promotes the nuclear translocation of the liver X receptor alpha (LXRα) and increases the expression of LXRα-targeted lipogenic genes. LXRα knockout attenuates the accumulation of FFA and TG caused by NgBR deficiency. In addition, we elucidated the mechanisms by which NgBR bridges the AMP-activated protein kinase α (AMPKα) signaling pathway with LXRα nuclear translocation and LXRα-mediated lipogenesis. Our study demonstrates that NgBR is a specific negative regulator for LXRα–dependent hepatic lipogenesis and suggests that loss of NgBR is a potential trigger for inducing hepatic steatosis.