Autophagy is a tightly regulated, programmed mechanism to eliminate damaged organelles and proteins from a cell to maintain homeostasis. Cisplatin, a chemotherapeutic agent, accumulates in the proximal tubules of the kidney and causes dose-dependent nephrotoxicity, which may involve autophagy. In the kidney, cisplatin induces the protective antioxidant heme oxygenase-1 (HO-1). In this study, we examined the relationship between autophagy and HO-1 during cisplatin-mediated acute kidney injury (AKI). In wild-type primary proximal tubule cells (PTC), we observed a time-dependent increase in autophagy after cisplatin. In HO-1 Ϫ/Ϫ PTC, however, we observed significantly higher levels of basal autophagy, impaired progression of autophagy, and increased apoptosis after cisplatin. Restoring HO-1 expression in these cells reversed the autophagic response and inhibited apoptosis after treatment with cisplatin. In vivo, although both wild-type and HO-1-deficient mice exhibited autophagosomes in the proximal tubules of the kidney in response to cisplatin, HO-1-deficient mice had significantly more autophagosomes, even in saline-treated animals. In addition, ecdysone-induced overexpression of HO-1 in cells led to a delay in autophagy progression, generated significantly lower levels of reactive oxygen species, and protected against cisplatin cytotoxicity. These findings demonstrsate that HO-1 inhibits autophagy, suggesting that the heme oxygenase system may contain therapeutic targets for AKI.

Recent data suggest that either excessive or deficient levels of protein S-nitrosylation may contribute to disease. Disruption of S-nitrosothiol (SNO) homeostasis may result not only from altered nitric oxide (NO) synthase activity but also from alterations in the activity of denitrosylases that remove NO groups. A subset of patients with familial amyotrophic lateral sclerosis (ALS) have mutations in superoxide dismutase 1 (SOD1) that increase the denitrosylase activity of SOD1. Here, we show that the increased denitrosylase activity of SOD1 mutants leads to an aberrant decrease in intracellular protein and peptide S-nitrosylation in cell and animal models of ALS. Deficient S-nitrosylation is particularly prominent in the mitochondria of cells expressing SOD1 mutants. Our results suggest that SNO depletion disrupts the function and͞or subcellular localization of proteins that are regulated by S-nitrosylation such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and thereby contributes to ALS pathogenesis. Repletion of intracellular SNO levels with SNO donor compounds rescues cells from mutant SOD1-induced death. These results suggest that aberrant depletion of intracellular SNOs contributes to motor neuron death in ALS, and raises the possibility that deficient S-nitrosylation is a general mechanism of disease pathogenesis. SNO donor compounds may provide new therapeutic options for diseases such as ALS that are associated with deficient S-nitrosylation.regulates a diverse array of biological processes by modifying critical cysteine residues on proteins (S-nitrosylation). Proteins acquire NO groups from a pool of intracellular S-nitrosylated peptides. The importance of Snitrosylation in physiology raises the possibility that dysregulated S-nitrosylation contributes to pathophysiology. Indeed, recent data suggest that excessive NO production and protein S-nitrosylation may contribute to disease. For instance, the E3 ligase parkin is aberrantly S-nitrosylated due to increased NO production in Parkinson's disease (1, 2). S-nitrosylation inhibits parkin activity, leading to the accumulation of misfolded proteins that may contribute to the death of dopaminergic neurons. Similarly, increased NO production during cerebral ischemia increases the Snitrosylation of matrix metalloproteinase-9 (MMP-9) (3). Snitrosylation of MMP-9 leads to the formation of a stable sulfinic or sulfonic acid derivative that is irreversibly activated, triggering neuronal apoptosis.Deficient S-nitrosylation has also been linked to disease pathogenesis. Specifically, increased activity of the denitrosylase Snitrosoglutathione (GSNO) reductase in asthmatic lungs depletes levels of the endogenous bronchodilator GSNO, leading to airway hyperresponsivity (4). Likewise, in sickle cell anemia, the sickle cell hemoglobin is deficient in the intramolecular and intermolecular transfer of NO moieties. Consequently, red blood cell membrane S-nitrosothiol (SNO) levels are decreased and hypoxic vasodilation is impaired, leading to vasoocclusion in hypoxic...

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radicalmediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.

SYNPOSIS Prototypical electrophiles such as the lipid 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) are well recognized for their therapeutic potential. Electrophiles modify signaling proteins in both the cytosol and mitochondrion which results in diverse cellular responses including cytoprotective effects and, at high doses, cell death. These findings led us to the hypothesis that targeting electrophiles to specific compartments in the cell can be used to fine-tune their biological effects. To examine this, we synthesized a novel mitochondrially-targeted analog of 15d-PGJ2 (mito-15d-PGJ2) and tested its effects on redox cell signaling. Mito-15d-PGJ2 caused profound defects in mitochondrial bioenergetics and mitochondrial membrane depolarization when compared to 15d-PGJ2. We also found that mito-15d-PGJ2 modified different members of the electrophile responsive proteome, was more potent at initiating intrinsic apoptotic cell death and was less effective than 15d-PGJ2 at upregulating the expression of heme oxygenase-1 and glutathione. These data demonstrate the feasibility of modulating the biological effects of electrophiles by targeting the pharmacophore to mitochondria.

When replete with zinc and copper, amyotrophic lateral sclerosis (ALS)-associated mutant SOD proteins can protect motor neurons in culture from trophic factor deprivation as efficiently as wild-type SOD. However, the removal of zinc from either mutant or wild-type SOD results in apoptosis of motor neurons through a copper-and peroxynitrite-dependent mechanism. It has also been shown that motor neurons isolated from transgenic mice expressing mutant SODs survive well in culture but undergo apoptosis when exposed to nitric oxide via a Fas-dependent mechanism. We combined these two parallel approaches for understanding SOD toxicity in ALS and found that zincdeficient SOD-induced motor neuron death required Fas activation, whereas the nitric oxide-dependent death of G93A SODexpressing motor neurons required copper and involved peroxynitrite formation. Surprisingly, motor neuron death doubled when Cu,Zn-SOD protein was either delivered intracellularly to G93A SOD-expressing motor neurons or co-delivered with zinc-deficient SOD to nontransgenic motor neurons. These results could be rationalized by biophysical data showing that heterodimer formation of Cu,Zn-SOD with zinc-deficient SOD prevented the monomerization and subsequent aggregation of zinc-deficient SOD under thiol-reducing conditions. ALS mutant SOD was also stabilized by mutating cysteine 111 to serine, which greatly increased the toxicity of zinc-deficient SOD. Thus, stabilization of ALS mutant SOD by two different approaches augmented its toxicity to motor neurons. Taken together, these results are consistent with copper-containing zinc-deficient SOD being the elusive "partially unfolded intermediate" responsible for the toxic gain of function conferred by ALS mutant SOD.Mutations to copper/zinc superoxide dismutase (SOD) 4 are the most common genetic cause of the familial form of amyotrophic lateral sclerosis (ALS) (1, 2). Although transgenic expression of these mutant SOD genes in mice and rats is sufficient to produce a progressive motor neuron disease that mimics human pathology (3), the toxic mechanisms remain obscure. Primary motor neuron cultures have proven to be a powerful model to elucidate the toxic gain of function conferred by ALS mutations to SOD and to determine the underlying cell death pathways. Motor neurons isolated from transgenic mice carrying ALS mutant SOD are fully viable in culture (4, 5) but undergo apoptosis after incubation with low, physiologically relevant concentrations of exogenous nitric oxide (50 -100 nM). This remains the most direct evidence that mutant SOD can become directly toxic to motor neurons but also demonstrates that expression of mutant SOD alone may not be sufficient to be toxic to motor neurons.The concentrations of nitric oxide used to induce death of ALS-SOD-expressing motor neurons were not injurious to motor neurons isolated from transgenic mice expressing wildtype SOD and are even trophic to nontransgenic motor neurons (6). Over the past decade, we have accumulated considerable evidence that the reacti...

. Mitochondria-targeted heme oxygenase-1 decreases oxidative stress in renal epithelial cells.

Background: Heat shock protein 90 (Hsp90) is a pro-survival molecular chaperone that is nitrated only in pathological conditions. Results: Hsp90 nitrated on tyrosine 33 down-regulates mitochondrial activity. Conclusion: Differential nitration states of Hsp90 regulate distinct aspects of cellular metabolism. Significance: This is the first demonstration of a site-specific-nitrated protein regulating mitochondrial metabolism.

SYNOPSIS Recently, a number of steps in the progression of metastatic disease have been shown to be regulated by redox signaling. Electrophilic lipids affect redox signaling through the post-translational modification of critical cysteine residues in proteins. However, the therapeutic potential as well as the precise mechanisms of action of electrophilic lipids in cancer cells is poorly understood. In this study, we investigate the effect of the electrophilic prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) on metastatic properties of breast cancer cells. 15d-PGJ2 was shown to decrease migration, stimulate focal adhesion disassembly and cause extensive F-actin reorganization at low concentrations (0.03-0.3 μM). Importantly, these effects seem to be independent of PPARγ and modification of actin or Keap1, which are known protein targets of 15d-PGJ2 at higher concentrations. Interestingly, the p38 inhibitor SB203580 was able to prevent both 15d-PGJ2-induced F-actin reorganization and focal adhesion disassembly. Taken together, our results suggest that electrophiles such as 15d-PGJ2 are potential anti-metastatic agents which exhibit specificity for migration and adhesion pathways at low concentrations where there are no observed effects on Keap1 or cytotoxicity.