No abstract

Recently, it has been reported that mitochondria possess a novel pathway for nitric oxide (NO) synthesis. This pathway is induced when cells experience hypoxia, is nitrite (NO 2 ؊ )-dependent, is independent of NO synthases, and is catalyzed by cytochrome c oxidase (Cco). It has been proposed that this mitochondrially produced NO is a component of hypoxic signaling and the induction of nuclear hypoxic genes. In this study, we examine the NO 2 ؊ -dependent NO production in yeast engineered to contain alternative isoforms, Va or Vb, of Cco subunit V. Previous studies have shown that these isoforms have differential effects on oxygen reduction by Cco, and that their genes (COX5a and COX5b, respectively) are inversely regulated by oxygen. Here, we find that the Vb isozyme has a higher turnover rate for NO production than the Va isozyme and that the Vb isozyme produces NO at much higher oxygen concentrations than the Va isozyme. We have also found that the hypoxic genes CYC7 and OLE1 are induced to higher levels in a strain carrying the Vb isozyme than in a strain carrying the Va isozyme. Together, these results demonstrate that the subunit V isoforms have differential effects on NO 2 ؊ -dependent NO production by Cco and provide further support for a role of Cco in hypoxic signaling. These findings also suggest a positive feedback mechanism in which mitochondrially produced NO induces expression of COX5b, whose protein product then functions to enhance the ability of Cco to produce NO in hypoxic/anoxic cells.hypoxia ͉ mitochondria ͉ reactive oxygen species ͉ nitrite ͉ yeast I t has been known for quite some time that the mitochondrial respiratory chain is capable of generating reactive oxygen species (ROS) that account for much of the oxidative stress experienced by cells (1-3). The levels of these ROS increase when electron flow through the respiratory chain is inhibited by respiratory inhibitors (4-6) or altered by uncoupling electron transport from oxidative phosphorylation (7,8). Several studies have shown that exposure of cells and tissues to hypoxia increases ROS levels and oxidative stress (9-11). This increase in oxidative stress during exposure to hypoxia depends on a functional mitochondrial respiratory chain (10). It is currently unclear whether this increase is the result of increased generation or decreased destruction of ROS under hypoxic conditions. In yeast cells, the increase in ROS levels and oxidative stress is transient, as determined by shifting cells from normoxia to anoxia (10). During this shift, cells experience a continuum of decreasing oxygen concentrations and a transient increase in the levels of carbonylation of both mitochondrial and cytosolic protein, an increase in 8-OH-dG levels in mtDNA, and an increase in expression of the SOD1 gene. Many of the proteins that are carbonylated during a shift from normoxia to anoxia are the same proteins that are carbonylated when cells are exposed to menadione, a redox recycling agent that produces elevated intracellular levels of superoxide (12). ...

No abstract

Most small molecule drugs act on living systems by physically interacting with specific proteins and modulating target function. Identification of drug binding targets, within the complex milieu of the human proteome, remains a challenging task of paramount importance in drug discovery. Existing approaches for target identification employ complex workflows with limited throughput. Here, we present the isothermal shift assay (iTSA), a mass spectrometry method for proteome-wide identification of drug targets within lysates or living cells. Compared with prevailing methods, iTSA uses a simplified experimental design with increased statistical power to detect thermal stability shifts that are induced by small molecule binding. Using a pan-kinase inhibitor, staurosporine, we demonstrate improved performance over commonly used thermal proteome profiling methods, identifying known targets in cell lysates and living cells. We also demonstrate the identification of both known targets and additional candidate targets for the kinase inhibitor harmine in cell and tissue lysates.

Eukaryotic cells respond to low oxygen concentrations by upregulating hypoxic and downregulating aerobic nuclear genes (hypoxic signaling). Most of the oxygen-regulated genes in yeast require the mitochondrial respiratory chain for their up- or downregulation when cells experience hypoxia. Although it was shown previously that the mitochondrial respiratory chain is required for the upregulation of some hypoxic genes in both yeast and mammalian cells, its underlying role in this process has been unclear. Recently, we have reported that mitochondria produce nitric oxide (NO(*)) when oxygen becomes limiting. This NO(*) production is nitrite (NO(2) (-))-dependent, requires an electron donor, and is carried out by cytochrome c oxidase in a pH-dependent fashion. We call this activity Cco/NO(*) and incorporate it into a new model for hypoxic signaling. In addition, we have found that some of the NO(*) produced by Cco/NO(*) is released from cells, raising the possibility that mitochondrially generated NO(*) also functions in extracellular hypoxic signaling pathways.