Expression and function of Yno1, a yeast NADPH oxidase

In two recent publications (Rinnerthaler et al. 2012; Leadsham et al. 2013) we have shown that the yeast (S. cerevisiae) ORF, YGL160W, codes for a genuine NADPH oxidase producing the superoxide radical anion, wich we named Yno1p. The enzyme is located in the perinuclear and cortical ER. Enzyme activity in vitro (purified microsomes) is NADPH dependent and is inhibited by DPI. Overexpression of Yno1p causes apoptotic cell death and a marked increase of the bud index in stationary cells. The Yno1p deletion mutant is resistant to oxidative stress by external H2O2 and hypersensitive to the well known cancer drugs, wiskostatin and latrunculinB. Both drugs inhibit remodeling of the actin cytoskeleton, which is needed in every cell division cycle of yeast, but also during stress, glucose starvation, cell cycle arrest and return to growth. A low non-toxic concentration of H2O2 can completely counteract this inhibition. In the subsequent paper (Leadsham et al. 2013), we showed that Yno1 is responsible for the major part of ROS production in respiratory deficient cox4 mutant cells in the post-diauxic phase. Yno1 is degraded by ERAD in wild type cells at post-diauxie (after glucose exhaustion), but is stabilized and active in cox4 mutant cells, a process which induces cell killing and requires the translocation of active Ras2 to mitochondria and the activity of the Bdf1 bromodomain transcription factor. A third paper relevant for this project (Reddi et al. 2012) shows that the yeast casein kinases Yck1 and Yck2 are downstream effectors of the H2O2 signal created by Yno1 with Sod1 which represses respiration on glucose media. Based on these findings our working hypothesis is that the physiological role of Yno1 is to create reactive oxygen species (ROS) as molecular signals needed for the redox-mediated regulation of the actin cytoskeleton in answer to glucose and/or glucose consumption. Redox regulation of the cytoskeleton and internal signaling through NADPH oxidases have not been previously investigated in yeast. Our aims in this project are: i) to identify and quantitate the signaling ROS, which we expect to be H2O2 interaction of Yno1p with Sod1p will be tested in this respect; ii) to test the hypothesis that Yno1p acts as a signal enhancer in conjunction with mitochondria; and iii) to identify protein modules which can act in the transmission mechanism to the actin cytoskeleton of the signal produced by Yno1p. We are investigating here a new mode of regulation of the cyoskeleton and of the cell cycle which potentially can be important for understanding and improving tumor therapy directed to the cytoskeleton.

In this project we found that a conserved eukaryotic NADPH oxidase oft he endoplasmic reticulum through production of hydrogen peroxide as a second messenger can regulate the actin cytoskeleton depending on growth conditions and in particular during the formation of pseudohyphae. In cancer cells the orthologous NADPH oxidase is responsible for the mobility of the cells (which depends on the actin cytoskeleton), and therefore, for the process of metastasis. This enzyme is therefore a new and promising target for cancer therapy.