Functional Characterizations Of The Gene Product-OLA1(Obg Like ATPase 1)

Glutathione (L-γ-glutamyl-L-cysteinyl-glycine; GSH), the most prevalent non-protein thiol in mammalian cells and the most abundant low molecular weight peptide present in eukaryotic cells. Today the implication of GSH in many cellular functions has been recognized. GSH acts as a reducing agent and an antioxidant, is involved in the metabolism of xenobiotics and different cell molecules, is a free-radical scavenger, and has a role in cell-cycle regulation and microtubular-related mechanisms. Due to these functions and to the roles of reactive oxygen (ROS) and nitrogen (RNS) species in cell signaling and in many human pathological processes, alterations in GSH levels and metabolism have been associated with different human diseases, including cancer, neurodegenerative diseases, acquired immune deficiency syndrome (AIDS), aging, cystic fibrosis, liver diseases, heart attack, stroke, seizure, diabetes, sickle cell anemia, and kwashiorkor.In order to further study the effects of lack of glutathione on cellular physiological activities, we developed mice deficient in the heavy subunit ofγ-glutamyl cysteine synthetase. We have successfully isolated cell lines from homozygous mutant blastocysts by culturing them in medium containing GSH. SuchγGCS-deficient cells grew for only a few days in the absence of GSH or NAC: cellular GSH drops to an undetectable level within 24 hours and cells eventually die. The microarray was applied to study the gene expression changes during this process. We have identified 32 candidate clones the expression of which either increases or decreases at least twofold 24 hours after the withdrawal of GSH. Database analysis suggested that these genes fell within the functional categories of cell cycle, apoptosis, metabolism, and unknown function. In the present study, we have performed functional characterizations on one of the gene products, originally named GTPBP9 or PTD004, and recently defined as a new member of the Obg-like ATPase family-OLA1, in human cells.OLA1, as the homolog of the E. coli P-loop NTPase YchF, is a member of the Obg-related family of GTPases belonging to the TRAFAC (translation factors) class. The deduced 396-amino acid protein consists of an N-terminal G domain, flanked on either side by an inserted coiled-coil, and a C-terminal TGS domain. It is a conserved gene and ubiquitinly expressed in many cell lines.When OLA1 was knocked down with short interference RNA (siRNA), cells gained more resistance to the cytotoxicity of diamide, a glutathione oxidation agent, and tert-butyl hydroperoxide (tBH), a peroxide oxidant. On the other hand, when OLA1 is overexpressed by plasmid transfection, cells became more sensitive to these stressors. However, knockdown of OLA1 exhibited no changes in the sensitivity of cells to mitochondria- or DNA-damaging agents including antimycin A, etoposide, and doxorubincine. Moreover, no notable difference in cell proliferation and baseline apoptosis was seen in the OLA1-knockdown cells.We further investigated the levels of reactive oxygen species (ROS) in the Ola1-knockdown cells by flow cytometry after DCFDA staining, and confirmed a significant decrease in ROS production of intracellular ROS when the cells were briefly challenged with tBH, as compared with the cells transfected with non-targeting siRNA. Meanwhile we have shown that the OLA1-knockdown cells maintained a higher level of reduced glutathione from the very beginning of the treatment of either diamide or tBH. In order to understand why the cells possess more reduced glutathione and/or can better sustain the oxidation of glutathione, we performed a genome-wide expression assay using the Agilent microarrays. Surprisingly, the knockdown of OLA1 caused only a minimal genomic response, and neither genes encoding antioxidant enzymes and enzymes for producing antioxidants (such as glutathione) were found to have any detectable changes at the mRNA level. Moreover, when de novo protein synthesis was blocked by cycloheximide in OLA1-knockdown cells, they continued to demonstrate increased resistance to both tBH and diamide. These data demonstrate that OLA1 suppresses the antioxidant response through nontranscriptional mechanisms.Moreover, we found that OLA1 played a very important role in cancer metastasis. Reactive oxygen species (ROS) are proposed to be signaling intermediates involved in metastatic processes including cell migration and invasion. We found that siRNA-mediated knockdown of OLA1 significantly inhibited cell migration and invasion in breast cancer cell line MBA-MD-231, as assessed by in vitro cell migration and invasion assays, respectively. Knockdown of OLA1 caused no changes in cell growth but affected ROS production. In wound-healing assays, decreased ROS in OLA1-knockdown cells were in situ associated with the cells' decreased motile morphology. Further, treatment of N-acetylcysteine, a general ROS scavenger, blunted the motility and invasiveness of MDA-MB-231, similar to the effect of OLA1-knockdown. These results suggest that knockdown of OLA1 inhibits breast cancer cell migration and invasion through a mechanism that involves the modulation of intracellular ROS levels. Taken together, our data suggest that OLA1 is a novel negative regulator specifically for the cellular anti-oxidative response system, and that this regulation is through a post-translational mechanism (e.g., enzymatic activity modulation and protein degradation). And knockdown of OLA1 could impair the cell migration and invasion ability, meaning a potential clinical application. Further, since the OLA1 protein is an ATPase, a common druggable structure, the above beneficial effects associated with the OLA1-knockdown may implicate OLA1 as a drug target for anti-oxidative and anti-metastasis therapy.