No abstract

hYVH1 [human orthologue of YVH1 (yeast VH1-related phosphatase)] is an atypical dual-specificity phosphatase that is widely conserved throughout evolution. Deletion studies in yeast have suggested a role for this phosphatase in regulating cell growth. However, the role of the human orthologue is unknown. The present study used MS to identify Hsp70 (heat-shock protein 70) as a novel hYVH1-binding partner. The interaction was confirmed using endogenous co-immunoprecipitation experiments and direct binding of purified proteins. Endogenous Hsp70 and hYVH1 proteins were also found to co-localize specifically to the perinuclear region in response to heat stress. Domain deletion studies revealed that the ATPase effector domain of Hsp70 and the zinc-binding domain of hYVH1 are required for the interaction, indicating that this association is not simply a chaperone-substrate complex. Thermal phosphatase assays revealed hYVH1 activity to be unaffected by heat and only marginally affected by non-reducing conditions, in contrast with the archetypical dual-specificity phosphatase VHR (VH1-related protein). In addition, Hsp70 is capable of increasing the phosphatase activity of hYVH1 towards an exogenous substrate under non-reducing conditions. Furthermore, the expression of hYVH1 repressed cell death induced by heat shock, H2O2 and Fas receptor activation but not cisplatin. Co-expression of hYVH1 with Hsp70 further enhanced cell survival. Meanwhile, expression of a catalytically inactive hYVH1 or a hYVH1 variant that is unable to interact with Hsp70 failed to protect cells from the various stress conditions. The results suggest that hYVH1 is a novel cell survival phosphatase that co-operates with Hsp70 to positively affect cell viability in response to cellular insults.

Oxidative stress leads to mitochondrial dysfunction, which triggers the opening of the permeability transition pores (PTP) and the release of pro-apoptotic factors causing apoptotic cell death. In a limited number of cell systems, anti-oxidants and free-radical scavengers have been shown to block this response. We have previously reported that coenzyme Q(10) (CoQ(10)), an electron carrier in the mitochondrial respiratory chain, is involved in the reactive oxygen species (ROS) removal and prevention of oxidative stress-induced apoptosis in neuronal cells. However, the mechanism of this protection has not been fully elucidated. In the present study we investigated the effects of CoQ(10) on the mitochondrial events characteristic to apoptosis, especially on the function of pro-apoptotic protein Bax. Our results demonstrated that following a brief exposure of two human cell lines (fibroblasts and HEK293 cells) to H(2)O(2) the intracellular levels of ROS and the association of Bax with the mitochondria significantly increased and the cells underwent apoptosis. Both of these events, as well as the release of cytochrome c from the mitochondria, were blocked by a 24 h pre-treatment with CoQ(10). It is therefore believed that CoQ(10) prevented the collapse of the mitochondrial membrane potential in response to the H(2)O(2) treatment. Recombinant Bax protein alone caused the ROS generation and release of cytochrome c from isolated mitochondria and, again, CoQ(10) inhibited these Bax-induced mitochondrial dysfunctions.