In mammals, the overall homology of PHGPx compared with the other Sec- containing GPxs is less than 40 %, yet they share conserved motifs that include the catalytic triad of Sec, Gln and Trp. This conserved triad forms a catalytic centre in which the selenol group of Sec is stabilized and activated by hydrogen bonds provided by the Gln and Trp residues (Maiorino et al., 1995). An initial mutational and biochemical approach by Maiorino et al. revealed that the conversion of Sec to Cys causes a marked decrease of PHGPx activity by about three orders of magnitude in the recombinant protein (Maiorino et al., 1995). Also mutations of Gln and Trp of the
Discussion (PHGPx)
catalytic triad reduced PHGPx enzyme activity, highlighting the importance of the proposed catalytic centre.
The lentiviral add-back system for wild type and various mutant forms of PHGPx in the PHGPx null background proved to be an ideal tool for mutagenesis studies on a cellular level, with main focus on the active site Sec. MEFs were reconstituted with PHGPx mutants, containing Ser and Cys substitutions for Sec. The Ser-containing mutant of PHGPx did not protect PHGPx-deficient cells from cell death, demonstrating that Sec or Cys at this position is absolutely essential for PHGPx function. Surprisingly, the Cys-containing PHGPx mutant fully rescued MEFs from cell death after Cre-mediated disruption of the endogenous PHGPx alleles and no difference in susceptibility towards peroxides was detected. Of note, the translational insertion of Sec is a complex process with a rather low efficiency, ranging around 4 % compared to the incorporation of other amino acids (Suppmann et al., 1999). Consequently, the expression levels of Sec/Ser and Sec/Cys mutants were significantly higher compared to wild type PHGPx. These findings demonstrate that despite its dramatically reduced enzyme activity (Maiorino et al., 1995), the Sec/Cys PHGPx mutant can fully substitute wild type PHGPx in vitro.
This surprising finding raises the question why mammalian cells express selenoproteins at all, when Cys-containing variants are apparently capable to replace Sec-containing wild type counterparts, at least for normal cell function. Certainly, findings obtained from in vitro cell culture studies can not be entirely translated to the
far more complex system of a whole organism. At present, it can only be speculated that the Sec-containing wild type PHGPx exerts cellular functions beyond its activity as antioxidant enzyme, e.g. during embryonic and tissue development, stress conditions, and sperm development. This hypothesis will be addressed in our laboratory by the production of knockin mice, harbouring the Cys-containing mutant of PHGPx. Moreover, the impact of PHGPx on cellular processes may vary significantly between different cell types. To address this question, ongoing studies in my laboratory will unravel, whether the Cys-containing variant is also able to replace wild type PHGPx in other in vitro PHGPx knockout systems, such as ES cells,
Discussion (PHGPx)
Since H2O2 and other hydroperoxides are now considered as not mere toxic compounds, but have drawn great attention as important second messengers, it has been speculated that PHGPx regulates various cellular processes by sensing and transducing the redox tone of the cell. Further mutational studies, using the inducible knockout system, may gain novel insights into these enzymatic mechanisms of PHGPx. In this respect, the nine Cys residues in the PHGPx gene are of major interest, since certain Cys residues may form a redox couple or also in cooperation with the Sec.
One putative mechanism involves redox sensor and transducer reactions, as initially described for the yeast PHGPx homologue, GPx3 (Delaunay et al., 2002). Upon increasing H2O2 levels, yeast GPx3 senses and transduces the stress signal to the transcription factor Yap1. The peroxidase function of yeast GPx3 was shown to involve the oxidation of Cys36 and Cys82 to form a transient intra-molecular Cys36-SS- Cys82 bridge. Subsequently, Yap1 is activated by yeast GPx3, involving a transient disulfide bond between GPx3 Cys36 (Sec46 homologue in
mus musculus) and Cys598
of Yap1. So far, no equal mechanism has been reported for mammalian PHGPx. But this cellular PHGPx knockout/knockin tool will prove most suitable to investigate this putative H2O2 sensing and transducing mechanism of mammalian PHGPx.
In the cell, ROS are not only generated as detrimental side products of the mitochondrial respiration, but are also produced for essential physiological functions. H2O2 production is catalysed by NADPH oxidases such as NOXs (NADPH oxidase) and DUOXs (dual oxidase), (Rueckschloss et al., 2003) mainly for the regulation of detoxification processes and inflammatory responses. Upon cellular infection, neutrophils and phagocytic cells produce large amounts of ROS in order to kill invading bacteria. Yet, NOX isoforms were also found in a number of non-phagocytic cells and tissue, indicating that the generation of ROS is a rather general feature of all somatic cells than being restricted to phagocytic cells (Lambeth, 2004). In this regard, H2O2 seems to be the major component of receptor mediated ROS production, which can be stimulated by cytokines or growth factors, such as transforming growth factor-β1 (TGF-β1) (Thannickal and Fanburg, 1995), interleukin- 1 (Meier et al., 1989), tumor necrosis factor α (TNFα) (Lo et al., 1996), platelet- derived growth factor (PDGF) (Krieger-Brauer and Kather, 1995; Sundaresan et al.,
Discussion (PHGPx)
1995), epidermal growth factor (EGF) (Bae et al., 1997) and basic fibroblast growth factor (bFGF) (Krieger-Brauer and Kather, 1995; Lo et al., 1996). ROS have been shown to activate mitogen-activated protein kinases (MAPK) (Sundaresan et al., 1995) and protein kinase C (Konishi et al., 1997), most likely by the specific inactivation of phosphatases (Lee et al., 1998). This has been nicely demonstrated by Kamata et al., showing that JNK-inactivating phosphatases are inhibited by ROS, by converting the catalytically active Cys to sulfenic acid (Kamata et al., 2005). Initially, Meng et al. demonstrated a reversible inactivation of multiple protein tyrosine phosphatases by H2O2 in vivo (Meng et al., 2002). Only recently, the established
cellular PHGPx knockout system was used in our laboratory, to address a putative impact of PHGPx on PDGFβ receptor signalling. By using this system, it has been shown that PHGPx antagonizes PDGFβ receptor signalling in MEFs, most likely by regulating lipid-associated ROS levels. Thereby, lipid-associated ROS transiently oxidize and thus inactivate counteracting protein tyrosine phosphatases (Conrad et al., in preparation).
ROS have also been reported to act as physiological mediators of transcriptional control by activating transcription factors, such as activation protein-1 (AP-1) (Meyer et al., 1993) and NF-κB (Schreck et al., 1991). Interestingly, PHGPx overexpression has been shown to inhibit the expression of NF-κB target genes, such as IL-1 mediated induction of VCAM-1 in smooth muscle cells (Banning et al., 2004) and TNF-induced COX2 expression in L929 cells (Heirman et al., 2006), by dampening intracellular ROS levels.
In conclusion, PHGPx may be involved in various cellular processes which have been masked by the lethal effects, caused by to the complete removal of PHGPx. The presented in vitro system, including the efficient reconstitution of PHGPx
mutants, provides an ideal tool for further investigations on PHGPx functions. This may provide additional knowledge to the herein identified function of PHGPx, as a regulator of a distinct cell death-inducing pathway by sensing and transducing oxidative stress inside the cell.
Discussion (xCT)