The PON gene family consists of three members, PON1, PON2, and PON3. All PON proteins have been implicated in the pathogenesis of inflammatory diseases, including atherosclerosis, Alzheimer's, Parkinson's, diabetes, and cancer. We have shown that stably transfected cells overexpressing PON2 exhibit significantly lower levels of intracellular oxidative stress when exposed to H2
). In addition, studies in mice suggest that PON2 protects against atherosclerosis (21
) by modulating intracellular oxidative stress. We have previously reported that PON2 is localized to mitochondrial membranes and may play a role in mitochondrial oxidative stress (3
); however, the mechanism(s) how PON2 modulates oxidative stress is still elusive and has been the focus of a great deal of research in recent years.
Accumulating evidence from in vitro
, in vivo
, and epidemiological studies suggests an important role for oxidative stress in the pathogenesis of atherosclerosis. Mitochondria are the major source of free radical-related oxidative stress. Decreased activity of mitochondrial ETC complexes activity results in increased production of ROS (2
), which play an important role in the development of many inflammatory diseases, including atherosclerosis. Our results suggest that PON2 deficiency under atherogenic conditions disrupts the activities of ETC complexes I
III, resulting in the production of superoxide that leads to enhanced mitochondrial oxidative stress. Further, mitochondrial function is impaired in PON2-def peritoneal macrophages, based on multiple indications, including oxygen consumption, ATP synthesis, and superoxide production, which are all direct measures of the efficacy of respiratory complex activity. In addition, mitochondrial superoxide was significantly elevated in the arotas of PON2- def mice. All these parameters indicate mitochondrial dysfunction in PON2-def in a manner that aggravates atherosclerosis lesions.
We initially identified Rieske iron sulfur protein as an interacting partner of PON2 by yeast two-hybrid screen (data not shown). This protein is localized to the mitochondria, as a subunit of respiratory complex III, where superoxide is primarily generated via
the Q cycle (13
). In the current investigation, we found that PON2 indeed localizes in the mitochondria, preferentially localizing to the IMM. Consistent with our study, McDonald et al.
) isolated the IMM and identified 154 previously unreported proteins that included PON2 (but not PON1 nor PON3). The IMM plays a vital role in the regulation of energy metabolism, oxidative stress, and apoptosis. Since the IMM harbors the respiratory complexes of the ETC, we subsequently examined whether PON2 interacted with any of the respiratory complexes and found that, among the four respiratory complexes, PON2 was found to be potentially associated with complex III. It is well established that within the IMM, superoxide is continuously generated and released into the matrix at complex I and released both into the matrix and intermembrane space at complex III (15
). The steady state concentration of superoxide in the mitochondrial matrix has been shown to be approximately 10-fold higher than that in the cytosolic and nuclear spaces (2
). All other ROS such as peroxynitrate, hydrogen peroxide, and hydroxy radicals are formed from superoxide. Therefore, as major sources of ROS production, the IMM could be a major target of ROS attack. Accordingly, the effects of ROS would be expected to be greatest at the level of IMM constituents, including the complexes of the respiratory chain and phospholipid(s) constituents particularly rich in unsaturated fatty acids, including cardiolipin (10
). Although mitochondria possess antioxidant enzymes, they are not positioned to protect the immediate surroundings of the respiratory complexes. For example, Mn superoxide dismutase (SOD) and glutathione peroxidase are located on the matrix side, whereas CuSOD resides both in cytoplasm and in the inner mitochondrial membrane space (15
). Perhaps PON2 on the inner membrane serves to reduce and inactivate some of the oxidized lipids as soon as they are formed, and at their source.
While the immunoprecipitation data demonstrate association of PON2 with complex III of the IMM (), it is feasible that PON2 is binding directly to the fatty acid tail of CoQ10
that associates with complex III. It has been reported that 32% of the CoQ10
in the mitochondria is associated with membrane proteins (12
) and that species with longer life-span contain higher amounts of protein-bound CoQ10
. X-ray crystallography studies suggest that fatty acid tail side chains are likely to fit into the substrate pocket of PON enzymes (8
has a fatty acid tail, is synthesized in the IMM, and plays a role in the bioenergetics pathway. Ubisemiquinone is a free radical that results from the process of dehydrogenation of hydroquinone to quinone. This unstable ubisemiquinone (involved in Q cycle) can donate a single electron to molecular oxygen to produce superoxide and reduce the ETC activity (13
). Our lipid protein binding studies suggest that CoQ10
binds rPON2 with an affinity of KD
M. Indeed, PON2-def mice show enhanced mitochondrial superoxide compare to control with impaired mitochondrial dysfunction. It has been widely accepted that steady-state concentration of ubisemiquinone was increased in the IMM by antimycin in a manner that produces superoxide, and our in vitro
studies further suggest that overexpression of PON2 reduces the superoxide level induced by antimycin. It is therefore plausible that PON2 maintains the respiratory chain by promoting the sequestration of the unstable reactive intermediate ubisemiquinone, thereby preventing the superoxide production. Supporting our hypothesis, previously, it has been shown that mitochondrial superoxide is inversely related to the amount of CoQ10
bound to membrane proteins (12
It is well established that oligomycin inhibits ATP synthase by blocking its proton channel (Fo subunit, which is required for oxidative phosphorylation of adenosine diphosphate to ATP) and leads to an increase in the proton gradient, which in turn reduces the respiratory activity and oxidative phosphorylation leading to mitochondrial dysfunction (11
). Interestingly, PON2 overexpression restores the ATP level, which further supports our hypothesis that PON2 protects the mitochondria from adverse conditions.
Despite the role in mitochondrial function, we did not see a severe phenotype in PON2-def mice as was reported for the MnSOD knockout mouse model. It should be noted that in the MnSOD knockout mouse model, superoxide can not be converted to hydrogen peroxide and the accumulating mitochondrial superoxide promotes the formation of peroxynitrite and hydroxyl radicals that result in a severe phenotype, which occurs only in the homozygous mice and not heterozygous mice. In contrast, in PON2-def model, we speculate that the downstream pathways needed to convert superoxide into water including MnSOD, Glutathione Peroxidase, and Catalase are intact. Moreover, PON2-def mouse is not a total knockout. We have previously reported that the PON2-def mouse is deficient in PON2 and that tissue expression of PON2 is roughly 5–10% that of wild-type mice (21
). Finally, it has been reported previously that mitochondrial dysfunction does not always correlate with severity in phenotype (1
Accumulation of unfolded proteins in the endoplasmic reticulum causes oxidative stress via
the unfolded protein response (UPR) (28
). Horke et al.
) reported that overexpression of PON2 protects vascular cells from UPR-induced oxidative stress and apoptosis. Recent work from various laboratories suggests that under pathophysiological conditions there is a crosstalk between endoplasmic reticulum and mitochondria via
both calcium-dependent and -independent pathways, which ultimately affects the function of mitochondria (28
). Studies are underway in our laboratory to address and understand the role of PON2 in this respect.
In conclusion, our data suggest that PON2 is an IMM protein that plays an important role in the redox mechanisms of the respiratory chain. We report here for the first time that PON2-deficiency causes mitochondrial dysfunction, which may, in part, be responsible for the aggravated atherosclerosis observed in PON2-def mice.