4E). Overexpressing MsrA was sufficient to increase specifically the activity of complex-IV of the respiratory chain, while activity of complex-II and mitochondrial content were unaffected. Thus, MsrA likely enhances ATP synthesis by increasing complex-IV activity. Such contribution of MsrA to energy metabolism is independent of its function in protection from elevated oxidative stress but contributes to routine but vital photoreceptor support by RPE cells. oocytes, a process reversed by MsrA overexpression . Methionine oxidation contributes to the activation of calcium/calmodulin-dependent protein kinase II suggesting a possible role for reversible oxidation in signal transduction pathways . Identification of specific MsrA substrates and cellular processes controlled by MsrA remains an active area of investigation. Survival and functionality in vision of photoreceptor neurons in the retina require continuous support by the neighboring retinal pigment epithelium (RPE)1 (reviewed in . Like photoreceptors, mammalian RPE cells are post-mitotic and subjected to a lifetime of photo-oxidative stress. Most RPE functions are dependent on sufficient availability of ATP generated by oxidative phosphorylation in mitochondria. Mitochondrial defects severely impair the functions of the RPE and in cell culture [10, 11]. Decline in mitochondrial activity is associated with aging of the human RPE and the development of age-related macular degeneration (AMD) . The molecular mechanisms controlling mitochondrial ATP synthesis efficiency in RPE cells have not yet been extensively studied. Earlier reports have shown a role for MsrA in protection of FRAP2 RPE cells from excess oxidative stress (reviewed in ). In rat retina, MsrA is abundant in the RPE . In monkey retina, MsrA levels are highest in the RPE in the macular region of the retina where RPE cells (+)-JQ1 must support a particularly high number of tightly packed cone photoreceptors . In human retina, MsrA localizes to the RPE and in part to drusen deposits beneath the RPE that are associated with AMD . RPE cells in culture respond to moderate levels of experimental oxidative stress by increasing MsrA expression. Acutely reducing MsrA of RPE cells by (+)-JQ1 gene silencing enhances cytotoxicity of oxidative stress [3, 14]. We hypothesized that MsrA may support the routine functions of unstressed RPE cells. (+)-JQ1 Whether or not MsrA fulfills functions in RPE cells other than protection from acute oxidative damage has not yet been directly investigated. The continuous clearance of shed photoreceptor outer segment fragments (POS) by phagocytosis and their prompt and complete digestion are among critical RPE responsibilities. POS phagocytosis employs the RPE F-actin cytoskeleton and its phago-lysosomal organelles all of which must be intact and dynamic [15, 16]. POS phagocytosis is a costly process that requires ATP synthesis by RPE mitochondria . Sensitive experimental uptake assays can accurately and with high sensitivity quantify phagocytic binding and engulfment of purified POS by RPE cells in culture. In this study, we characterized the effects of specifically decreasing or increasing MsrA on the phagocytic function of RPE cells in culture reasoning that even moderate changes in RPE function will affect RPE phagocytosis. We compared the effects of altered MsrA expression on phagocytic activity and cell viability in the presence of hydrogen peroxide, trolox antioxidant, or mitochondrial respiratory chain inhibitors. We determined that MsrA promotes phagocytic function by increasing the activity of complex-IV of the respiratory chain and as a result mitochondrial ATP synthesis, regardless of the levels of oxidative stress. Conversely, MsrA protection from damage by hydrogen peroxide was unaffected by mitochondrial inhibition. Thus, MsrA supports RPE function by independently supporting mitochondrial ATP synthesis and counteracting oxidative damage. Materials and methods All reagents were purchased from Sigma (St. Louis, MO) or Invitrogen (Carlsbad, CA) unless otherwise (+)-JQ1 specified. Antibodies Opsin.
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