Pavel P. Philippov

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals

Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6′-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS-induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age_induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 μM SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases.

The presence of a calcium‐sensitive p26‐containing complex in bovine retina rod cells

A protein with apparent M r, 26 kDa (p26 [(1991) Biokhnya 56, 225‐228] or recoverin [(1991) Science 251, 915‐918]) was suggested to activate GC in a calcium dependent manner in bovine retina rod cells [(1991) Science 251, 915‐918; (1991) EMBO J, 10, 793‐798]. However, according to our present data homogeneous p26 preparations do not activate the enzyme. At the same time we have revealed a complex of p26 with an unidentified protein, presumably RK, in bovine ROS. Calcium favours formation of the complex whereas EGTA addition (which corresponds to a low free Ca2+ concentration) leads to its dissociation.

Transducin GTPase provides for rapid quenching of the cGMP cascade in rod outer segments.

The role of transducin GTPase in rapid cGMP phosphodiesterase quenching was studied by simultaneous registration of GTP hydrolysis and phosphodiesterase activity in the same rod outer segments (ROS) preparation. The results thus obtained allow the conclusion that: (i) phosphodiesterase quenching coincides with transducin‐bound GTP hydrolysis independently of ROS concentration; (ii) an increase in the ROS concentration results in the acceleration of cascade quenching due to the existence of a GTPase accelerating mechanism in ROS; (iii) approximation to physiological conditions (protein concentration, temperature) provides a transducin GTPase rate equal to 1–2 turnovers per second i.e., sufficiently high for satisfying the real rate of photoresponse reversion in dark‐adapted rods.

The effect of rhodopsin phosphorylation on the light-dependent activation of phosphodiesterase from bovine rod outer segments

ATP quenches light‐dependent phosphodiesterase (PDE) activation in rod outer segments presumably due to rhodopsin phosphorylation. Here we compared the efficiency of phosphorylated and non‐phosphorylated rhodopsins as PDE activators in a reconstituted cell‐free system. It is shown that the ability of phosphorylated membranes to activate this enzyme is supressed compared with non‐phosphorylated ones.

N-Myristoylation of recoverin enhances its efficiency as an inhibitor of rhodopsin kinase

Recoverin, a recently identified member of the EF‐hand superfamily of Ca2+‐binding proteins, is capable to inhibit rhodopsin phosphorylation by rhodopsin kinase at high but not at low free [Ca2+]. The N‐terminal glycine residue of retinal recoverin is heterogeneously acylated with myristoyl or related N‐acyl group. To clarify the role of the N‐terminal acylation of recoverin in its inhibitory action upon rhodopsin phosphorylation, we compared the efficiency of myristoylated and non‐myristoylated forms of recombinant recoverin as inhibitors of rhodopsin kinase activity. We have found that rhodopsin phosphorylation by purified rhodopsin kinase, which does not depend on free [Ca2+] in the absence of recoverin, is regulated by Ca2+ in the presence of both forms of the recombinant protein. EC50 values for Ca2+ are the same (2 μM) for the myristoylated and non‐myristoylated forms; the Hill coefficients of 1.7 and 0.9, respectively, indicate that the effect is cooperative with respect to Ca2+ only for myristoylated recoverin. In the presence of Ca2+, both forms of recoverin taken at saturated concentrations cause an almost equal inhibition of rhodopsin phosphorylation. However, the inhibitory action of the myristoylated form occurs at much lower its concentrations than that of the non‐myristoylated form (EC50 are 0.9 and 6.5 μM, respectively).

Recoverin mediates the calcium effect upon rhodopsin phosphorylation and cGMP hydrolysis in bovine retina rod cells

Rhodopsin phosphorylation and in consequence cGMP hydrolysis in bovine rod outer segments are Ca2+ dependent in the presence of ATP. The level of rhodopsin phosphorylation decreases and the lifetime of active phosphodiesterase increases when the free [Ca2+] is raised from <1 nM to about 1 μM; in both cases the half‐maximal effect was observed at 140–170 nM of free Ca2+. Antibodies to recoverin reverse both effects at high [Ca2+] but have no influence at low [Ca2+]. We conclude that the Ca2+ effects observed are mediated by recoverin which inhibits rhodopsin kinase at a high Ca2+ level.

Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer Therapy

Mitochondria are indispensable for energy metabolism, apoptosis regulation, and cell signaling. Mitochondria in malignant cells differ structurally and functionally from those in normal cells and participate actively in metabolic reprogramming. Mitochondria in cancer cells are characterized by reactive oxygen species (ROS) overproduction, which promotes cancer development by inducing genomic instability, modifying gene expression, and participating in signaling pathways. Mitochondrial and nuclear DNA mutations caused by oxidative damage that impair the oxidative phosphorylation process will result in further mitochondrial ROS production, completing the “vicious cycle” between mitochondria, ROS, genomic instability, and cancer development. The multiple essential roles of mitochondria have been utilized for designing novel mitochondria‐targeted anticancer agents. Selective drug delivery to mitochondria helps to increase specificity and reduce toxicity of these agents. In order to reduce mitochondrial ROS production, mitochondria‐targeted antioxidants can specifically accumulate in mitochondria by affiliating to a lipophilic penetrating cation and prevent mitochondria from oxidative damage. In consistence with the oncogenic role of ROS, mitochondria‐targeted antioxidants are found to be effective in cancer prevention and anticancer therapy. A better understanding of the role played by mitochondria in cancer development will help to reveal more therapeutic targets, and will help to increase the activity and selectivity of mitochondria‐targeted anticancer drugs. In this review we summarized the impact of mitochondria on cancer and gave summary about the possibilities to target mitochondria for anticancer therapies. J. Cell. Physiol. 231: 2570–2581, 2016.

Ca2+-Dependent Control of Rhodopsin Phosphorylation: Recoverin And Rhodopsin Kinase

Over many years until the middle of the 1980s, the main problem in vision research had been the mechanism of transducing the visual signal from photobleached rhodopsin to the cationic channels in the plasma membrane of a photoreceptor to trigger the electrophysiological response of the cell. After cGMP was proven to be the secondary messenger, the main intriguing question has become the mechanisms of negative feedback in photoreceptors to modulate their response to varying conditions of illumination. Although the mechanisms of light-adaptation are not completely understood, it is obvious that Ca2+ plays a crucial role in these mechanisms and that the effects of Ca2+ can be mediated by several Ca2+-binding proteins. One of them is recoverin. The leading candidate for the role of an intracellular target for recoverin is believed to be rhodopsin kinase, a member of a family of G-protein-coupled receptor kinases. The present review considers recoverin, rhodopsin kinase and their interrelationships in the in vitro as well as in vivo contexts.

Calcium-sensitive control of rhodopsin phosphorylation in the reconstituted system consisting of photoreceptor membranes, rhodopsin kinase and recoverin

Rhodopsin phosphorylation in the reconstituted system consisting of urea‐washed photoreceptor membranes, rhodopsin kinase and recoverin is regulated by Ca2+: the process takes place at low [Ca2+] but is suppressed at high [Ca2+]. In the absence of recoverin, rhodopsin kinase is active irrespective of the cation concentration used. Hence, recoverin is an inhibitor (at high [Ca2+]) but not an activator of rhodopsin kinase. Based jointly on these data obtained on the reconstituted system and on our preceding experiments on rod outer segments suspension, one may conclude that (i) the function of recoverin in retina rod cells is the Ca2+‐sensitive control of rhodopsin phosphorylation and (ii) the presence of recoverin is essential and sufficient to provide rhodopsin kinase with the Ca2+ sensitivity.

Mechanism of rhodopsin kinase regulation by recoverin.

Recoverin is suggested to inhibit rhodopsin kinase (GRK1) at high [Ca2+] in the dark state of the photoreceptor cell. Decreasing [Ca2+] terminates inhibition and facilitates phosphorylation of illuminated rhodopsin (Rh*). When recoverin formed a complex with GRK1, it did not interfere with the phosphorylation of a C‐terminal peptide of rhodopsin (S338‐A348) by GRK1. Furthermore, while GRK1 competed with transducin on interaction with rhodopsin and thereby suppressed GTPase activity of transducin, recoverin in the complex with GRK1 did not influence this competition. Constructs of GRK1 that encompass its N‐terminal, catalytic or C‐terminal domains were used in pull‐down assays and surface plasmon resonance analysis to monitor interaction. Ca2+‐recoverin bound to the N‐terminus of GRK1, but did not bind to the other constructs. GRK1 interacted with rhodopsin also by its N‐terminus in a light‐dependent manner. No interaction was observed with the C‐terminus. We conclude that inhibition of GRK1 by recoverin is not the result of their direct competition for the same docking site on Rh*, although the interaction sites of GRK1/Rh* and GRK1/recoverin partially overlap. The N‐terminus of GRK1 is recognized by Rh* leading to a conformational change which moves the C‐terminus of Rh* into the catalytic kinase groove. Ca2+‐recoverin interacting with the N‐terminus of GRK1 prevents this conformational change and thus blocks Rh* phosphorylation by GRK1.