Ivan I. Senin

An attempt to prevent senescence: A mitochondrial approach

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1=SkQR1>SkQ3>MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H(2)O(2)-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H(2)O(2) or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53(-/-) mice, 5 nmol/kgxday SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.

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.

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.

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.

Tuning of a Neuronal Calcium Sensor

Recoverin is a Ca2+-regulated signal transduction modulator expressed in the vertebrate retina that has been implicated in visual adaptation. An intriguing feature of recoverin is a cluster of charged residues at its C terminus, the functional significance of which is largely unclear. To elucidate the impact of this segment on recoverin structure and function, we have investigated a mutant lacking the C-terminal 12 amino acids. Whereas in myristoylated recoverin the truncation causes an overall decrease in Ca2+ sensitivity, results for the non-myristoylated mutant indicate that the truncation primarily affects the high affinity EF-hand 3. The three-dimensional structure of the mutant has been determined by x-ray crystallography. In addition to significant changes in average coordinates compared with wild-type recoverin, the structure provides strong indication of increased conformational flexibility, particularly in the C-terminal domain. Based on these observations, we propose a novel role of the C-terminal segment of recoverin as an internal modulator of Ca2+ sensitivity.

Low titre autoantibodies against recoverin in sera of patients with small cell lung cancer but without a loss of vision.

To date, many authors have described the presence of autoantibodies against various neuronal proteins, paraneoplastic antigens (PNA), in a serum of patients with different kinds of malignant tumors located outside the nervous system. These autoantibodies may cross-react with the corresponding PNA or their epitopes present in neurons and thus initiate the development of a variety of neurological disorders, paraneoplastic syndromes (PNS), even though the primary tumor and its metastases have not invaded the nervous system. Cancer-associated retinopathy (CAR) is a rare ocular PNS induced by autoantibodies against several retinal antigens, one of which is a photoreceptor calcium-binding protein, recoverin. Only several CAR patients with a few kinds of cancer (endothelial carcinoma, breast cancer, epithelial ovarian carcinoma) have so far been found to contain autoantibodies against recoverin in their sera. As for lung cancer, the majority of CAR cases mediated by anti-recoverin autoantibodies have been revealed in patients with the most malignant lung cancer, small cell lung carcinoma (SCLC), and only one similar case has been described for a patient with non-small lung carcinoma. The common feature of all these anti-recoverin-positive patients, irrespective of the type of cancer, is the presence of both the CAR syndrome and high titres (as a rule, more than 1:1000) of the underlying autoantibodies in their serum. In this study, we have used recombinant myristoylated recoverin to screen serum samples of 50 patients with SCLC by Western blot and revealed 5 individuals with low titres of anti-recoverin antibodies, who have no manifestation of a loss of vision. To our knowledge, this is the first report on the presence of low titre autoantibodies against recoverin in a serum of patients with cancer, but without visual dysfunction.

Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase

NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.