Dry eye syndrome (DES) is an age‐related condition increasingly detected in younger people of risk groups, including patients who underwent ocular surgery or long‐term general anesthesia. Being a multifactorial disease, it is characterized by oxidative stress in the cornea and commonly complicated by ocular surface inflammation. Polyetiologic DES is responsive to SkQ1, a mitochondria‐targeted antioxidant suppressing age‐related changes in the ocular tissues. Here, we demonstrate safety and efficacy of topical administration of SkQ1 at a dosage of 7.5 μM for the prevention of general anesthesia‐induced DES in rabbits. The protective action of SkQ1 improves clinical state of the ocular surface by inhibiting apoptotic and prenecrotic changes in the corneal epithelium. The underlying mechanism involves the suppression of the oxidative stress supported by the stimulation of intrinsic antioxidant activity and the activity of antioxidant enzymes, foremost glutathione peroxidase and glutathione reductase, in the cornea. Furthermore, SkQ1 increases antioxidant activity and stability of the tear film and produces anti‐inflammatory effect exhibited as downregulation of TNF‐α and IL‐6 and pronounced upregulation of IL‐10 in tears. Our data suggest novel features of SkQ1 and point to its feasibility in patients with DES and individuals at risk for the disease including those subjected to general anesthesia.

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.

Light-induced oxidation of lipids and proteins provokes retinal injuries and results in progression of degenerative retinal diseases, such as, for instance, iatrogenic photic maculopathies. Having accumulated over years retinal injuries contribute to development of age-related macular degeneration (AMD). Antioxidant treatment is regarded as a promising approach to protecting the retina from light damage and AMD. Here, we examine oxidative processes induced in rabbit retina by excessive light illumination with or without premedication using mitochondria-targeted antioxidant SkQ1 (10-(6’-plastoquinonyl)decyltriphenyl-phosphonium). The retinal extracts obtained from animals euthanized within 1–7 days post exposure were analyzed for H₂O₂, malondialdehyde (MDA), total antioxidant activity (AOA), and activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) using colorimetric and luminescence assays. Oxidation of visual arrestin was monitored by immunoblotting. The light exposure induced lipid peroxidation and H₂O₂ accumulation in the retinal cells. Unexpectedly, it prominently upregulated AOA in retinal extracts although SOD and GPx activities were compromised. These alterations were accompanied by accumulation of disulfide dimers of arrestin revealing oxidative stress in the photoreceptors. Premedication of the eyes with SkQ1 accelerated normalization of H₂O₂ levels and redox-status of lipids and proteins, contemporarily enhancing AOA and, likely, sustaining normal activity of GPx. Thus, SkQ1 protects the retina from light-induced oxidative stress and could be employed to suppress oxidative damage of proteins and lipids contributing to AMD.

Neuronal calcium sensor-1 (NCS-1) protein is abundantly expressed in the central nervous system and retinal neurons, where it regulates many vital processes such as synaptic transmission. It coordinates three calcium ions by EF-hands 2-4, thereby transducing Ca²⁺ signals to a wide range of protein targets, including G protein-coupled receptors and their kinases. Here, we demonstrate that NCS-1 also has Zn²⁺-binding sites, which affect its structural and functional properties upon filling. Fluorescence and circular dichroism experiments reveal the impact of Zn²⁺ binding on NCS-1 secondary and tertiary structure. According to atomic absorption spectroscopy and isothermal titration calorimetry studies, apo-NCS-1 has two high-affinity (4 × 10⁶ M^-1) and one low-affinity (2 × 10⁵ M^-1) Zn²⁺-binding sites, whereas Mg²⁺-loaded and Ca²⁺-loaded forms (which dominate under physiological conditions) bind two zinc ions with submicromolar affinity. Metal competition analysis and circular dichroism studies suggest that Zn²⁺-binding sites of apo- and Mg²⁺-loaded NCS-1 overlap with functional EF-hands of the protein. Consistently, high Zn²⁺ concentrations displace Mg²⁺ from the EF-hands and decrease the stoichiometry of Ca²⁺ binding. Meanwhile, one of the EF-hands of Zn²⁺-saturated NCS-1 exhibits a 14-fold higher calcium affinity, which increases the overall calcium sensitivity of the protein. Based on QM/MM molecular dynamics simulations, Zn²⁺ binding to Ca²⁺-loaded NCS-1 could occur at EF-hands 2 and 4. The high-affinity zinc binding increases the thermal stability of Ca²⁺-free NCS-1 and favours the interaction of its Ca²⁺-loaded form with target proteins, such as dopamine receptor D2R and GRK1. In contrast, low-affinity zinc binding promotes NCS-1 aggregation accompanied by the formation of twisted rope-like structures. Altogether, our findings suggest a complex interplay between magnesium, calcium and zinc binding to NCS-1, leading to the appearance of multiple conformations of the protein, in turn modulating its functional status.