Dmitry V. Zinchenko

Oxidation mimicking substitution of conservative cysteine in recoverin suppresses its membrane association

Recoverin belongs to the family of intracellular Ca2+-binding proteins containing EF-hand domains, neuronal calcium sensors (NCS). In photoreceptor outer segments, recoverin is involved into the recovery of visual cycle via Ca2+-dependent interaction with disk membranes and inhibition of rhodopsin kinase. The function of a conservative within NCS family Cys residue in the inactive EF-loop 1 remains unclear, but previous study has shown its vulnerability to oxidation under mild oxidizing conditions. To elucidate the influence of oxidation of the conservative Cys39 in recoverin the properties of its C39D mutant, mimicking oxidative conversion of Cys39 into sulfenic, sulfinic or sulfonic acids have been studied using intrinsic fluorescence, circular dichroism, and equilibrium centrifugation methods. The C39D substitution results in essential changes in structural, physico-chemical and physiological properties of the protein: it reduces α-helical content, decreases thermal stability and suppresses protein affinity for photoreceptor membranes. The latter effect precludes proper functioning of the Ca2+-myristoyl switch in recoverin. The revealed significance of oxidation state of Cys39 for maintaining the protein functional status shows that it may serve as redox sensor in vision and suggests an explanation of the available data on localization and light-dependent translocation of recoverin in rod photoreceptors.

Light-induced disulfide dimerization of recoverin under ex vivo and in vivo conditions

Despite vast knowledge of the molecular mechanisms underlying photochemical damage of photoreceptors, linked to progression of age-related macular degeneration, information on specific protein targets of the light-induced oxidative stress is scarce. Here, we demonstrate that prolonged intense illumination (halogen bulb, 1500 lx, 1-5 h) of mammalian eyes under ex vivo (cow) or in vivo (rabbit) conditions induces disulfide dimerization of recoverin, a Ca(2+)-dependent inhibitor of rhodopsin kinase. Western blotting and mass spectrometry analysis of retinal extracts reveals illumination time-dependent accumulation of disulfide homodimers of recoverin and its higher order disulfide cross-linked species, including a minor fraction of mixed disulfides with intracellular proteins (tubulins, etc.). Meanwhile, monomeric bovine recoverin remains mostly reduced. These effects are accompanied by accumulation of disulfide homodimers of visual arrestin. Histological studies demonstrate that the light-induced oxidation of recoverin and arrestin occurs in intact retina (illumination for 2 h), while illumination for 5 h is associated with damage of the photoreceptor layer. A comparison of ex vivo levels of disulfide homodimers of bovine recoverin with redox dependence of its in vitro thiol-disulfide equilibrium (glutathione redox pair) gives the lowest estimate of redox potential in rod outer segments under illumination from -160 to -155 mV. Chemical crosslinking and dynamic light scattering data demonstrate an increased propensity of disulfide dimer of bovine recoverin to multimerization/aggregation. Overall, the oxidative stress caused by the prolonged intense illumination of retina might affect rhodopsin desensitization via concerted disulfide dimerization of recoverin and arrestin. The developed herein models of eye illumination are useful for studies of the light-induced thiol oxidation of visual proteins.

Amino acid sequences of two immune-dominant epitopes of recoverin are involved in Ca2+/recoverin-dependent inhibition of phosphorylation of rhodopsin

Antibodies AB60–72 and AB80–92 against two immune-dominant epitopes of photoreceptor Ca2+-binding protein recoverin, 60-DPKAYAQHVFRSF-72 and 80-LDFKEYVIALHMT-92, which can be exposed in a Ca2+-dependent manner, were obtained. The presence of AB60–72 or AB80–92 results in a slight increase in Ca2+-affinity of recoverin and does not affect significantly a Ca2+-myristoyl switch mechanism of the protein. However in the presence of AB60–72 or AB80–92 recoverin loses its ability to interact with rhodopsin kinase and consequently to perform a function of Ca2+-sensitive inhibitor of rhodopsin phosphorylation in photoreceptor cells.

Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs

Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1–19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, ‘black’ and ‘gray’ clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

The LKEKK synthetic peptide as a ligand of rat intestinal epithelial cell membranes

A tritium-labeled synthetic LKEKK pentapeptide corresponding to the sequences 16–20 of human thymosin-α1 and 131–135 of human interferon-α2 was obtained with a specific activity of 28 Ci/mmol. [3H]LKEKK was found to bind with high affinity (Kd 3.7 ± 0.3 nM) to the membranes isolated from epithelial cells of rat small intestinal mucosa. The trypsin treatment of the membranes did not affect the binding, thus supporting the nonprotein nature of the peptide receptor. The binding of the labeled peptide was inhibited by unlabeled thymosin-α1, interferon-α2, and cholera toxin B subunit (Ki 4.2 ± 0.4, 3.5 ± 0.3, and 4.7 ± 0.3 nM respectively). The pentapeptide did not affect the adenylate cyclase activity within the concentration range of 1–1000 nM.

Ca 2+ -Myristoyl Switch in Neuronal Calcium Sensor-1: A Role of C-Terminal Segment

NCS1 (neuronal calcium sensor-1) is a Ca(2+)-myristoyl switch protein of the NCS protein family involved in synaptic plasticity and neurotransmission via Ca(2+)-dependent regulation of dopamine D2 receptor and associated Gprotein coupled receptor kinase (GRK)-2. Overexpression of NCS1 in synaptic terminals results in accumulation of membrane-bound protein and its redundant regulatory activity associated with neurological disorders. Here, we have demonstrated that bovine photoreceptors contain NCS1 that is capable of a partially irreversible interaction with isolated photoreceptor membranes and implicated in Ca(2+)-dependent binding and regulation of GRK1 in vitro. Using NCS1- recoverin C-terminal chimeric construct (NR), it was found that the Ca(2+)-myristoyl switch of NCS1 is affected by its C-terminal segment downstream the fourth EF-loop of the protein, which is variable within the NCS family. NR retains structural stability and sensitivity to Ca(2+), but interacts with photoreceptor membranes with lower affinity in a Ca(2+)- dependent fully reversible manner and displays altered GRK1 modulation. These data combined with fluorescent probing of surface hydrophobicity of NCS1, NR and recoverin suggest that the C-terminal segment of NCS1 regulates reuptake of myristoyl group under Ca(2+)-free conditions and participates in organization of the target-binding pocket of the protein. We point out a putative role of NCS1 in photoreceptors as a modulator of GRK activity and propose targeting of the C-terminal segment of NCS1 as an appropriate way for selective suppression of excessive membrane accumulation and aberrant activity of the protein in neurons associated with central nervous system dysfunctions.

Ca2+-dependent regulatory activity of recoverin in photoreceptor raft structures: The role of caveolin-1

Recoverin is a Ca2+-binding protein implicated in the Ca2+-dependent regulation of desensitization of visual receptor rhodopsin in vertebrate retinal rods. Here we report that Ca2+ sensitivity of recoverin regulating rhodopsin phosphorylation increases in the presence of the photoreceptor membranes enriched in raft structures. The observed effect is mediated by a key protein component of raft structures caveolin-1. The presence of recombinant fragment Phe81-Arg101 of the caveolin-1 cytoplasmic domain enhances Ca2+ affinity of recoverin, therefore affecting its Ca2+-dependent regulatory activity.

Regulatory function of the c-terminal segment of guanylate cyclase-activating protein 2

Neuronal responses to Ca2+-signals are provided by EF-hand-type neuronal Ca2+-sensor (NCS) proteins, which have similar core domains containing Ca2+-binding and target-recognizing sites. NCS proteins vary in functional specificity, probably depending on the structure and conformation of their non-conserved C-terminal segments. Here, we investigated the role of the C-terminal segment in guanylate cyclase activating protein-2, GCAP2, an NCS protein controlling the Ca2+-dependent regulation of photoreceptor guanylate cyclases. We obtained two chimeric proteins by exchanging C-terminal segments between GCAP2 and its photoreceptor homolog recoverin, a Ca2+-sensor controlling rhodopsin kinase (RK) activity. The exchange affected neither the structural integrity of GCAP2 and recoverin nor the Ca2+-sensitivity of GCAP2. Intrinsic fluorescence, circular dichroism, biochemical studies and hydrophobic dye probing revealed Ca2+-dependent conformational transition of the C-terminal segment of GCAP2 occurring in the molecular environment of both proteins. In Ca2+-GCAP2, the C-terminal segment was constrained and its replacement provided the protein with approximately two-fold inhibitory activity towards RK, suggesting that the segment contributes to specific target recognition by interfering with RK-binding. Upon Ca2+-release, it became less constrained and more available for phosphorylation by cyclic nucleotide-dependent protein kinase. The transition from the Ca2+-bound to the apo-state exposed hydrophobic sites in GCAP2, and was associated with its activating function without affecting its dimerization. The released C-terminal segment participated further in photoreceptor membrane binding making it sensitive to phosphorylation. Thus, the C-terminal segment in GCAP2 confers target selectivity, facilitates membrane binding and provides sensitivity of the membrane localization of the protein to phosphorylation by signaling kinases.

Point amino acid substitutions in the Ca2+-binding sites of recoverin. III. A mutant with the fourth reconstructed Ca2+-binding site

Unlike wild type recoverin with only two (the second and the third) functioning Ca+2-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca+2-binding site. This site was reconstructed from the fourth potential Ca+2-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca+2-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca2+ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild-type recoverin.

α 1 -Thymosin, α 2 -interferon, and the LKEKK syntetic peptide inhibit the binding of the B subunit of the cholera toxin to intestinal epithelial cell membranes

The 125I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (Kd = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α2-interferon (IFN-α2), α1-thymosin, (TM-α1), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α1 and the 131–135 sequence of human IFN-α2 (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (Ki > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α1, IFN-α2, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.