C-Terminal Region of Caveolin-3 Contains a Stretch of Amino Acid Residues Capable of Diminishing Symptoms of Experimental Autoimmune Encephalomyelitis but Not Rheumatoid Arthritis Modeled in Rats.

A short synthetic peptide from the C-terminal part of the caveolin-3 structure was tested for experimental autoimmune encephalomyelitis (EAE) treatment in rats. The structure-function similarity established between the novel synthetic peptide of pCav3 and the well-known immunomodulator immunocortin determined pCav3's ability to reduce EAE symptoms in Dark Agouti (DA) rats injected with pCav3 (500 µg/kg). pCav3 was found to interfere with the proliferation of lymphocytes extracted from the LNs of DA rats primed with homogenate injection, with IC50 = 0.42 µM (2.35 mcg/mL). pCav3 affected EAE in a very similar manner as immunocortin. The high degree of homology between the amino acid sequences of pCav3 and immunocortin corresponded well with the therapeutic activities of both peptides, as demonstrated on EAE. The latter peptide, possessing a homologous structure to pCav3, was also tested on EAE to explore whether there were structural restrictions between these peptides implied by the MHC-involved cell machinery. Consequently, immunocortin was further examined with a different autoimmune disease model, collagen-induced arthritis (CIA), established in Sprague-Dawley rats. CIA was established using an intentionally different genetic platform than EAE. Based on the results, it was concluded that the effectiveness of pCav3 and immunocortin peptides in EAE rat model was almost identical, but differed in the rat model of rheumatoid arthritis; thus, efficacy may be sensitive to the MHC type of animals used to establish the autoimmune disease model.

Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1.

Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca2+-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1-101), or its short scaffolding domain (81-101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca2+-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca2+-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca2+-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca2+-signals thereby maintaining efficient phototransduction recovery and light adaptation.

Efficacy of Synthetic Peptide Corresponding to the ACTH-Like Sequence of Human Immunoglobulin G1 in Experimental Autoimmune Encephalomyelitis.

Peptide immunocortin sequence corresponds to the amino acid residues 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain. Since immunocortin was shown previously to inhibit phagocytosis in peritoneal macrophages and ConA-induced T-lymphocytes proliferation in culture, we suggested that immunocortin administering may be of use for patients with self-immune syndrome. Immunocortin in concentration 10 µM inhibited proliferation of both antigen (myelin)-induced and ConA-induced LN lymphocytes isolated from the lymph nodes of Dark Agouti (DA) rats immunized with chorda shear. The biological trials of the synthetic immunocortin were carried out on the DA rats with induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. These in vivo experiments have shown that intraperitoneal injections of immunocortin in a daily dosage 100 µg per animal reduced symptoms of EAE in DA rats.

Immunosuppressant Peptide Abu-TGIRIS-Abu-NH2 and its Application for Treatment of Multiple Sclerosis.

Immunosuppressant peptide immunocortin for the first time was described in 1993. It corresponds to residues 11-20 of human Ig heavy chain (conserved motif of VH domain). There are no data about production of immunocortin by proteolysis of Ig in vivo. Synthetic immunocortin in concentration ~ 10-9 M suppresses phagocytosis in peritoneal macrophages, ConA-dependent blast transformation of rat lymphocytes, exhibits ACTH-like neurotropic activity and was suggested as a potential drug for treatment of a multiple sclerosis (MS). Here, we report a sequence and method of synthesis of Abu-TGIRIS-Abu-NH2 (Abu, alpha-aminobutyric acid), an artificial analogue of immunocortin. Biological trials of peritoneally injected Abu-TGIRIS-Abu-NH2 gave an evidence of its better efficacy versus immunocortin in a test for suppression of the experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats.

Binding of cholera toxin B subunit to intestinal epithelial cells.

We have prepared 125I-labeled cholera toxin B subunit (125I-labeled CT-B, a specific activity of 98Ci/mmol) and found that it binds to rat IEC-6 and human Caco-2 intestinal epithelial cells with high affinity (Kd 3.6 and 3.7nM, respectively). The binding of labeled protein was completely inhibited by unlabeled thymosin-α1 (TM-α1), interferon-α2 (IFN-α2), and the synthetic peptide LKEKK that corresponds to residues 16-20 in TM-α1 and 131-135 in IFN-α2, but was not inhibited by the synthetic peptide KKEKL with inverted amino acid sequence (Ki>10µM). Thus, TM-α1, IFN-α2, and the peptide: LKEKK bind with high affinity and specificity to the cholera toxin receptor on IEC-6 and Caco-2 cells. It was found that CT-B and the peptide: LKEKK at concentrations of 10-1000nM increased in a dose-dependent manner the nitric oxide production and the soluble guanylate cyclase activity in IEC-6 and Caco-2 cells.

Interaction of cholera toxin B subunit with T and B lymphocytes.

We have prepared 125I-labeled cholera toxin B subunit (125I-labeled CT-B, a specific activity of 98Ci/mmol) and found that its binding to T and B lymphocytes from the blood of healthy donors was high-affinity (Kd 2.8 and 3.0nM, respectively). The binding of labeled protein was completely inhibited by unlabeled thymosin-α1 (TM-α1), interferon-α2 (IFN-α2), and the synthetic peptide LKEKK that corresponds to residues 16-20 in TM-α1 and 131-135 in IFN-α2, but was not inhibited by the synthetic peptide KKEKL with inverted amino acid sequence (Ki>10µM). Thus, TM-α1, IFN-α2, and the peptide: LKEKK bind with high affinity and specificity to CT-B receptor on donor blood T and B lymphocytes. It was found that CT-B and the peptide: LKEKK at concentrations of 10-1000nM increased in a dose-dependent manner the soluble guanylate cyclase activity in T and B lymphocytes.

Abnormal pigment epithelium-derived factor processing in progressive myopia.

Pigment Epithelium-Derived Factor (PEDF) is a secreted glycoprotein belonging to the family of non-inhibitory serpins. It is known, that in cases of complicated myopia, the content of PEDF in aqueous humor of the anterior chamber is significantly reduced. Here we examined a bulk of Tenon's capsule samples obtained from various groups of myopes, to examine PEDF processing in progressive myopia. We have analyzed the distribution of full length PEDF50 and its truncated form PEDF45 in the soluble and insoluble fractions extracted from Tenon's capsule of myopic and control (non-myopic) patients using SDS-polyacrylamide gel electrophoresis, as well as monitored the proteolytic degradation of PEDF ex vivo by enzyme-linked immunosorbent assay. These results were complemented by PEDF mRNA analysis in correspondent tissues by using qPCR and immunohistochemistry analysis of PEDF distribution in normal and myopic specimens. We found that in the Tenon's capsule of patients suffering from a high myopia the level of "soluble" 45 kDa PEDF reduced by 2-fold, while the content of "insoluble" 50 kDa form of PEDF was increased by 4-fold compared to controls. Excessive amount of PEDF50 in myopic specimens have been shown to correlate with the abrogated PEDF processing rather than with an increase of its expression. Moreover, immunohistochemical staining of the myopic Tenon's capsule tissue sections revealed the halo of deposited PEDF50 in the fibroblast extracellular space. These findings suggest that in myopia limited proteolysis of PEDF is altered or abrogated. Accumulation of full-length PEDF insoluble aggregates in the fibroblast intercellular space may affect cell survival and consequently causes the destructive changes in the extracellular matrix of the eye connective tissues. As a result, the abrogation of full-length PEDF normal processing can be an important mechanism leading to biomechanical destabilization of the scleral capsule and myopia progression.

Anxiolytic activity of the neuroprotective peptide HLDF-6 and its effects on brain neurotransmitter systems in BALB/c and C57BL/6 mice.

This study is focused on a new amide derivative of the peptide HLDF-6 (Thr-Gly-Glu-Asn-His-Arg). This hexapeptide is a fragment of Human Leukaemia Differentiation Factor (HLDF). It displays a broad range of nootropic and neuroprotective activities. We showed, for the first time, that the peptide HLDF-6-amide has high anxiolytic activity. We used 'open field' and 'elevated plus maze' tests to demonstrate anxiolytic effects of HLDF-6-amide (0.1 and 0.3 mg/kg intranasally), which were comparable to those of the reference drug diazepam (0.5 mg/kg). Five daily equipotent doses of HLDF-6-amide selectively mitigated anxiety and increased the density of NMDA receptors in the hippocampus of stress-susceptible BALB/c mice, and had no effect on stress-resilient C57BL/6 mice. The subchronic administration of HLDF-6-amide showed no effect on the density of GABAA and nicotine receptors but was accompanied by a nonselective decrease of the 5-HT2A serotonin receptor density in frontal cortex of both strains. The mechanism of the specific anxiolytic activity of HLDF-6-amide may include its action on the NMDA-glutamatergic receptor system of the hippocampus and on serotonin 5-HT2A-receptors in the prefrontal cortex. The psychotropic activity of HLDF-6-amide is promising for its introduction to medical practice as a highly effective anxiolytic medicine for mental and neurological diseases.

Comparative study of the neuroprotective and nootropic activities of the carboxylate and amide forms of the HLDF-6 peptide in animal models of Alzheimer's disease.

A comparative study of the neuroprotective and nootropic activities of two pharmaceutical substances, the HLDF-6 peptide (HLDF-6-OH) and its amide form (HLDF-6-NH2), was conducted. The study was performed in male rats using two models of a neurodegenerative disorder. Cognitive deficit in rats was induced by injection of the beta-amyloid fragment 25-35 (ßA 25-35) into the giant-cell nucleus basalis of Meynert or by coinjection of ßA 25-35 and ibotenic acid into the hippocampus. To evaluate cognitive functions in animals, three tests were used: the novel object recognition test, the conditioned passive avoidance task and the Morris maze. Comparative analysis of the data demonstrated that the neuroprotective activity of HLDF-6-NH2, evaluated by improvement of cognitive functions in animals, surpassed that of the native HLDF-6-OH peptide. The greater cognitive/ behavioral effects can be attributed to improved kinetic properties of the amide form of the peptide, such as the character of biodegradation and the half-life time. The effects of HLDF-6-NH2 are comparable to, or exceed, those of the reference compounds. Importantly, HLDF-6-NH2 exerts its effects at much lower doses than the reference compounds.

Recoverin as a redox-sensitive protein.

Recoverin is a member of the neuronal calcium sensor (NCS) family of EF-hand calcium binding proteins. In a visual cycle of photoreceptor cells, recoverin regulates activity of rhodopsin kinase in a Ca2+-dependent manner. Like all members of the NSC family, recoverin contains a conserved cysteine (Cys38) in nonfunctional EF-hand 1. This residue was shown to be critical for activation of target proteins in some members of the NCS family but not for interaction of recoverin with rhodopsin kinase. Spectrophotometric titration of Ca2+-loaded recoverin gave 7.6 for the pKa value of Cys38 thiol, suggesting partial deprotonation of the thiol in vivo conditions. An ability of recoverin to form a disulfide dimer and thiol-oxidized monomer under mild oxidizing conditions was found using SDS-PAGE in reducing and nonreducing conditions and Ellman's test. Both processes are reversible and modulated by Ca2+. Although formation of the disulfide dimer takes place only for Ca2+-loaded recoverin, accumulation of the oxidized monomer proceeds more effectively for apo-recoverin. The Ca2+ modulated susceptibility of the recoverin thiol to reversible oxidation may be of potential importance for functioning of recoverin in photoreceptor cells.

The Stimulating Effect of the beta-Endorphin-Like Peptide Immunorphin on the Human T-Lymphoblastoid Cell Line Jurkat Is Mediated by a Non-Opioid Receptor for beta-Endorphin.

It was shown that beta-endorphin and the synthetic decapeptide SLTCLVKGFY that corresponds to the amino acid sequence 364-373 of the human IgG heavy chain (referred to as immunorphin) is able to stimulate growth of the human T-lymphoblastoid cell line Jurkat. The antagonist of opioid receptors naloxone did not inhibit the stimulating effect of the peptides. Studies on [(3)H]-immunorphin binding to Jurkat cell receptors have demonstrated that it binds with high affinity to naloxone-insensitive receptors (K(d) = 1.3 nM; n = 5.2 x 10(5)). Unlabeled beta-endorphin and the 6-10 fragment of immunorphin completely inhibited the labeled ligand specific binding to naloxone-insensitive receptors on T lymphocytes (K(i) = 1.4 x 10(-7) and 3.7 x 10(-5) M, respectively). Thus, beta-endorphin and immunorphin share the naloxone-insensitive receptors on human T-lymphoblastoid cell line Jurkat.

Recoverin is a zinc-binding protein.

Recoverin is an N-myristoylated 23 kDa calcium-binding protein from retina, which modulates the Ca2+-sensitive deactivation of rhodopsin via Ca2+-dependent inhibition of rhodopsin kinase. It was shown by intrinsic and bis-ANS probe fluorescence, circular dichroism, and differential scanning calorimetry that myristoylated recombinant recoverin interacts specifically with zinc ions. Similar to the calcium binding, the binding of zinc to Ca2+-loaded recoverin additionally increases its alpha-helical content, hydrophobic surface area, and environmental mobility/polarity of its tryptophan residues. In contrast to the calcium binding, the binding of zinc decreases thermal stability of the Ca2+-loaded protein. Zn2+-titration of recoverin, traced by bis-ANS fluorescence, reveals binding of a single Zn2+ ion per protein molecule. It was shown that the double-mutant E85Q/E121Q with inactivated Ca2+-binding EF-hands 2 and 3 (Alekseev, A. M.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, S. A.; Suchkov, D. V.; Vaganova, S. A.; Senin, I. I.; Zargarov, A. A.; Lipkin, V. M.; Akhtar, M.; Philippov, P. P. FEBS Lett. 1998, 440, 116-118), which can be considered as an analogue of the apo-protein, binds Zn2+ ion as well. Apparent zinc equilibrium binding constants evaluated from spectrofluorimetric Zn2+-titrations of the protein are 1.4 x 10(5) M(-1) (dissociation constant 7.1 microM) for Ca2+-loaded wild-type recoverin and 3.3 x 10(4) M(-1) (dissociation constant 30 microM) for the E85Q/E121Q mutant (analogue of apo-recoverin). Study of the binding of wild-type recoverin to ROS membranes showed a zinc-dependent increase of its affinity for the membranes, without regard to calcium content, suggesting further solvation of a protein myristoyl group upon Zn2+ binding. Possible implications of these findings to the functioning of recoverin are discussed.

Beta-endorphin-like peptide SLTCLVKGFY reduces the production of 11-oxycorticosteroids by rat adrenal cortex through nonopioid beta-endorphin receptors.

Beta-endorphin-like peptide immunorphin (SLTCLVKGFY), a selective agonist of nonopioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid beta-endorphin receptors on rat adrenal cortex membranes (Kd = 31.6 +/- 0.2 nM, Bmax = 37.4 +/- 2.2 pmol/mg protein). Immunorphin at concentrations of 10(-9) to 10(-6) M was found to inhibit the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunorphin at doses of 10-100 microg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.

Synthetic peptide SLTCLVKGFY competes with beta-endorphin for naloxone-insensitive binding sites on rat brain membranes.

The synthetic decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain and its synthetic fragment VKGFY were found to compete with 125I-labeled beta-endorphin for high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K(i)=1.18+/-0.09 and 1.58+/-0.11 nM, respectively). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to [Met(5)]enkephalin and [Leu(5)]enkephalin as well. The K(d) values characterizing the specific binding of 125I-labeled immunorphin and its fragment Val-Lys-Gly-Phe-Tyr to these binding sites were determined to be 2.93+/-0.27 nM and 3.17+/-0.29 nM, respectively.