Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C atom movements of up to 8 A ° upon channel gating, and predicts the location of the leucine– isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Oxidative impairment of plasma and skeletal muscle sarcoplasmic reticulum in rats with adjuvant arthritis - effects of pyridoindole antioxidants

OBJECTIVES: To study possible oxidation of proteins and lipids in plasma and sarcoplasmic reticulum (SR) from skeletal muscles and to assess the effects of pyridoindole antioxidants in rats with adjuvant arthritis (AA) and to analyze modulation of Ca-ATPase activity from SR (SERCA). METHODS: SR was isolated by ultracentrifugation, protein carbonyls in plasma and SR were determined by ELISA. Lipid peroxidation was analyzed by TBARS determination and by mass spectrometry. ATPase activity of SERCA was measured by NADH-coupled enzyme assay. Tryptophan fluorescence was used to analyze conformational alterations. RESULTS: Increase of protein carbonyls and lipid peroxidation was observed in plasma of rats with adjuvant arthritis. Pyridoindole antioxidant stobadine and its methylated derivative SMe1 decreased protein carbonyl formation in plasma, effect of stobadine was significant. Lipid peroxidation of plasma was without any effect of pyridoindole derivatives. Neither protein oxidation nor lipid peroxidation was identified in SR from AA rats. SERCA activity from AA rats increased significantly, stobadine and SMe1 diminished enzyme activity. Ratio of tryptophan fluorescence intensity in SR of AA rats increased and was not influenced by antioxidants. CONCLUSION: Plasma proteins and lipids were oxidatively injured in rats with AA; antioxidants exerted protection only with respect to proteins. In SR, SERCA activity was altered, apparently induced by its conformational changes, as supported by study of tryptophan fluorescence. Stobadine and SMe1 induced a decrease of SERCA activity, elevated in AA rats, but they did not affect conformational changes associated with tryptophan fluorescence

Structural insights into the human RyR2 N terminal region involved in cardiac arrhythmias

Human ryanodine receptor 2 hRyR2 mediates calcium release from the sarcoplasmic reticulum, enabling cardio myocyte contraction. The N terminal region of hRyR2 amino acids 1 606 is the target of gt;30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near physiological conditions, as well as a homology model of the hRyR2 N terminal region, are presented. The N terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix amino acids 410 437 plays a prominent stabilizing role. Importantly, the anion binding site reported for the mouse RyR2 N terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix R420W, I419F and I419F R420W which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N terminus adopts a globular shape with a prominent tail that is likely to involve residues 545 606, which are unresolved in the crystal structure. Docking the N terminal domains into cryo electron microscopy maps of the closed and open RyR1 conformations reveals C[alpha] atom movements of up to 8 upon channel gating, and predicts the location of the leucine isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surfac

N-feruloylserotonin in preventive combination therapy with methotrexate reduced inflammation in adjuvant arthritis.

Many of disease-modifying anti-rheumatic drugs often have side effects at high doses and/or during long-term administration. Increased efficacy without increased toxicity is expected for combination therapy of rheumatoid arthritis (RA). The aim of the study was to examine the effect of N-feruloylserotonin (N-f-5HT) and methotrexate (MTX) in monotherapy and in combination therapy on disease progression and inflammation in arthritic rats. Adjuvant arthritis was induced by intradermal injection of Mycobacterium butyricum in incomplete Freund's adjuvant in Lewis rats. The experiment included healthy animals, arthritic animals without any drug administration, arthritic animals with administration of N-f-5HT in the oral daily dose of 15 mg/kg b.w., arthritic animals with administration of MTX in the oral dose of 0.3 mg/kg b.w. twice a week and arthritic animals treated with the combination of N-f-5HT and MTX. N-f-5HT in monotherapy reduced only activation of NF-jB and did not have any significant effect on other parameters monitored. Low-dose treatment of MTX decreased the level of IL-1b and MCP-1 on day 14 and activation of NF-jB in liver without significant effect on other parameters. N-f-5HT and MTX combination showed both the anti-arthritic (hind paw volume and arthritic score) and anti-inflammatory effect (plasmatic levels of IL-1b, IL-17, MCP-1, CRP, and activation of NF-jB in liver). In combination with MTX, N-f-5HT markedly potentiated the therapeutic effect of MTX low dose, which resulted in significant improvement of all parameters measured. The findings showed that the combination therapy simultaneously decreased multiple markers of inflammation, a result crucial for future therapy of RA

The role of redox imbalance in relation to immunological process in adjuvant arthritis

The model of adjuvant arthritis (AA) is well characterized concerning the immunological processes involved. However knowledge on the participation of redox imbalance in the progression of AA is scarce. The link between oxidative stress (OS) and immunological status in arthritis should be more precisely investigated. In our experiments, we focused on AA development in the time domain. AA was induced in rats by a single intradermal injection of Mycobacterium butyricum in incomplete Freund’s adjuvant. All experiments included healthy animals (HC) and arthritic animals not treated. We confirmed that the clinical parameters hind paw volume and body weight became significantly modified starting around day 14 after AA induction and this change was maintained until the end of the experiment (day 28). We obtained a good agreement of clinical parameters with parameters of OS. Measurements of plasmatic protein carbonyls revealed damage of proteins caused by OS. Progression of lipid peroxidation in AA was described by analysis of TBARS, HNE- /MDA-protein adducts and F2 isoprostane levels in plasma. Total antioxidant status analyzed in plasma was decreased significantly in both the acute (day 21) and the subchronic phase (day 28) of AA. Further we focused on evaluating CoQ9 plasmatic levels. The arthritic process increased significantly the level of CoQ9 in comparison to HC. Evidently, the arthritic process stimulates the synthesis of CoQ9 and its transport to plasma. In the model of AA, we observed already on experimental day 7 that AA was accompanied by an increased number of neutrophils in blood and by a more pronounced spontaneous as well as phorbol myristate acetate stimulated chemiluminescence. As the changes in neutrophils occur before the clinical parameter HPV starts to be increased, for further evaluation of neutrophil functionality we chose experimental day 7. The functionality of peripheral blood neutrophils in AA was described by phagocytosis, oxidative burst and metabolic activity. Both phagocytosis and oxidative burst were increased due to arthritis, and that despite the decreased metabolic activity. Analysis of OS in tissue showed changed GGT activity in spleen and joint homogenate accompanied by increased chemiluminescence. The OS parameters were in close relationship with time profiles of selected cytokines, chemokine MCP-1 and of C-reactive protein levels in plasma. Measurements of the immunoregulatory index in peripheral blood and lipoxygenase tissue activity (lung, liver) were also included. Our observations have evidenced the importance of pharmacological regulation of redox imbalance in arthritis. (VEGA 2/0090/08, COST B35, APVV-51-017905, APVV-21-055205