Conformation-dependent stability of junctophilin 1 (JP1) and ryanodine receptor type 1 (RyR1) channel complex is mediated by their hyper-reactive thiols.

Junctophilin 1 (JP1), a 72-kDa protein localized at the skeletal muscle triad, is essential for stabilizing the close apposition of T-tubule and sarcoplasmic reticulum membranes to form junctions. In this study we report that rapid and selective labeling of hyper-reactive thiols found in both JP1 and ryanodine receptor type 1 (RyR1) with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, a fluorescent thiol-reactive probe, proceeded 12-fold faster under conditions that minimize RyR1 gating (e.g. 10 mM Mg2+) compared with conditions that promote high channel activity (e.g. 100 microM Ca2+, 10 mM caffeine, 5 mM ATP). The reactivity of these thiol groups was very sensitive to oxidation by naphthoquinone, H2O2, NO, or O2, all known modulators of the RyR1 channel complex. Using preparative SDS-PAGE, in-gel tryptic digestion, high pressure liquid chromatography, and mass spectrometry-based peptide sequencing, we identified 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin-thioether adducts on three cysteine residues of JP1 (101, 402, and 627); the remaining five cysteines of JP1 were unlabeled. Co-immunoprecipitation experiments demonstrated a physical interaction between JP1 and RyR1 that, like thiol reactivity, was sensitive to RyR1 conformation and chemical status of the hyper-reactive cysteines of JP1 and RyR1. These findings support a model in which JP1 interacts with the RyR1 channel complex in a conformationally sensitive manner and may contribute integral redox-sensing properties through reactive sulfhydryl chemistry.

Lobocyclamides A-C, lipopeptides from a cryptic cyanobacterial mat containing Lyngbya confervoides.

The structures of lipopeptides lobocyclamides A (1), B (2), and C (3) were solved using a combination of mass spectrometry, 2D NMR spectroscopy, and degradative analysis. Lobocyclamides B and C are the first peptides reported with the unusual amino acid 4-hydroxythreonine and also incorporate the rare homologous long-chain beta-amino acids 3-aminooctanoic acid and 3-aminodecanoic acid, respectively. The absolute configurations of the amino acid residues in each compound were assigned, after acid hydrolysis, by either direct chiral HPLC comparison with authentic standards or by prior derivatization by Marfey's method and reversed-phase HPLC. Both compounds exhibited moderate antifungal activity against a panel of Candida spp., including two fluconazole-resistant strains. When tested as a mixture, lobocyclamides A and B displayed synergistic in vitro antifungal activity, a phenomenon noted earlier for the related peptides laxaphycins A and B.