Cav-1 Scaffolding Domain Peptide Research Articles

PBN inhibits pro-inflammatory cytokines, and amplifies the anti-inflammatory cytokine interleukin-10 in μ-ray irradiated rats: [formula omitted], G. L. Wallis, C. A. Stewart, Y. Kotake, Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA

Nitrative stress is considered as an important pathological process of hepatic ischemia and reperfusion injury but its regulating mechanisms are largely unknown. In this study, we tested the hypothesis that caveolin-1 (Cav-1), a plasma membrane scaffolding protein, could be an important cellular signaling against hepatic I/R injury through inhibiting peroxynitrite (ONOO−)-induced cellular damage. Male wild-type mice and Cav-1 knockout (Cav-1−/−) were subjected to 1 h hepatic ischemia following 1, 6 and 12 h of reperfusion by clipping and releasing portal vessels respectively. Immortalized human hepatocyte cell line (L02) was subjected to 1 h hypoxia and 6 h reoxygenation and treated with Cav-1 scaffolding domain peptide. The major discoveries included: (1) the expression of Cav-1 in serum and liver tissues of wild-type mice was time-dependently elevated during hepatic ischemia-reperfusion injury. (2) Cav-1 scaffolding domain peptide treatment inhibited cleaved caspase-3 expression in the hypoxia-reoxygenated L02 cells; (3) Cav-1 knockout (Cav-1−/−) mice had significantly higher levels of serum transaminases (ALT&AST) and TNF-α, and higher rates of apoptotic cell death in liver tissues than wild-type mice after subjected to 1 h hepatic ischemia and 6 hour reperfusion; (4) Cav-1−/− mice revealed higher expression levels of iNOS, ONOO− and 3-nitrotyrosine (3-NT) in the liver than wild-type mice, and Fe-TMPyP, a representative peroxynitrite decomposition catalyst (PDC), remarkably reduced level of ONOO− and 3-NT and ameliorated the serum ALT, AST and TNF-α levels in both wild-type and Cav-1−/− mice. Taken together, we conclude that Cav-1 could play a critical role in preventing nitrative stress-induced liver damage during hepatic ischemia-reperfusion injury.

Interaction between Caveolin-1 and the Reductase Domain of Endothelial Nitric-oxide Synthase: CONSEQUENCES FOR CATALYSIS

Endothelial nitric-oxide synthase (eNOS) is targeted to caveoli through interaction with caveolin-1 (cav-1). cav-1 binding to a consensus site in the eNOS oxygenase domain is proposed to antagonize calmodulin (CaM) binding and thereby inhibit eNOS nitric oxide (NO) synthesis. To study the mechanism, we examined how cav-1 scaffolding domain peptide (amino acids 82-101; cav-1P) would affect NO synthesis, NADPH oxidation, cytochrome c reduction, and ferricyanide reduction by full-length eNOS or its isolated oxygenase and reductase domains. Cav-1P equivalently inhibited NO synthesis and NADPH oxidation by full-length eNOS in a manner reversible by CaM but did not affect NADPH-independent NO synthesis by full-length eNOS or its oxygenase domain, indicating inhibition required the reductase domain. Similar concentrations of cav-1P inhibited cytochrome c reduction by full-length eNOS or the reductase domain (amino acids 492-1205) in a CaM-reversible manner, indicating cav-1P interaction with reductase or full-length eNOS are equivalent. Ferricyanide reduction was unaffected by cav-1P in all cases. Immunoblotting showed that full-length eNOS, eNOS oxygenase, and eNOS reductase all bound to an immobilized glutathione S-transferase-cav-1 fusion protein. Thus, cav-1 interacts independently with both oxygenase and reductase domains of eNOS. The reductase interaction occurs independent of a cav-1 binding motif, is CaM-reversible, and is of sufficient affinity to match cav-1P inhibition of NO synthesis by full-length eNOS. We propose that cav-1 binding to eNOS reductase compromises its ability to bind CaM and to donate electrons to the eNOS heme, thereby inhibiting NO synthesis.