Curcumin protects the developing lung against long-term hyperoxic injury.

Curcumin, a potent anti-inflammatory and antioxidant agent, modulates peroxisome proliferator-activated receptor-γ signaling, a key molecule in the etiology of bronchopulmonary dysplasia (BPD). We have previously shown curcumin's acute protection against neonatal hyperoxia-induced lung injury. However, its longer-term protection against BPD is not known. Hypothesizing that concurrent treatment with curcumin protects the developing lung against hyperoxia-induced lung injury long-term, we determined if curcumin protects against hyperoxic neonatal rat lung injury for the first 5 days of life, as determined at postnatal day (PND) 21. One-day-old rat pups were exposed to either 21 or 95% O2 for 5 days with or without curcumin treatment (5 mg/kg) administered intraperitoneally one time daily, following which the pups grew up to PND21 in room air. At PND21 lung development was determined, including gross and cellular structural and functional effects, and molecular mediators of inflammatory injury. To gain mechanistic insights, embryonic day 19 fetal rat lung fibroblasts were examined for markers of apoptosis and MAP kinase activation following in vitro exposure to hyperoxia for 24 h in the presence or absence of curcumin (5 µM). Curcumin effectively blocked hyperoxia-induced lung injury based on systematic analysis of markers for lung injury (apoptosis, Bcl-2/Bax, collagen III, fibronectin, vimentin, calponin, and elastin-related genes) and lung morphology (radial alveolar count and alveolar septal thickness). Mechanistically, curcumin prevented the hyperoxia-induced increases in cleaved caspase-3 and the phosphorylation of Erk1/2. Molecular effects of curcumin, both structural and cytoprotective, suggest that its actions against hyperoxia-induced lung injury are mediated via Erk1/2 activation and that it is a potential intervention against BPD.

Met-Gly-Cys motif from G-protein alpha subunit cannot direct palmitoylation when fused to heterologous protein.

To determine whether the N-terminal Met-Gly-Cys motif from G-protein alpha subunit can direct palmitoylation of protein, we have generated heterologous fusion proteins containing the first 10 amino acids of Gi1 alpha and Gs alpha using tumor necrosis factor as a model protein and determined their ability to incorporate palmitate using in vitro and in vivo expression systems. DNA sequences coding for the N-terminal 10 amino acids of Gi1 alpha and Gs alpha were fused to the 5'-end of the cDNA coding for the mature domain of tumor necrosis factor (TNF) to give Gi1 alpha-TNF and Gs alpha-TNF cDNA. In vitro translation of the mRNA coding for the Gi1 alpha-TNF cDNA gave rise to an N-myristoylated fusion TNF with a molecular mass of 18 kDa as determined by the incorporation of [3H]myristic acid and by immunoprecipitation with anti-TNF antibody. In contrast, no incorporation of fatty acid was detected for Gs alpha-TNF. Baculovirus expression of the Gi1 alpha-TNF cDNA in Sf-9 cells gave rise to an N-myristoylated but not palmitoylated fusion TNF. This myristoylation was inhibited by replacement of Gly-2 with Ala but not Cys-3 with Ala, indicating the acylation reaction is entirely dependent on the N-myristoylation signal (Met-Gly-X-X-X-Ser) and Cys-3 is not involved. As is the case with in vitro translation, no incorporation of fatty acid was detected for Gs alpha-TNF. These results indicated that unlike the myristoylation signal Met-Gly-X-X-X-Ser/Thr/Cys, the Met-Gly-Cys motif found in G-protein alpha subunits itself is not sufficient to direct palmitoylation even if Gly-2 is myristoylated after removal of initiating Met. Thus, another structural determinant is implicated in this modification.

In vitro synthesis of an N-myristoylated fusion protein that binds to the liposomal surface.

To increase the efficiency of association of tumor necrosis factor (TNF), a hydrophilic model protein, with liposomes, an N-myristoylation signal sequence was linked to the N-terminus of TNF by gene fusion. A DNA sequence coding for the N-myristoylation signal of Rasheed leukemia virus-gag protein was fused to be 5'-end of the cDNA coding for the mature domain of TNF to give N-myristoylated fusion TNF cDNA. In vitro translation of the mRNA coding for this fusion cDNA using rabbit reticulocyte lysate gave rise to an N-myristoylated fusion TNF with a molecular mass of 18 kDa as determined by the incorporation of [3H]myristic acid and by immunoprecipitation with anti-TNF antibody. Replacement of Gly2 in the myristoylation signal with Ala entirely inhibited the incorporation of [3H]-myristic acid into the fusion protein. A liposome binding assay using Ficoll density gradient centrifugation revealed that incubating the N-myristoylated fusion TNF with dipalmitoyl phosphatidylcholine-liposomes caused the complete binding of the protein to the liposomes, whereas much less of the nonmyristoylated counterpart bound. Thus, N-myristoylated fusion TNF, with high affinity for liposomes, was synthesized by the in vitro translation/transcription system.