| Research Abstract |
For the project described above we have been supported by the Ministry of Education, Science, Sports and Culture. We have obtained the following results including the hypothesis and have published some of them.
(1) By constructing an expression plasmid for a mutant pre-IL-1alpha lacking phosphorylation sites, we obtained cells producing unphosphorylated pre-IL-1alpha. Although calcium-dependent proteolytic processing of the pre-IL-1alpha could be observed in cell lysates, proteolytic processing was not induced by treatment with calcium ionophore in intact cells.
(2) PSA-3 cells are a mutant requiring phosphatidylserine (PS), the content of which are decreased in the absence of PS.We obtained PSA-3 cells harboring pre-IL-1alpha expression vector. Irrespective of the presence or absence of PS,pre-IL-1alpha was proteolytically processed and released by these cells.
(3) IL-1alpha, but not IL-1beta, caused an increase in the permeability of liposomes composed of PS,at neutral and acidic pHs, as demonstrated by measuring the efflux of calcein. On the other hand, liposomes composed of phosphatidylcholine showed no increase in permeability. IL-1alpha also induced the efflux of fluorescent dextran with an average mol.mass of 40kDa.
(4) We made a hypothesis for processing and release of IL-1alpha. According the hypothesis, upon phosphorylation, pre-IL-1alpha binds to the inner surface of plasma membrane through a calcium-dependent interaction with PS,and is processed by activated calpain with preferential release of mature IL-1alpha.
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