The functional relationships between phosphoinositides and sphingolipids have not been well characterized to date. ISP-1/myriocin is a potent inhibitor of sphingolipid biosynthesis and induces severe growth defects in eukaryotic cells because of the sphingolipid deprivation. We characterized a novel multicopy suppressor gene of ISP-1-mediated cell death in yeast, MSS4. MSS4 encodes a phosphatidylinositol-4-phosphate 5-kinase that synthesizes phosphatidylinositol (4,5)-bisphosphate (PI4,5P 2 ). We demonstrate here that ISP-1 treatment of yeast causes defects both in the activity and subcellular localization of Mss4. The effect of the Mss4 defect on the downstream signaling was examined, because interaction between the Mss4 product, PI4,5P 2 , and the pleckstrin-homology domain of Rom2 mediates recruitment of Rom2 to the membrane, which is the crucial step for subsequent Rho1/2 activation. Indeed, failure of Rom2 recruitment was observed in ISP-1-treated cells as well as in csg2-deleted cells, which have reduced mannosylated inositolphosphorylceramide. These data suggested that proper sphingolipids are required for the signaling pathway involving Mss4.
The obstacles to the development of therapeutic aptamers for systemic inflammatory diseases, such as nuclease degradation and renal clearance, have not been fully overcome. Here, we report a novel PEGylation method, sbC-PEGylation, which improves the pharmacokinetic properties of RNA aptamers that act against interleukin-17A (IL-17A) in mice and monkeys. sbC-PEGylated aptamers were synthesized by coupling the symmetrical branching molecule 2-cyanoethyl-N,N-diisopropyl phosphoroamidite to the 5′ end of the aptamer, before conjugating two polyethylene glycol (PEG) molecules to the aptamer. Pharmacokinetic studies showed that compared with conventionally PEGylated aptamers, the sbC-PEGylated aptamer exhibited excellent stability in the blood circulation of mice and monkeys. In addition, one of the sbC-PEGylated aptamers, 17M-382, inhibited the interleukin-6 (IL-6) production induced by IL-17A in NIH3T3 cells in a concentration-dependent manner, and the half-maximal inhibitory concentration of sbC-PEGylated 17M-382 was two times lower than that of non-PEGylated 17M-382. Furthermore, the intraperitoneal administration of sbC-PEGylated 17M-382 significantly inhibited the IL-6 production induced by IL-17A in a mouse air pouch model. Our findings suggest that the novel PEGylation method described in this study, sbC-PEGylation, could be used to develop anti-IL-17A aptamers as a therapeutic option for systemic inflammatory disease.
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