Inhibition of aberrant and constitutive phosphorylation of the high-molecular-mass neurofilament subunit by CEP-1347 (KT7515), an inhibitor of the stress-activated protein kinase signaling pathway

Previous studies have implicated stress-activated protein kinases (SAPKs) in aberrant phosphorylation of the high-molecular-mass neurofilament subunit (NFH). We now present direct evidence for this involvement using CEP-1347, a specific inhibitor of SAPK activation. Inhibition by this drug of stress-induced phosphorylation of NFH and the middle-molecular-mass neurofilament subunit in the perikaryon of dorsal root ganglion (DRG) neurons paralleled the decrease in levels of activated SAPKs and was essentially complete at 1 microM: CEP-1347. In addition, a role for SAPKs in the constitutive phosphorylation of NFH was demonstrated. Longterm treatment of unstressed DRG neurons with CEP-1347 lowered the steady-state phosphorylation level of NFH in neurites. No such effect was seen in neurons treated with PD 098059, which blocks activation of extracellular signal-regulated kinase 1/2. DRG neurites were shown to contain high basal levels of activated SAPKs. These included a 55-kDa SAPK whose activation was completely abolished at 0.05 microM: CEP-1347 and a 45-kDa SAPK that was not affected by the drug. These results indicate that SAPKs are involved in both stress-induced and constitutive phosphorylation of NFH. The differing responses of SAPKs in neurites and cell bodies to CEP-1347 inhibition further suggest the presence of different signaling pathways in the two neuronal compartments.     

Inhibitors of Trypsin-Like Serine Proteases Inhibit Processing of the Caspase Nedd-2 and Protect PC12 Cells and Sympathetic Neurons from Death Evoked by Withdrawal of Trophic Support

Abstract: Rat pheochromocytoma (PC12) cells and sympathetic neurons undergo apoptotic cell death upon withdrawal of trophic support. We have shown previously that selective cysteine aspartase (caspase) inhibitors protect PC12 cells and sympathetic neurons from such death, and that the caspase Nedd-2 is required for this type of death to occur. We now show that 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and N ^α- p -tosyl- l -lysine chloromethyl ketone (TLCK), agents that inhibit another class of proteases, the trypsin-like serine proteases, also suppress cell death in this paradigm. The site of action of these agents is upstream of the caspases, because the CPP32-like and Nedd-2-cleaving activities that are induced upon withdrawal of trophic support in PC12 cells are inhibited when AEBSF and TLCK are applied to the cells. Both agents inhibit thymidine incorporation in PC12 cells at concentrations similar to those that promote survival, raising the possibility that they may promote survival in neuronal cells through inhibition of aberrant activation of cell cycle components.     

质谱鉴定PC12细胞蛋白酶体抑制剂PSI-诱导性包含体中的LBs蛋白质

路易(小)体(Lewy body, LB）构成特征的蛋白质组份（protein content）在体外形成的路易(小)体样包含体（Lewy body-like inclusion）或聚集体（aggresome）中能够获得鉴定．通过蛋白质组学方法鉴定LB蛋白质组分是一种新的途径.10 µmol/L人工合成蛋白酶体抑制剂PSI（proteasomal inhibitor）作用PC12细胞48 h使其产生PSI诱导性包含体（PSI-induced inclusions）. 为了在体外指明可能的LB蛋白质组分,通过生物化学分级分离、双向电泳（two-dimensional electrophoresis,2-D）和肽质量指纹鉴定（identification via peptide mass fingerprints,PMF）的蛋白质组学方法,鉴定了2个涉及突触递质合成的蛋白质、6个26 S蛋白酶体亚基、2个细胞骨架蛋白、2个线粒体蛋白、1个抗氧化蛋白和7个分子伴侣蛋白和（或）分子伴侣样蛋白等20个LB蛋白质组分.结果提示,当PC12细胞发生蛋白酶体抑制时,这20个LB蛋白质组分可能被富集到PSI诱导性包含体中．