Molecular mechanism of cellular processing of transmembrane tumor necrosis factor

检测人跨膜肿瘤坏死因子（pro-TNF）翻译后蛋白修饰。编码前tnf的cDNA在COS-1细胞和Sf9细胞中均有表达，并尝试用[^3H]肉豆蔻或[^3H]棕榈酸进行代谢标记。从转染细胞分泌的17 kDa成熟TNF未被标记，而26 kDa前TNF被[^3H]棕榈酸特异性标记。前tnf的[^3H]棕榈酸标记被羟胺处理消除，表明标记是由于半胱氨酸残基通过硫酯键棕榈酰化。对原tnf先导序列中两个半胱氨酸残基的定点诱变表明，原tnf的棕榈酰化仅发生在位于原tnf跨膜结构域和细胞质结构域交界的Cys-47上。因此，tnf前体通过其蛋白跨膜结构域和脂质锚点与质膜相互作用。

Human transmembrane tumor necrosis factor (pro-TNF) was examined for posttranslational protein modification. The cDNA coding for pro-TNF was expressed in both COS-1 cells and Sf9 cells and metabolic labeling with [^3H]myristic or [^3H]palmitic acid was attempted. The 17 kDa mature TNF secreted from the transfected cells was not labeled, whereas the 26 kDa pro-TNF was specifically labeled with [^3H]palmitic acid. The [^3H]palmitic acid labeling of pro-TNF was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation of a cysteine residue via a thiocster bond. Site-directed mutagenesis of the two cysteine residues residing in the leader sequence of pro-TNF demonstrated that palmitoylation of pro-TNF occurs solely at Cys-47, located at the boundary between the transmembrane and cytoplasmic domains of pro-TNF.Thus, pro-TNF interacts with the plasma membrane via both its proteinaceous transmembrane domain and a lipid anchor.