Dunbar J D, Song H Y, Guo D, Wu L W, Donner D B
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202, USA.
J Immunol. 1997 May 1;158(9):4252-9.
A protein that binds the intracellular domain of the type 1 TNFR (TNFR-1IC) has been identified by two-hybrid cloning. The 97-kDa TNFR-associated protein, TRAP2, shows sequence identity with internal amino acid sequences from subunit 2 of the 26S protease. TRAP2 antiserum recognizes subunit 2 of the 26S protease, which is consistent with the identity of these proteins. TRAP2 antiserum interacted with the 97-kDa protein in HeLa cell lysates and cytosol, the latter observation showing that TRAP2 resides in the same cellular compartment as TNFR-1IC. A fusion of glutathione-S-transferase and TNFR-1IC (GST-TNFR-1IC) precipitated TRAP2 from a HeLa cell lysate; conversely, GST-TRAP2 precipitated TNFR-1 from such a lysate. These observations show that the proteins interact in the cellular milieu. After in vitro transcription/translation and 35S labeling, TRAP2 was precipitated from a cellfree system by GST-TNFR-1IC, showing that TNFR-1IC and TRAP2 interact directly. TRAP2 was also precipitated from the cellfree translation system by a GST fusion containing the N-terminal half of TNFR-1IC, but not by a GST fusion containing the C-terminal half of TNFR-1IC that contains a "death domain" that plays an obligatory role in signaling cytotoxicity. The ability of deletion mutants of TNFR-1IC to interact with TRAP2 was tested using the two-hybrid system. This also showed that the amino acid sequences that mediate binding reside outside of the death domain in TNFR-1IC. The demonstration that a subunit of the 26S protease binds TNFR-1 may identify a novel TNF-signaling pathway.
通过双杂交克隆已鉴定出一种与1型肿瘤坏死因子受体细胞内结构域（TNFR-1IC）结合的蛋白质。97 kDa的肿瘤坏死因子受体相关蛋白TRAP2与26S蛋白酶亚基2的内部氨基酸序列具有序列同一性。TRAP2抗血清可识别26S蛋白酶的亚基2，这与这些蛋白质的同一性相符。TRAP2抗血清与HeLa细胞裂解物和胞质溶胶中的97 kDa蛋白质相互作用，后一观察结果表明TRAP2与TNFR-1IC存在于同一细胞区室中。谷胱甘肽-S-转移酶与TNFR-1IC的融合体（GST-TNFR-1IC）从HeLa细胞裂解物中沉淀出TRAP2；相反，GST-TRAP2从这样的裂解物中沉淀出TNFR-1。这些观察结果表明这些蛋白质在细胞环境中相互作用。在体外转录/翻译和35S标记后，GST-TNFR-1IC从无细胞系统中沉淀出TRAP2，表明TNFR-1IC和TRAP2直接相互作用。含有TNFR-1IC N端一半的GST融合体也从无细胞翻译系统中沉淀出TRAP2，但含有TNFR-1IC C端一半且包含在细胞毒性信号传导中起关键作用的“死亡结构域”的GST融合体则不能沉淀出TRAP2。使用双杂交系统测试了TNFR-1IC缺失突变体与TRAP2相互作用的能力。这也表明介导结合的氨基酸序列位于TNFR-1IC的死亡结构域之外。26S蛋白酶的一个亚基与TNFR-1结合的证明可能确定了一条新的肿瘤坏死因子信号通路。
