The serine protease inhibitors TLCK and TPCK react with the RB-binding core of HPV-18 E7 protein and abolish its RB-binding capability.
The serine protease inhibitors TLCK and TPCK react with the RB-binding core of HPV-18 E7 protein and abolish its RB-binding capability.
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丝氨酸蛋白酶抑制剂 TLCK 和 TPCK 与 HPV-18 E7 蛋白的 RB 结合核心发生反应，并消除其 RB 结合能力。
DOI：
10.1006/viro.1996.0149
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Schlegel,R
中科院分区：
文献类型：
--
作者：
Stoppler,H;Stoppler,MC;Adduci,A;Koval,D;Schlegel,R
与宫颈癌相关的人乳头瘤病毒（例如，HPV-16和HPV-18）表达E7癌蛋白，其介导原代生殖器角质形成细胞的永生化和啮齿动物细胞的转化。105个氨基酸的HPV-18 E7蛋白含有两个锌指以及一个保守的氨基末端基序（Rb结合核心），其结合并改变视网膜母细胞瘤易感基因产物（Rb）的相互作用。我们在这里报告，两个丝氨酸蛋白酶抑制剂，甲苯磺酰基-L-赖氨酸氯甲基酮（TLCK）和甲苯磺酰基-L-苯丙氨酸氯甲基酮（TPCK），与HPV-18 E7蛋白反应，并产生了改变形式。E7蛋白的化学修饰最初是在从哺乳动物细胞中提取和免疫沉淀的过程中观察到的，但也可以使用网织红细胞裂解物体外表达的E7蛋白来检测。更重要的是，TLCK和TPCK能够修改E7蛋白在活的角质形成细胞后，他们添加到培养基中。位点特异性突变表明，E7 Rb结合核心（Leu-X-Cys-X-Glu）含有半胱氨酸残基，这是必不可少的这种修饰和TLCK/TPCK依赖性改变E7蛋白废除其结合Rb的能力。这些研究表明，E7蛋白可以被一类特定的蛋白酶抑制剂失活，并且这些试剂可能对体内E7功能的调节是有用的，此外，这些结果表明，在评价E7/细胞蛋白相互作用的实验中应用这些常用的蛋白酶抑制剂时必须小心。
The human papillomaviruses associated with cervical cancer (e.g., HPV-16 and HPV-18) express an E7 oncoprotein which mediates the immortalization of primary genital keratinocytes and the transformation of rodent cells. The 105-amino-acid HPV-18 E7 protein contains two zinc fingers as well as a conserved amino-terminal motif (Rb-binding core) which binds and alters the interactions of the retinoblastoma susceptibility gene product (Rb). We report here that two serine protease inhibitors, tosyl-L-lysine chloromethyl ketone (TLCK) and tosyl-L-phenylalanine chloromethyl ketone (TPCK), reacted with and generated an altered form of the HPV-18 E7 protein. Chemical modification of the E7 protein was initially observed during its extraction and immunoprecipitation from mammalian cells but could also be detected using E7 protein expressedin vitroby reticulocyte lysates. More importantly, TLCK and TPCK were able to modify E7 protein in live keratinocytes following their addition to the culture medium. Site-specific mutagenesis demonstrated that the E7 Rb-binding core (Leu-X-Cys-X-Glu) contained a cysteine residue which was essential for this modification and that the TLCK/TPCK-dependent alteration of E7 protein abolished its ability to bind Rb. These studies indicate that the E7 protein can be inactivated by a specific class of protease inhibitors and that such reagents may be useful for pharmacologically regulating E7 functionin vivo.In addition, these results demonstrate that care must be taken when applying these commonly used protease inhibitors in experiments evaluating E7/cellular protein interactions.