Mammalian HSP60 is a major target for an immunosuppressant mizoribine
Mammalian HSP60 is a major target for an immunosuppressant mizoribine
复制标题
DOI：
10.1074/jbc.274.49.35147
复制
发表时间：
1999-12-03
影响因子：
4.8
通讯作者：
Tashima, Y
中科院分区：
文献类型：
--
作者：
Itoh, H;Komatsuda, A;Tashima, Y
据报道，免疫抑制剂环孢菌素A或FK506与细胞内的亲免素结合，这些亲免素与分子伴侣HSP70或HSP90形成复合物。虽然咪唑立宾已作为免疫抑制剂应用于临床，但该剂的亲免素尚未完全了解。我们使用咪唑立宾亲和柱层析和猪肾胞质溶胶研究了它们的特异性结合蛋白，通过增加柱洗脱液中的咪唑立宾，通过 SDS-聚丙烯酰胺凝胶电泳检测到两种主要蛋白（分子量为 60 和 43 kDa）。根据这些蛋白质的氨基酸序列分析，分别用 HSP60 和胞质肌动蛋白鉴定出 60-kDa 和 43-kDa 的咪唑立宾结合蛋白。大量的肌动蛋白也被核苷酸从亲和柱上洗脱下来，但在相同条件下洗脱出非常少量的HSP60。另一方面，HSP60作为洗脱液中的主要蛋白质被优先洗脱，其次是核苷酸，其次是咪唑立宾。洗脱液中也检测到肌动蛋白，但蛋白质的量非常低。这些结果表明HSP60与咪唑立宾具有高亲和力，并且在表面等离子共振分析中也观察到了相互作用。尽管HSP60或GroE在体外促进柠檬酸合酶的重折叠，但咪唑立宾干扰HSP60的伴侣活性。在不同类型的咪唑立宾亲和柱上，HSP60或肌动蛋白识别咪唑立宾的NH2基团，并且该基团可能是该试剂的官能团。
It has been reported that immunosuppressant cyclosporin A or FK506 binds to immunophilins in the cell and that these immunophilins make a complex with molecular chaperones HSP70 or HSP90, Although mizoribine has been used clinically as an immunosuppressant, immunophilins of the agent have not yet been fully understood. We investigated their specific binding proteins using mizoribine affinity column chromatography and porcine kidney cytosols, By increasing mizoribine in the eluant from the column, two major proteins (with molecular masses of 60 and 43 kDa) were detected by SDS-polyacrylamide gel electrophoresis. Based on the amino acid sequence analysis of these proteins, 60- and 43-kDa mizoribine-binding proteins were identified with HSP60 and cytosolic actin, respectively. A considerable amount of actin was also eluted from the affinity column by nucleotides, but a very low quantity of HSP60 was eluted under the same conditions. On the other hand, HSP60 was eluted as a major protein in the eluant that was eluted preferentially, with nucleotide followed by mizoribine. Actin was also detected in the eluant, but the quantity of the protein was very low. These results indicated that HSP60 has high affinity to mizoribine, and the interaction was also observed on surface plasmon resonance analysis. Although HSP60 or GroE facilitated refolding of citrate synthase in vitro, mizoribine interfered with the chaperone activity of HSP60. On different types of mizoribine affinity columns, HSP60 or actin recognized the NH2 group of mizoribine, and this group may be a functional group of the agent.