School of Pharmacy, China Medical University, Taichung, Taiwan.
Biochem Biophys Res Commun. 2010 Jul 16;398(1):26-31. doi: 10.1016/j.bbrc.2010.06.004. Epub 2010 Jun 9.
Amplification and overexpression of murine double minute (MDM2) has been observed in several human cancers. Some chemotherapeutic agents cause MDM2 ubiquitination and degradation in a proteasome-dependent system. In addition to the proteasome system, chaperone-mediated autophagy (CMA) is a lysosomal pathway for selective misfolded protein degradation. Molecular chaperone heat shock cognate 70 protein (Hsc70) recognizes the misfolded proteins, which are then delivered to lysosome-associated membrane protein type 2A (LAMP2A) for lysosomal degradation. Our previous study reported that hispolon was able to induce cell apoptosis and downregulate MDM2 expression. In this study, our results showed that the proteasome inhibitor, MG132, could not inhibit hispolon-induced MDM2 downregulation. In contrast, both inhibition of lysosomes with NH(4)Cl and inhibition of LAMP2A using siRNA partially attenuated hispolon-induced MDM2 downregulation. To determine whether Hsc70 recognizes MDM2 on amino acids 135-141, SMP14 antibody was used to compete with Hsc70 for interaction with MDM2. After Hsc70 knockdown, SMP14 antibody immunoprecipitated increased MDM2. We also found that hispolon induced increased association of Hsp70, Hsc70, Hsp90 and LAMP2A with MDM2. This association was inhibited in cells pretreated with geldanamycin (GA), an Hsp90 inhibitor. GA also attenuated hispolon-induced MDM2 downregulation. Meanwhile, inhibition of Hsc70 using siRNA attenuated hispolon-induced MDM2 downregulation. Our study provides the first example of the ability of hispolon to mediate MDM2 downregulation in lysosomes through the CMA pathway.
在几种人类癌症中已经观察到鼠双微体（MDM2）的扩增和过表达。一些化疗药物在蛋白酶体依赖性系统中引起 MDM2 的泛素化和降解。除了蛋白酶体系统外，伴侣介导的自噬（CMA）是一种溶酶体途径，用于选择性降解错误折叠的蛋白质。分子伴侣热休克同源物 70 蛋白（Hsc70）识别错误折叠的蛋白质，然后将其递送至溶酶体相关膜蛋白 2A（LAMP2A）进行溶酶体降解。我们之前的研究报告称，千里光碱能够诱导细胞凋亡并下调 MDM2 表达。在这项研究中，我们的结果表明，蛋白酶体抑制剂 MG132 不能抑制千里光碱诱导的 MDM2 下调。相反，用氯化铵抑制溶酶体和用 siRNA 抑制 LAMP2A 均可部分减弱千里光碱诱导的 MDM2 下调。为了确定 Hsc70 是否在氨基酸 135-141 上识别 MDM2，我们使用 SMP14 抗体与 Hsc70 竞争与 MDM2 的相互作用。在 Hsc70 敲低后，SMP14 抗体免疫沉淀增加了 MDM2。我们还发现千里光碱诱导 Hsp70、Hsc70、Hsp90 和 LAMP2A 与 MDM2 的结合增加。在用 HSP90 抑制剂格尔德霉素（GA）预处理的细胞中，这种结合受到抑制。GA 还减弱了千里光碱诱导的 MDM2 下调。同时，用 siRNA 抑制 Hsc70 减弱了千里光碱诱导的 MDM2 下调。我们的研究提供了第一个例子，表明千里光碱能够通过 CMA 途径介导溶酶体中的 MDM2 下调。
