Kao T-Y, Chiu Y-C, Fang W-C, Cheng C-W, Kuo C-Y, Juan H-F, Wu S-H, Lee A Y-L
Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu 300, Taiwan.
1] Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu 300, Taiwan [2] National Institute of Cancer Research, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan.
Cell Death Dis. 2015 Feb 12;6(2):e1642. doi: 10.1038/cddis.2015.9.
Human Lon protease is a mitochondrial matrix protein with several functions, including protein degradation, mitochondrial DNA (mtDNA) binding, and chaperone activity. Lon is currently emerging as an important regulator of mitochondria-contributed tumorigenesis due to its overexpression in cancer cells. To understand the mechanism of increased Lon in tumor cells, we studied the interactome to identify the chaperone Lon-associated proteins by proteomics approaches using the cells overexpressing Lon. In the present study, we designed a method connecting co-immunoprecipitation (Co-IP) to in-solution digestion for the shotgun mass spectrometry. We identified 76 proteins that were putative Lon-associated proteins that participated in mitochondrial chaperone system, cellular metabolism and energy, cell death and survival, and mtDNA stability. The association between Lon and NDUFS8 or Hsp60-mtHsp70 complex was confirmed by Co-IP and immunofluorescence co-localization assay. We then found that the protein stability/level of Hsp60-mtHsp70 complex depends on the level of Lon under oxidative stress. Most importantly, the ability of increased Lon-inhibited apoptosis is dependent on Hsp60 that binds p53 to inhibit apoptosis. These results suggest that the mechanism underlying cell survival regulated by Lon is mediated by the maintenance of the protein stability of Hsp60-mtHsp70 complex. This new knowledge of chaperone Lon interactome will allow us to better understand the cellular mechanism of Lon in mitochondrial function and of its overexpression in enhancing cell survival and tumorigenesis.
人源Lon蛋白酶是一种线粒体基质蛋白，具有多种功能，包括蛋白质降解、线粒体DNA（mtDNA）结合和伴侣活性。由于Lon在癌细胞中过表达，它目前正成为线粒体介导的肿瘤发生的重要调节因子。为了了解肿瘤细胞中Lon增加的机制，我们通过蛋白质组学方法，利用过表达Lon的细胞研究了相互作用组，以鉴定与伴侣蛋白Lon相关的蛋白质。在本研究中，我们设计了一种将免疫共沉淀（Co-IP）与溶液内消化相连接的方法，用于鸟枪法质谱分析。我们鉴定出76种蛋白质，它们是假定的与Lon相关的蛋白质，参与线粒体伴侣系统、细胞代谢与能量、细胞死亡与存活以及mtDNA稳定性。通过Co-IP和免疫荧光共定位分析证实了Lon与NDUFS8或Hsp60-mtHsp70复合物之间的关联。然后我们发现，在氧化应激下，Hsp60-mtHsp70复合物的蛋白质稳定性/水平取决于Lon的水平。最重要的是，Lon增加抑制细胞凋亡的能力取决于与p53结合以抑制凋亡的Hsp60。这些结果表明，Lon调节细胞存活的机制是由维持Hsp60-mtHsp70复合物的蛋白质稳定性介导的。伴侣蛋白Lon相互作用组的这一新知识将使我们能够更好地理解Lon在 mitochondrial功能中的细胞机制，以及其在增强细胞存活和肿瘤发生中的过表达机制。
