Muscle Health Research Centre, School of Kinesiology and Health Science, York University , Toronto, Ontario , Canada.
Am J Physiol Cell Physiol. 2018 Oct 1;315(4):C516-C526. doi: 10.1152/ajpcell.00275.2017. Epub 2018 Jun 27.
The mitochondrial unfolded protein response (UPR) is a protein quality control mechanism that strives to achieve proteostasis in the face of misfolded proteins. Because of the reliance of mitochondria on both the nuclear and mitochondrial genomes, a perturbation of the coordination of these genomes results in a mitonuclear imbalance in which holoenzymes are unable to assume mature stoichiometry and thereby activates the UPR. Thus, we sought to perturb this genomic coordination by using a systemic antisense oligonucleotide (in vivo morpholino) targeted to translocase of the inner membrane channel subunit 23 (Tim23), the major channel of the inner membrane. This resulted in a 40% reduction in Tim23 protein content, a 32% decrease in matrix-destined protein import, and a trend to elevate reactive oxygen species (ROS) emission under maximal respiration conditions. This import defect activated the C/EBP homologous protein (CHOP) branch of the UPR, as evident from increases in caseinolytic mitochondrial matrix peptidase proteolytic subunit (ClpP) and chaperonin 10 (cpn10) but not the activating transcription factor 5 (ATF5) arm. Thus, in the face of proteotoxic stress, CHOP and ATF5 could be activated independently to regain proteostasis. Our second aim was to investigate the role of proteolytically derived peptides in mediating retrograde signaling. Peptides released from the mitochondrion following basal proteolysis were isolated and incubated with import reactions. Dose- and time-dependent effect of peptides on protein import was observed. Our data suggest that mitochondrial proteolytic byproducts exert an inhibitory effect on protein import, possibly to reduce excessive protein import as a potential negative feedback mechanism. The inhibition of import into the organelle also serves a retrograde function, possibly via ROS emission, to modify nuclear gene expression and ultimately improve folding capacity.
线粒体未折叠蛋白反应（UPR）是一种蛋白质质量控制机制，旨在面对错误折叠的蛋白质时维持蛋白质的稳定。由于线粒体依赖于核基因组和线粒体基因组，因此这两个基因组之间协调的干扰会导致线粒体-核失衡，使全酶无法达到成熟的比例，从而激活 UPR。因此，我们试图通过使用靶向内膜通道亚基 23（Tim23）的系统反义寡核苷酸（体内形态发生素）来干扰这种基因组协调，Tim23 是内膜的主要通道。这导致 Tim23 蛋白含量减少 40%，基质靶向蛋白导入减少 32%，最大呼吸条件下活性氧（ROS）发射呈上升趋势。这种导入缺陷激活了 UPR 的 C/EBP 同源蛋白（CHOP）分支，这从酪蛋白水解线粒体基质肽酶水解亚基（ClpP）和伴侣蛋白 10（cpn10）的增加而明显看出，但 ATF5 臂没有增加。因此，在面对蛋白毒性应激时，CHOP 和 ATF5 可以独立激活以恢复蛋白质稳定。我们的第二个目标是研究蛋白水解衍生肽在介导逆行信号中的作用。在基础蛋白水解后从线粒体释放的肽被分离并与导入反应孵育。观察到肽对蛋白导入的剂量和时间依赖性影响。我们的数据表明，线粒体蛋白水解副产物对蛋白导入施加抑制作用，可能是作为一种潜在的负反馈机制来减少过度的蛋白导入。这种对细胞器的导入的抑制也具有逆行功能，可能通过 ROS 发射来修饰核基因表达，并最终提高折叠能力。
