Kadokura Hiroshi, Harada Nanshi, Yamaki Satoshi, Hirai Naoya, Tsukuda Ryusuke, Azuma Kota, Amagai Yuta, Nakamura Daisuke, Yanagitani Kota, Taguchi Hideki, Kohno Kenji, Inaba Kenji
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan.
Institute for Research Initiatives, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.
iScience. 2024 Oct 16;27(11):111189. doi: 10.1016/j.isci.2024.111189. eCollection 2024 Nov 15.
Localization to the endoplasmic reticulum (ER) and subsequent disulfide bond formation are crucial processes governing the biogenesis of secretory pathway proteins in eukaryotes. Hence, comprehending the mechanisms underlying these processes is important. Here, we have engineered firefly luciferase (FLuc) as a tool to detect deficiencies in these processes within mammalian cells. To achieve this, we introduced multiple cysteine substitutions into FLuc and targeted it to the ER. The reporter exhibited FLuc activity in response to defects in protein localization or disulfide bond formation within the ER. Notably, this system exhibited outstanding sensitivity, reproducibility, and convenience in detecting abnormalities in these processes. We applied this system to observe a protein translocation defect induced by an inhibitor of HIV receptor biogenesis. Moreover, utilizing the system, we showed that modulating LMF1 levels dramatically impacted the ER's redox environment, confirming that LMF1 plays some critical role in the redox control of the ER.
定位于内质网（ER）并随后形成二硫键是真核生物中分泌途径蛋白生物合成的关键过程。因此，理解这些过程背后的机制很重要。在这里，我们构建了萤火虫荧光素酶（FLuc）作为一种工具来检测哺乳动物细胞中这些过程的缺陷。为了实现这一点，我们在FLuc中引入了多个半胱氨酸替换并将其靶向内质网。该报告基因在响应内质网内蛋白质定位或二硫键形成缺陷时表现出FLuc活性。值得注意的是，该系统在检测这些过程中的异常方面表现出出色的敏感性、可重复性和便利性。我们应用该系统观察了HIV受体生物合成抑制剂诱导的蛋白质转运缺陷。此外，利用该系统，我们表明调节LMF1水平会显著影响内质网的氧化还原环境，证实LMF1在内质网的氧化还原控制中起关键作用。
