Bell Katherine S, Ko Sebastian, Ali Sam, Bognar Brett, Khmelkov Michael, Rau Nick, Peng Oscar K, Eyuboglu Mavi, Paine John, Tong Andy, Saria Anuj, Agrawal Siddharth, Davies Kelvin J A, Tower John
Molecular and Computational Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
Leonard Davis School of Gerontology, Ethel Percy Andrus Gerontology Center, University of Southern California, Los Angeles, CA 90089, USA.
Insects. 2025 May 31;16(6):583. doi: 10.3390/insects16060583.
Conditional transgenic systems and multi-copy target transgenes were used to produce transient fluorescent protein expression in adult , with the goal of developing an in vivo assay of protein turnover. Free-moving flies were assayed at multiple time points using video, and decay in fluorescence was used to calculate protein half-life. Additional experiments involved image capture of anesthetized flies. The half-life of eGFP was increased by the proteasome inhibitor bortezomib, both in vivo and in vitro, indicating proteasomal degradation of eGFP. The accumulation of eGFP in vivo was decreased by the protein synthesis inhibitor cycloheximide, without affecting half-life. The half-lives of several fluorescent proteins were determined, using both tissue-general and tissue-specific expression, in flies of both sexes and varying ages. Typical half-life values varied by fluorescent protein. DsRED showed a greater half-life than eGFP, and little if any degradation was detected for mCherry. Half-life also varied by tissue, with greater eGFP half-life observed in muscle relative to other tissues. Increased half-life with age was detected for DsRED but not for eGFP. Limited effects were observed for sex and female mating status. Taken together, the data indicate the in vivo assays are promising tools for the study of protein degradation regulated by protein sequence, subcellular compartment, tissue and small molecules.
利用条件转基因系统和多拷贝靶转基因在成虫中产生瞬时荧光蛋白表达，目的是开发一种蛋白质周转的体内检测方法。使用视频在多个时间点对自由活动的果蝇进行检测，并利用荧光衰减来计算蛋白质半衰期。额外的实验包括对麻醉果蝇的图像采集。蛋白酶体抑制剂硼替佐米在体内和体外均增加了eGFP的半衰期，表明eGFP通过蛋白酶体降解。蛋白质合成抑制剂环己酰亚胺降低了eGFP在体内的积累，但不影响半衰期。利用组织普遍表达和组织特异性表达，在不同年龄和性别的果蝇中测定了几种荧光蛋白的半衰期。典型的半衰期值因荧光蛋白而异。DsRED的半衰期比eGFP长，而mCherry几乎未检测到降解。半衰期也因组织而异，相对于其他组织，在肌肉中观察到eGFP的半衰期更长。检测到DsRED的半衰期随年龄增加，但eGFP没有。性别和雌性交配状态的影响有限。综上所述，这些数据表明体内检测方法是研究由蛋白质序列、亚细胞区室、组织和小分子调节的蛋白质降解的有前途的工具。
