Intra-FCY1: a novel system to identify mutations that cause protein misfolding.
Intra-FCY1: a novel system to identify mutations that cause protein misfolding.
复制标题
DOI：
10.3389/fgene.2023.1198203
复制
发表时间：
2023
影响因子：
3.7
通讯作者：
Geiler-Samerotte, K.
中科院分区：
文献类型：
--
作者：
Quan, N.;Eguchi, Y.;Geiler-Samerotte, K.
关键词：
蛋白质错误折叠是细胞内常见的现象。编码序列的大多数突变增加了编码蛋白质错误折叠的倾向。这些错误折叠的分子可能对细胞产生毁灭性的影响。尽管蛋白质错误折叠在人类疾病和蛋白质进化中的重要性，但仍有一些基本问题尚未得到解答，例如，哪些突变导致最多的错误折叠？这些问题很难部分回答，因为我们缺乏高通量方法来比较不同突变的不稳定影响。评估突变蛋白在体内稳定性的常用系统通常依赖于必需蛋白作为传感器，但错误折叠的蛋白质可以破坏必需蛋白的功能，足以杀死细胞。这使得使用这些系统难以识别和比较导致蛋白质错误折叠的突变。在这里，我们提出了一种新的体内系统命名为内FCY 1，我们用来识别突变，导致错误折叠的模型蛋白[黄色荧光蛋白（YFP）]在酿酒酵母。Intra-FCY 1系统利用酵母胞嘧啶脱氨酶Fc γ 1（一种毒性蛋白）的两个互补片段，其中插入了YFP。当YFP折叠时，Fc γ 1片段结合在一起以重建其功能，在含有5-氟胞嘧啶的培养基中赋予毒性并阻碍生长。但是，使YFP错误折叠的突变消除了Fc γ 1的毒性，因此具有错误折叠的YFP变体的菌株在生长竞争实验中出现的频率很高。这使得这种菌株更容易研究。内-FCY 1系统取消了感兴趣的蛋白质的定位，因此可以应用于研究不同细胞蛋白质的突变形式的相对稳定性。在这里，我们证实这种方法可以识别导致错误折叠的新突变，突出了Intra-FCY 1阐明蛋白质序列和稳定性之间关系的潜力。
Protein misfolding is a common intracellular occurrence. Most mutations to coding sequences increase the propensity of the encoded protein to misfold. These misfolded molecules can have devastating effects on cells. Despite the importance of protein misfolding in human disease and protein evolution, there are fundamental questions that remain unanswered, such as, which mutations cause the most misfolding? These questions are difficult to answer partially because we lack high-throughput methods to compare the destabilizing effects of different mutations. Commonly used systems to assess the stability of mutant proteins in vivo often rely upon essential proteins as sensors, but misfolded proteins can disrupt the function of the essential protein enough to kill the cell. This makes it difficult to identify and compare mutations that cause protein misfolding using these systems. Here, we present a novel in vivo system named Intra-FCY1 that we use to identify mutations that cause misfolding of a model protein [yellow fluorescent protein (YFP)] in Saccharomyces cerevisiae. The Intra-FCY1 system utilizes two complementary fragments of the yeast cytosine deaminase Fcy1, a toxic protein, into which YFP is inserted. When YFP folds, the Fcy1 fragments associate together to reconstitute their function, conferring toxicity in media containing 5-fluorocytosine and hindering growth. But mutations that make YFP misfold abrogate Fcy1 toxicity, thus strains possessing misfolded YFP variants rise to high frequency in growth competition experiments. This makes such strains easier to study. The Intra-FCY1 system cancels localization of the protein of interest, thus can be applied to study the relative stability of mutant versions of diverse cellular proteins. Here, we confirm this method can identify novel mutations that cause misfolding, highlighting the potential for Intra-FCY1 to illuminate the relationship between protein sequence and stability.
登录
查看更多内容
影响因子：
3.7
作者：
Geiler-Samerotte KA;Hashimoto T;Dion MF;Budnik BA;Airoldi EM;Drummond DA
通讯作者：
Drummond DA
影响因子：
48
作者：
Fowler, Douglas M.;Fields, Stanley
通讯作者：
Fields, Stanley
影响因子：
6
作者：
Buß O;Rudat J;Ochsenreither K
通讯作者：
Ochsenreither K
影响因子：
7.7
作者：
Flynn, Julia M.;Rossouw, Ammeret;Bolon, Daniel N. A.
通讯作者：
Bolon, Daniel N. A.
影响因子：
46.9
作者：
Cabantous, S;Terwilliger, TC;Waldo, GS
通讯作者：
Waldo, GS