Cis-suppression to arrest protein aggregation in mammalian cells.
Cis-suppression to arrest protein aggregation in mammalian cells.
复制标题
DOI：
10.1002/bit.25119
复制
发表时间：
2014-03
影响因子：
3.8
通讯作者：
Kwon, Inchan
中科院分区：
文献类型：
--
作者：
Gregoire, Simpson;Zhang, Shaojie;Costanzo, Joseph;Wilson, Kelly;Fernandez, Erik J.;Kwon, Inchan
蛋白质的错误折叠和聚集与许多人类疾病有关，并显著降低了在哺乳动物细胞中表达的蛋白质的产量。尽管了解和抑制哺乳动物细胞中的蛋白质聚集很重要，但目前还没有关于调节哺乳动物细胞中蛋白质错误折叠/聚集的蛋白质设计和选择策略的报道。在这项工作中，我们解决了哺乳动物细胞中突变诱导的蛋白质聚集所带来的特殊挑战。我们假设，一个额外的突变(S)可以被引入到一个容易聚集的蛋白质变体中，在空间上靠近原始突变，以抑制错误折叠和聚集(顺式抑制)。作为模式蛋白，我们选择了与家族性肌萎缩侧索硬化症相关的人铜锌超氧化物歧化酶突变体(SOD1A4V)，该突变体含有第4位丙氨酸到缬氨酸的突变。我们使用RosettaDesign程序确定SOD1A4V中的Phe20是导致SOD1A4V构象不稳定的关键残基。这些信息被用来合理地开发Phe20位点的候选突变池。经过两轮基于哺乳动物细胞的突变体筛选，选出了三个新的SOD1A4V突变体，它们在细胞内的聚集倾向显著降低。三个新的SOD1A4V变异体的稳定性增强，聚集倾向降低，通过细胞分级和体外稳定性分析得到验证。
Protein misfolding and aggregation are implicated in numerous human diseases and significantly lower production yield of proteins expressed in mammalian cells. Despite the importance of understanding and suppressing protein aggregation in mammalian cells, a protein design and selection strategy to modulate protein misfolding/aggregation in mammalian cells has not yet been reported. In this work, we address the particular challenge presented by mutation-induced protein aggregation in mammalian cells. We hypothesize that an additional mutation(s) can be introduced in an aggregation-prone protein variant, spatially near the original mutation, to suppress misfolding and aggregation (cis-suppression). As a model protein, we chose human copper, zinc superoxide dismutase mutant (SOD1A4V) containing an alanine to valine mutation at residue 4, associated with the familial form of amyotrophic lateral sclerosis. We used the program RosettaDesign to identify Phe20 in SOD1A4V as a key residue responsible for SOD1A4V conformational destabilization. This information was used to rationally develop a pool of candidate mutations at the Phe20 site. After two rounds of mammalian-cell based screening of the variants, three novel SOD1A4V variants with a significantly reduced aggregation propensity inside cells were selected. The enhanced stability and reduced aggregation propensity of the three novel SOD1A4V variants were verified using cell fractionation and in vitro stability assays.
登录
查看更多内容
影响因子：
4.8
作者：
Kabuta, Tomohiro;Suzuki, Yasuyuki;Wada, Keiji
通讯作者：
Wada, Keiji
DOI：
10.1016/j.molbrainres.2005.07.010
发表时间：
2005-10-31
期刊：
MOLECULAR BRAIN RESEARCH
影响因子：
--
作者：
Babetto, E;Mangolini, A;Cantoni, L
通讯作者：
Cantoni, L
DOI：
10.1073/pnas.97.19.10383
发表时间：
2000-09-12
影响因子：
11.1
作者：
Kuhlman, B;Baker, D
通讯作者：
Baker, D
影响因子：
3.5
作者：
Prudencio, Mercedes;Durazo, Armando;Borchelt, David R.
通讯作者：
Borchelt, David R.
DOI：
10.1073/pnas.0902505106
发表时间：
2009-05-12
影响因子：
11.1
作者：
Karch, Celeste M.;Prudencio, Mercedes;Borchelt, David R.
通讯作者：
Borchelt, David R.