Suppressing mutation-induced protein aggregation in mammalian cells by mutating residues significantly displaced upon the original mutation.
Suppressing mutation-induced protein aggregation in mammalian cells by mutating residues significantly displaced upon the original mutation.
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DOI：
10.1016/j.bej.2014.08.013
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发表时间：
2014-10-15
影响因子：
3.9
通讯作者：
Kwon I
中科院分区：
文献类型：
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作者：
Gregoire S;Glitzos K;Kwon I
天然或通过蛋白质工程故意引入野生型蛋白质的突变通常导致蛋白质聚集。特别是，哺乳动物细胞内的蛋白质聚集在疾病病理学和生物制剂生产中具有重要意义;使哺乳动物细胞内的蛋白质聚集调节成为非常重要的工程课题。以前，我们表明，半理性的设计方法可以用来减少细胞内聚集的蛋白质通过恢复的构象稳定性，降低了突变。然而，当没有合理的设计方法来提高构象稳定性时，这种方法的实用性有限。为了克服这一限制，我们研究了在原始突变后显著移位的残基的修饰是否是减少哺乳动物细胞中蛋白质聚集的有效方法。作为模型系统，使用在位置93处含有甘氨酸至丙氨酸突变的人铜、锌超氧化物歧化酶突变体（SOD 1G 93 A）。将一组突变引入到在G93 A突变后基本上被置换的残基中。通过使用基于细胞的聚集测定，我们鉴定了SOD 1G 93 A的几种新变体，其在哺乳动物细胞内具有降低的聚集倾向。我们的研究结果成功地证明，突变蛋白的聚集可以通过突变原始突变后显著移位的残基来抑制。
Mutations introduced to wild-type proteins naturally, or intentionally via protein engineering, often lead to protein aggregation. In particular, protein aggregation within mammalian cells has significant implications in the disease pathology and biologics production; making protein aggregation modulation within mammalian cells a very important engineering topic. Previously, we showed that the semi-rational design approach can be used to reduce the intracellular aggregation of a protein by recovering the conformational stability that was lowered by the mutation. However, this approach has limited utility when no rational design approach to enhance conformational stability is readily available. In order to overcome this limitation, we investigated whether the modification of residues significantly displaced upon the original mutation is an effective way to reduce protein aggregation in mammalian cells. As a model system, human copper, zinc superoxide dismutase mutant containing glycine to alanine mutation at position 93 (SOD1G93A) was used. A panel of mutations was introduced into residues substantially displaced upon the G93A mutation. By using cell-based aggregation assays, we identified several novel variants of SOD1G93A with reduced aggregation propensity within mammalian cells. Our findings successfully demonstrate that the aggregation of a mutant protein can be suppressed by mutating the residues significantly displaced upon the original mutation.