Enhancing the Conformational Stability of the cl-Par-4 Tumor Suppressor via Site-Directed Mutagenesis.
Enhancing the Conformational Stability of the cl-Par-4 Tumor Suppressor via Site-Directed Mutagenesis.
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通过定位的诱变增强CL-PAR-4肿瘤抑制器的构象稳定性。
DOI：
10.3390/biom13040667
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发表时间：
2023-04-12
期刊：
影响因子：
5.5
通讯作者：
中科院分区：
文献类型：
--
作者：
本质上无序的蛋白质在细胞信号中起着重要作用，而这些蛋白质的失调与几种疾病有关。前列腺细胞凋亡反应-4(PAR-4)是一种约40kodalton的促凋亡肿瘤抑制因子，是一种主要的内在紊乱蛋白，在多种癌症中均有下调表达。半胱氨酸天冬氨酸氨基转移酶裂解的PAR-4片段(CL-PAR-4)具有活性，通过抑制细胞存活途径发挥抑制肿瘤的作用。在这里，我们采用定点突变的方法创造了一个CL-PAR-4点突变(D313K)。用生物物理技术对表达和纯化的D313K蛋白进行了鉴定，并与野生型(WT)进行了比较。我们先前已经证明了WT CL-PAR-4在生理pH的高盐存在下获得了稳定、致密和螺旋构象。在这里，我们证明了D313K蛋白在盐的存在下达到了与WT相似的构象，但盐浓度大约低了两倍。这证明了在313位用碱性残基取代酸性残基减轻了二聚体伙伴之间的螺旋间电荷斥力，并有助于稳定结构构象。
Intrinsically disordered proteins play important roles in cell signaling, and dysregulation of these proteins is associated with several diseases. Prostate apoptosis response-4 (Par-4), an approximately 40 kilodalton proapoptotic tumor suppressor, is a predominantly intrinsically disordered protein whose downregulation has been observed in various cancers. The caspase-cleaved fragment of Par-4 (cl-Par-4) is active and plays a role in tumor suppression by inhibiting cell survival pathways. Here, we employed site-directed mutagenesis to create a cl-Par-4 point mutant (D313K). The expressed and purified D313K protein was characterized using biophysical techniques, and the results were compared to that of the wild-type (WT). We have previously demonstrated that WT cl-Par-4 attains a stable, compact, and helical conformation in the presence of a high level of salt at physiological pH. Here, we show that the D313K protein attains a similar conformation as the WT in the presence of salt, but at an approximately two times lower salt concentration. This establishes that the substitution of a basic residue for an acidic residue at position 313 alleviates inter-helical charge repulsion between dimer partners and helps to stabilize the structural conformation.
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影响因子：
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Guo, Q;Fu, WM;Mattson, MP
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