Generation of a novel allelic series of cryptochrome mutants via mutagenesis reveals residues involved in protein-protein interaction and CRY2-specific repression.
Generation of a novel allelic series of cryptochrome mutants via mutagenesis reveals residues involved in protein-protein interaction and CRY2-specific repression.
复制标题
通过诱变产生一系列新型等位基因隐花色素突变体揭示了参与蛋白质-蛋白质相互作用和 CRY2 特异性抑制的残基。
DOI：
10.1128/mcb.00641-09
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发表时间：
2009
影响因子：
5.3
通讯作者：
Green,CarlaB
中科院分区：
文献类型：
--
作者：
McCarthy,EllenaV;Baggs,JulieE;Geskes,JeanneM;Hogenesch,JohnB;Green,CarlaB
哺乳动物昼夜节律所必需的，并在分子钟内具有许多公认的生物化学作用。虽然研究nullCryalleles的作用已经提供了信息，但它们未能剖析出NullCry 1和NullCry 2蛋白的许多作用的相对重要性及其背后的分子机制。为了解决这一问题，我们通过随机诱变产生了一系列的Crymutants等位基因，然后通过基于细胞的筛选分离出具有异常抑制CLOCK-BMAL 1的突变体。我们鉴定了22个突变体，这些突变导致单个氨基酸取代，导致不同CRY功能的各种缺陷。为了说明这些新工具的广度和价值，我们提出了一个深入的分析这些突变体中的两个，E12 G354 D和E12 G351 D;前者显示时钟蛋白结合的缺陷，是由两个E12 G351 D抑制所需的，而相反，后者显示正常的结合功能，但表现出E12 G354 D特异性抑制表型。此外，尽管NIH 3 T3细胞中过表达G412导致节律幅度的剂量依赖性降低，但过表达G412 G351 D则消除了节律性。总之，这些独特等位基因的表征为更复杂地洞察CRY蛋白在昼夜节律中的多方面功能提供了新的机会。
CRYPTOCHOME proteins are necessary for mammalian circadian rhythms and have many well-established biochemical roles within the molecular clock. While studies examining the effect of nullCryalleles have been informative, they have failed to dissect out the relative importance of, and the molecular mechanisms behind, the many roles of the CRY1 and CRY2 proteins. To address this, we created an allelic series ofCrymutants through random mutagenesis, followed by a cell-based screen to isolate mutants with aberrant repression of CLOCK-BMAL1. We identified 22 mutants with mutations resulting in single amino acid substitutions which cause a variety of deficiencies in different CRY functions. To illustrate the breadth and value of these new tools, we present an in-depth analysis of two of these mutants, CRY2G354D and CRY2G351D; the former shows deficiency in clock protein binding and is required for repression by both CRYs, while in contrast, the latter displays normal binding function but exhibits a CRY2-specific repression phenotype. Further, while overexpression of CRY2 in NIH 3T3 cells caused a dose-dependent decrease in rhythm amplitude, overexpression of CRY2G351D abolished rhythmicity. In summary, characterization of these unique alleles provides new opportunities for more-sophisticated insight into the multifaceted functions of the CRY proteins in circadian rhythms.