Photo-sensitive degron variants for tuning protein stability by light.
Photo-sensitive degron variants for tuning protein stability by light.
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DOI：
10.1186/s12918-014-0128-9
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发表时间：
2014-11-18
影响因子：
--
通讯作者：
Taxis C
中科院分区：
文献类型：
--
作者：
Usherenko S;Stibbe H;Muscò M;Essen LO;Kostina EA;Taxis C
由蛋白酶体调节的蛋白质水解是真核细胞中用于控制细胞行为的基本机制之一。调节蛋白质稳定性的有效工具在分子水平上对选定的生物过程提供合成影响。利用光敏降解决定子（psd）模块实现了蛋白质稳定性的光遗传控制。该工程工具由来自拟南芥光致蛋白1的光感受器结构域光氧电压2（LOV 2）与诱导直接蛋白酶体降解的序列融合组成，所述蛋白酶体降解源自鼠鸟氨酸脱羧酶的羧基末端降解决定子。如果降解标签暴露于细胞质或细胞核中，则用psd模块标记的靶蛋白的丰度可以被蓝光调节。我们使用的模式生物酿酒酵母产生psd模块的变体，增加和减少在黑暗中的稳定性，或当暴露于蓝光使用位点特异性和随机诱变。这些变体的特征在于与荧光报告蛋白的融合，并且在暴露于蓝光的细胞中显示出6至75分钟的半衰期，在黑暗中显示出14至187分钟的半衰期。在蓝光下，10种变体显示出加速降解，4种变体与原始psd模块相比增加了稳定性。在酵母细胞中测量所选构建体的暗/亮比率表明，获得了两种变体，其比率是野生型psd模块中的两倍。光感受器变体特征的计算机模拟表明，在大多数情况下，行为的改变是由LOV 2结构域的光响应变化引起的。总的来说，突变分析导致psd模块变体，其通过蓝光在宽范围内提供蛋白质稳定性的调谐。两个变体显示出与原始构建体相比有深刻改进的特征。LOV 2结构域在光遗传学工具中的模块化使用也允许在合成和系统生物学中的其他应用的背景下使用突变体。本文的在线版本（doi：10.1186/s12918-014-0128-9）包含补充材料，可供授权用户使用。
Regulated proteolysis by the proteasome is one of the fundamental mechanisms used in eukaryotic cells to control cellular behavior. Efficient tools to regulate protein stability offer synthetic influence on molecular level on a selected biological process. Optogenetic control of protein stability has been achieved with the photo-sensitive degron (psd) module. This engineered tool consists of the photoreceptor domain light oxygen voltage 2 (LOV2) from Arabidopsis thaliana phototropin1 fused to a sequence that induces direct proteasomal degradation, which was derived from the carboxy-terminal degron of murine ornithine decarboxylase. The abundance of target proteins tagged with the psd module can be regulated by blue light if the degradation tag is exposed to the cytoplasm or the nucleus. We used the model organism Saccharomyces cerevisiae to generate psd module variants with increased and decreased stabilities in darkness or when exposed to blue light using site-specific and random mutagenesis. The variants were characterized as fusions to fluorescent reporter proteins and showed half-lives between 6 and 75 minutes in cells exposed to blue light and 14 to 187 minutes in darkness. In blue light, ten variants showed accelerated degradation and four variants increased stability compared to the original psd module. Measuring the dark/light ratio of selected constructs in yeast cells showed that two variants were obtained with ratios twice as high as in the wild type psd module. In silico modeling of photoreceptor variant characteristics suggested that for most cases alterations in behavior were induced by changes in the light-response of the LOV2 domain. In total, the mutational analysis resulted in psd module variants, which provide tuning of protein stability over a broad range by blue light. Two variants showed characteristics that are profoundly improved compared to the original construct. The modular usage of the LOV2 domain in optogenetic tools allows the usage of the mutants in the context of other applications in synthetic and systems biology as well. The online version of this article (doi:10.1186/s12918-014-0128-9) contains supplementary material, which is available to authorized users.
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