Site-specific methionine oxidation initiates calmodulin degradation by the 20S proteasome.
Site-specific methionine oxidation initiates calmodulin degradation by the 20S proteasome.
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DOI：
10.1021/bi802117k
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发表时间：
2009-04-07
期刊：
影响因子：
2.9
通讯作者：
Ferrington, Deborah A.
中科院分区：
文献类型：
--
作者：
Balog, Edward M.;Lockamy, Elizabeth L.;Thomas, David D.;Ferrington, Deborah A.
蛋白酶体是一种重要的细胞内蛋白酶，通过选择性降解特定蛋白质来调节信号转导和蛋白质质量控制等过程。靶向蛋白质降解的信号，统称为降解决定子，已被定义为参与细胞信号传导的许多蛋白质。然而，参与识别和降解氧化损伤的蛋白质的分子信号尚未完全确定。目前的研究使用生物化学和光谱测量来定义钙调素的性质，启动降解的20 S蛋白酶体。我们的实验方法涉及产生多个钙调蛋白突变体与特定的蛋氨酸取代亮氨酸。这种定点诱变策略允许Met定点氧化为Met亚砜。我们发现，氧化诱导的损失的二级结构，通过圆二色性，与降解率的野生型和突变体含有亮氨酸取代的C-末端。然而，没有观察到降解的突变体与蛋氨酸到亮氨酸取代的N-末端，这表明氧化诱导的结构展开的N-末端区域是必不可少的降解的20 S蛋白酶体。比较CaM突变体的热力学稳定性的实验有助于进一步本地化的氧化诱导的局灶性破坏的N-末端区域的残基51和72的关键站点。这项工作为降解决定子的概念带来了新的生物化学和结构清晰度，降解决定子是蛋白质的一部分，决定了蛋白质对蛋白酶体降解的敏感性。
The proteasome is a key intracellular protease that regulates processes, such as signal transduction and protein quality control, through the selective degradation of specific proteins. Signals that target a protein for degradation, collectively known as degrons, have been defined for many proteins involved in cell signaling. However, the molecular signals involved in recognition and degradation of proteins damaged by oxidation have not been completely defined. The current study used biochemical and spectroscopic measurements to define the properties in calmodulin that initiate degradation by the 20S proteasome. Our experimental approach involved the generation of multiple calmodulin mutants with specific Met replaced by Leu. This strategy of site-directed mutagenesis permitted site-selective oxidation of Met to Met sulfoxide. We found that the oxidation-induced loss of secondary structure, as measured by circular dichroism, correlated with the rate of degradation for wild-type and mutants containing Leu substitutions in the C-terminus. However, no degradation was observed for mutants with Met to Leu substitution in the N-terminus, suggesting that oxidation-induced structural unfolding in the N-terminal region is essential for degradation by the 20S proteasome. Experiments comparing the thermodynamic stability of CaM mutants helped to further localize the critical site of oxidation-induced focal disruption to residues 51 and 72 in the N-terminal region. This work brings new biochemical and structural clarity to the concept of the degron, the portion of a protein that determines its susceptibility to degradation by the proteasome.
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