Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1
Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1
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DOI：
10.1074/jbc.m600876200
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发表时间：
2006-12-29
影响因子：
4.8
通讯作者：
Hamilton, Susan L.
中科院分区：
文献类型：
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作者：
Aracena-Parks, Paula;Goonasekera, Sanjeewa A.;Hamilton, Susan L.
骨骼肌 Ca2+ 释放通道（兰尼碱受体 1 型 (RyR1)）是一种氧化还原传感器，易受可逆 S-亚硝基化、S-谷胱甘肽化和二硫键氧化的影响。到目前为止，Cys-3635 仍然是唯一被确定为与通道的氧化还原传感特性功能相关的半胱氨酸残基。我们证明，RyR1 缺失肌管中 C3635A-RyR1 突变体的表达改变了兰尼定受体对电压激活的敏感性，表明 Cys-3635 参与了电压门控兴奋-收缩耦合。然而，C3635A-RyR1 通道或野生型 RyR1 在人胚胎肾细胞中表达后，H2O2 处理可将 [H-3] 兰尼定结合增强至相同程度，表明除 Cys-3635 之外的半胱氨酸负责通道活性的氧化增强。结合使用蛋白质印迹和巯基定向荧光标记，我们发现之前显示参与二硫键形成的 RyR1 的两个大区域（氨基酸 1-2401 和 3120-4475）也是 S-亚硝基化和 S-谷胱甘肽化的主要位点。使用 RyR1 的选择性同位素编码亲和标签标记和基质辅助激光解吸/电离飞行时间质谱，我们在每个 RyR1 亚基的 100 个半胱氨酸中鉴定出 9 个内源修饰的半胱氨酸（Cys-36、Cys-315、Cys-811、Cys-906、Cys-1591、Cys-2326、 Cys-2363、Cys-3193 和 Cys-3635）和另外 3 个仅用外源氧化还原剂修饰的残基（Cys-253、Cys-1040 和 Cys-1303）。我们还确定了这些半胱氨酸可以经历的氧化还原修饰的类型。总之，我们已经确定了半胱氨酸的一个离散子集，它们可能参与 RyR1 对不同氧化还原修饰（S-亚硝基化、S-谷胱甘肽化和二硫化物氧化）的功能反应。
The skeletal muscle Ca2+-release channel ( ryanodine receptor type 1 (RyR1)) is a redox sensor, susceptible to reversible S-nitrosylation, S-glutathionylation, and disulfide oxidation. So far, Cys-3635 remains the only cysteine residue identified as functionally relevant to the redox sensing properties of the channel. We demonstrate that expression of the C3635A-RyR1 mutant in RyR1-null myotubes alters the sensitivity of the ryanodine receptor to activation by voltage, indicating that Cys-3635 is involved in voltage-gated excitation-contraction coupling. However, H2O2 treatment of C3635A-RyR1 channels or wildtype RyR1, following their expression in human embryonic kidney cells, enhances [H-3] ryanodine binding to the same extent, suggesting that cysteines other than Cys-3635 are responsible for the oxidative enhancement of channel activity. Using a combination of Western blotting and sulfhydryl-directed fluorescent labeling, we found that two large regions of RyR1 (amino acids 1-2401 and 3120-4475), previously shown to be involved in disulfide bond formation, are also major sites of both S-nitrosylation and S-glutathionylation. Using selective isotope-coded affinity tag labeling of RyR1 and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we identified, out of the 100 cysteines in each RyR1 subunit, 9 that are endogenously modified (Cys-36, Cys-315, Cys-811, Cys-906, Cys-1591, Cys-2326, Cys-2363, Cys-3193, and Cys-3635) and another 3 residues that were only modified with exogenous redox agents (Cys-253, Cys-1040, and Cys-1303). We also identified the types of redox modification each of these cysteines can undergo. In summary, we have identified a discrete subset of cysteines that are likely to be involved in the functional response of RyR1 to different redox modifications (S-nitrosylation, S-glutathionylation, and oxidation to disulfides).