Molecular pathology of dityrosine cross-links in proteins: Structural and functional analysis of four proteins
Molecular pathology of dityrosine cross-links in proteins: Structural and functional analysis of four proteins
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DOI：
10.1023/a:1015927907418
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发表时间：
2002-05-01
影响因子：
4.3
通讯作者：
Kanwar, R
中科院分区：
文献类型：
--
作者：
Balasubramanian, D;Kanwar, R
二酪氨酸键（DT）是两个酪氨酸之间氧化共价交联。DT交联越来越被认为是氧化应激、衰老和疾病的标志，并在多种病理中被检测到。虽然已经记录了DT交联蛋白，但DT连接对如此修饰的蛋白的结构和功能的影响尚不清楚。鉴于此，我们研究了四种不同功能蛋白的分子间dt二聚体的性质，即核糖核酸酶A、信号蛋白钙调蛋白和眼晶状体蛋白α -和α - b -晶体蛋白。我们发现DT是通过自由基反应和I型光敏（包括(OH)- o -）形成的。, 0 2(。而O-1(2)和NO2-（它们能修饰his， trp，更容易相遇）则不能。蛋白质表面的Tyr残基最容易和优先地形成DT键（分子内和分子间）。通过光谱监测，这些DT-二聚体的构象与母体单体相比没有明显改变，但DT交联分子的结构稳定性低于母体原生单体。dt二聚体在较低温度和较低浓度的尿素或氯化胍下变性。接下来研究了dt交联对这些蛋白生物活性的影响。核糖核酸酶A的dt二聚体的酶活性不是丧失而是降低。晶状体α -结晶蛋白的dt二聚化对伴侣蛋白样能力无显著影响；它抑制目标蛋白的自聚集和沉淀，就像母体一样，未修饰的α -晶体蛋白。然而，伽马b晶体蛋白的dt二聚化被认为导致更容易聚集和沉淀，这是白内障的一个感兴趣的点。以钙调素为例，我们可以生成分子间和分子内的DT交联，并研究DT二聚体和DT单体。dt -二聚体结合平滑肌轻链激酶和Ca2+，但比天然单体效率低，浓度范围宽。分子内dt单体在所有这些方面都较弱，可能是因为它在结构上更受约束。这些结果表明，球形蛋白的DT交联削弱了它们的结构稳定性，损害了（尽管没有取消）它们的生物活性，这两者都是病理相关的。分子内的DT交联似乎会导致更严重的结构和功能后果。
The dityrosine bond (DT) is an oxidative covalent cross-link between two tyrosines. DT cross-linking is increasingly identified as a marker of oxidative stress, aging and disease, and has been detected in diverse pathologies. While DT cross- linked proteins have been documented, the consequences of the DT link on the structure and function of the so modified proteins are yet to be understood. With this in view, we have studied the properties of intermolecular DT-dimers of four proteins of diverse functions, namely the enzyme ribonuclease A, the signal protein calmodulin, and the eye lens proteins alpha- and gamma B-crystallins. We find that DT is formed through radical reactions and type I photosensitization (including (OH)-O-., O-2(.-) and OONO-), but not by O-1(2) and NO2- (which modify his, trp and met more readily). Tyr residues on the surface of the protein make DT bonds (intra- and intermolecular) most readily and preferentially. The conformation of each of these DT-dimers, monitored by spectroscopy, is seen not to be significantly altered in comparison to that of the parent monomer, but the structural stability of the DT cross-linked molecule is lower than that of the parent native monomer. The DT-dimer is denatured at a lower temperature, and at lower concentrations of urea or guanidinium chloride. The effect of DT-cross-linking on the biological activities of these proteins was next studied. The enzymatic activity of the DT-dimer of ribonuclease A is not lost but lowered. DT-dimerization of lens alpha-crystallin did not significantly affect the chaperone-like ability; it inhibits the self-aggregation and precipitation of target proteins just as well as the parent, unmodified alpha-crystallin does. DT-dimerization of gamma B-crystallin is however seen to lead to more ready aggregation and precipitation, a point of interest in cataract. In the case of calmodulin, we could generate both intermolecular and intramolecular DT cross-linking, and study both the DT-dimer and DT-monomer. The DT-dimer binds smooth muscle light chain kinase and also Ca2+, but less efficiently and over a broad concentration range than the native monomer. The intramolecular DT-monomer is weaker in all these respects, presumably since it is structurally more constrained. These results suggest that DT cross-linking of globular proteins weakens their structural stability and compromises (though does not abolish) their biological activity, both of which are pathologically relevant. The intramolecular DT cross-link would appear to lead to more severe structural and functional consequences.