Stroylova Yulia Y, Zimny Jaroslaw, Yousefi Reza, Chobert Jean-Marc, Jakubowski Hieronim, Muronetz Vladimir I, Haertlé Thomas
Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
Biochim Biophys Acta. 2011 Oct;1814(10):1234-45. doi: 10.1016/j.bbapap.2011.05.017. Epub 2011 Jun 12.
Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. α(S1)-, β- and κ-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5, 2.1 and 1.3 homocysteyl residues per one β-, α(S1)-, and κ-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DLS and microscopic studies have revealed the formation of large aggregates of about 1-3μm. Homocysteinylation of α(S1)- and β-caseins results in formation of regular spheres. Homocysteinylated κ-casein forms thin unbranched fibrils about 400-800nm long. In case of κ-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and other structural features.
同型半胱氨酸水平升高会导致蛋白质中赖氨酸残基的N-同型半胱氨酸化，并且它们与多种人类病理状况相关。然而，赖氨酸残基同型半胱氨酸化的作用仍知之甚少。为了研究内在无序蛋白（IUP）同型半胱氨酸化的特征，使用牛酪蛋白作为模型。对表现出不同聚集和胶束形成的α(S1)-、β-和κ-酪蛋白用同型半胱氨酸硫内酯进行修饰，并研究它们的物理化学性质。估计同型半胱氨酸掺入效率分别为每一个β-、α(S1)-和κ-酪蛋白分子约1.5、2.1和1.3个同型半胱氨酸残基。使用诸如色氨酸、硫黄素T和ANS等内在和外在荧光标记，揭示了同型半胱氨酸化后酪蛋白结构的结构变化，其表现为β-折叠含量增加，在某些情况下这可能是淀粉样蛋白样转变的特征。圆二色光谱也显示同型半胱氨酸化酪蛋白的β-折叠含量增加。酪蛋白同型半胱氨酸化在所有情况下都会导致聚集。聚集体的大小和聚集速率取决于同型半胱氨酸硫内酯浓度和温度。动态光散射和显微镜研究揭示了形成约1-3μm的大聚集体。α(S1)-和β-酪蛋白的同型半胱氨酸化导致形成规则的球体。同型半胱氨酸化的κ-酪蛋白形成约400-800nm长的细的无分支纤维。就κ-酪蛋白而言，通过刚果红光谱证实了同型半胱氨酸化的淀粉样蛋白生成作用。综上所述，数据表明N-同型半胱氨酸化引发了天然酪蛋白性质的显著变化。对属于IUP蛋白家族的3种不同酪蛋白类型的淀粉样蛋白生成转变的比较表明，同型半胱氨酸化时淀粉样蛋白生成转变的效率取决于胶束化能力、额外的二硫键和其他结构特征。
