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首页> 外文期刊>Biochemistry >Enhanced Potency of Human Sonic Hedgehog by Hydrophobic Modification
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Enhanced Potency of Human Sonic Hedgehog by Hydrophobic Modification

机译：通过疏水改性增强人声刺猬的效能

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Post-translational modifications of the developmental signaling protein Sonic hedgehog (Shh) by a long-chain fatty acid at the N-terminus and cholesterol at the C-terminus greatly activate the protein in a cell-based signaling assay. To investigate the structural determinants of this activation phenomenon, hydrophobic and hydrophilic moieties have been introduced by chemical and mutagenic methods to the soluble N-terminal signaling domain of Shh and tested in both in vitro and in vivo assays. A wide variety of hydrophobic modifications increased the potency of Shh when added at the N-terminus of the protein, ranging from long-chain fatty acids to hydrophobic amino acids, with EC5o values from 99 nM for the unmodified protein to 0.6 nM for the myristoylated form. The N-myristoylated Shh was as active as the natural form having both N- and C-terminal modifications. The degree of activation appears to correlate with the hydrophobicity of the modification rather than any specific chemical feature of the adduct; moreover, substitution with hydrophilic moieties decreased activity. Hydrophobic modifications at the C-terminus of Shh resulted in only a 2—3-fold increase in activity, and no activation was found with hydrophobic modification at other surface positions. The N-terminal modifications did not appear to alter the binding affinity of the Shh protein for the transfected receptor protein, Patched, and had no apparent effect on structure as measured by circular dichroism, thermal denaturation, and size determination. Activation of Desert Hh through modification of its N-terminus was also observed, suggesting that this is a common feature of Hh proteins.
机译：N端的长链脂肪酸和C端的胆固醇对发育信号蛋白Sonic Hedgehog（Shh）的翻译后修饰在基于细胞的信号分析中极大地激活了该蛋白。为了研究这种激活现象的结构决定因素，已通过化学和诱变方法将疏水和亲水部分引入Shh的可溶性N末端信号传导域，并在体外和体内试验中进行了测试。当在蛋白质的N末端添加多种疏水修饰时，Shh的效力增强，范围从长链脂肪酸到疏水氨基酸，EC 50值从未修饰的蛋白质的99 nM到肉豆蔻酰化的0.6 nM形成。 N-肉豆蔻酰化的Shh与具有N-和C-末端修饰的天然形式一样活跃。活化程度似乎与修饰的疏水性有关，而不是与加合物的任何特定化学特征有关。此外，用亲水性部分取代降低了活性。在Shh的C端进行疏水修饰只会导致活性提高2-3倍，而在其他表面位置进行疏水修饰则没有发现活化。 N-末端修饰似乎并未改变Shh蛋白对转染的受体蛋白Patched的结合亲和力，并且通过圆二色性，热变性和大小确定，对结构没有明显影响。还观察到通过修饰其N末端来激活沙漠Hh，这表明这是H​​h蛋白的共同特征。

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