Zapun A, Grammatyka S, Déral G, Vernet T
Institut de Biologie Structurale J.-P. Ebel (CEA, CNRS, UJF), Laboratoire d'Ingénierie des Macromolécules, 41 rue Jules Horowitz, 38027 Grenoble, France.
Biochem J. 2000 Sep 15;350 Pt 3(Pt 3):873-81.
Gelsolin modulates the actin cytoskeleton in the cytoplasm and clears the circulation of stray filaments. In vitro, gelsolin cleaves, nucleates and caps actin filaments, activities that are calcium-dependent. Both cellular and secreted forms share a sequence of 730 residues comprising six homologous modules termed G1-G6. A disulphide bond is formed in secreted G2, whereas in the cytoplasm it remains reduced. A point mutation in G2 causes an amyloidosis with neurological, ophthalmological and dermatological symptoms. This mutation does not affect the cytoplasmic form, while the secreted form is proteolysed. As a first step towards understanding how gelsolin folds and functions in different cellular compartments, we have characterized at equilibrium the urea-induced unfolding of G1 and G2, with or without calcium and/or disulphide bond. G1 and G2 both exhibit two-state unfolding behaviour and are stabilized by calcium. The disulphide bond also contributes to the stability of G2. In the absence of Ca(2+) and disulphide bond, G2 adopts a non-native conformation, suggesting that folding of G2 in the cytoplasm relies on the presence of surrounding modules or other molecular partners.
凝溶胶蛋白可调节细胞质中的肌动蛋白细胞骨架，并清除循环中的游离细丝。在体外，凝溶胶蛋白可切割、成核并封端肌动蛋白丝，这些活性依赖于钙。细胞形式和分泌形式都有一段由730个残基组成的序列，包含六个称为G1 - G6的同源模块。分泌型G2中形成了二硫键，而在细胞质中它仍保持还原状态。G2中的一个点突变会导致具有神经、眼科和皮肤病学症状的淀粉样变性。这种突变不影响细胞质形式，而分泌形式会被蛋白水解。作为了解凝溶胶蛋白如何在不同细胞区室中折叠和发挥功能的第一步，我们在有或没有钙和/或二硫键的情况下，通过尿素诱导的平衡解折叠对G1和G2进行了表征。G1和G2均表现出两态解折叠行为，并由钙稳定。二硫键也有助于G2的稳定性。在没有Ca(2+)和二硫键的情况下，G2会采取非天然构象，这表明G2在细胞质中的折叠依赖于周围模块或其他分子伴侣的存在。
