From the Department of Biosciences, University of Salzburg, A-5020 Salzburg, Austria and.
the Center for Integrated Protein Science Munich, Technical University of Munich, D-85748 Munich, Germany.
J Biol Chem. 2018 Aug 24;293(34):13151-13165. doi: 10.1074/jbc.RA118.002154. Epub 2018 Jul 2.
Protein activity is often regulated by altering the oligomerization state. One mechanism of multimerization involves domain swapping, wherein proteins exchange parts of their structures and thereby form long-lived dimers or multimers. Domain swapping has been specifically observed in amyloidogenic proteins, for example the cystatin superfamily of cysteine protease inhibitors. Cystatins are twin-headed inhibitors, simultaneously targeting the lysosomal cathepsins and legumain, with important roles in cancer progression and Alzheimer's disease. Although cystatin E is the most potent legumain inhibitor identified so far, nothing is known about its propensity to oligomerize. In this study, we show that conformational destabilization of cystatin E leads to the formation of a domain-swapped dimer with increased conformational stability. This dimer was active as a legumain inhibitor by forming a trimeric complex. By contrast, the binding sites toward papain-like proteases were buried within the cystatin E dimer. We also showed that the dimers could further convert to amyloid fibrils. Unexpectedly, cystatin E amyloid fibrils contained functional protein, which inhibited both legumain and papain-like enzymes. Fibril formation was further regulated by glycosylation. We speculate that cystatin amyloid fibrils might serve as a binding platform to stabilize the pH-sensitive legumain and cathepsins in the extracellular environment, contributing to their physiological and pathological functions.
蛋白质的活性通常通过改变寡聚状态来调节。多聚化的一种机制涉及结构域交换，其中蛋白质交换其结构的部分，从而形成长寿命的二聚体或多聚体。结构域交换已在淀粉样蛋白原蛋白中被特异性观察到，例如半胱氨酸蛋白酶抑制剂的胱抑素超家族。胱抑素是双头抑制剂，同时靶向溶酶体组织蛋白酶和组织蛋白酶 L，在癌症进展和阿尔茨海默病中具有重要作用。尽管胱抑素 E 是迄今为止鉴定出的最有效的组织蛋白酶 L 抑制剂，但对于其寡聚化倾向一无所知。在这项研究中，我们表明胱抑素 E 的构象不稳定导致形成具有增加构象稳定性的结构域交换二聚体。该二聚体通过形成三聚体复合物作为组织蛋白酶 L 抑制剂发挥活性。相比之下，针对木瓜蛋白酶样蛋白酶的结合位点被埋藏在胱抑素 E 二聚体中。我们还表明，二聚体可以进一步转化为淀粉样纤维。出乎意料的是，胱抑素 E 淀粉样纤维含有功能性蛋白质，该蛋白质抑制组织蛋白酶 L 和木瓜蛋白酶样酶。纤维形成进一步受到糖基化的调节。我们推测胱抑素淀粉样纤维可能作为结合平台，稳定细胞外环境中对 pH 敏感的组织蛋白酶 L 和组织蛋白酶，从而促进其生理和病理功能。
