Faculty of Chemistry, Department of Medicinal Chemistry, University of Gdańsk Gdańsk, Poland.
Front Mol Neurosci. 2012 Jul 27;5:82. doi: 10.3389/fnmol.2012.00082. eCollection 2012.
Human cystatin C (hCC) is a small but very intriguing protein. Produced by all nucleated cells is found in almost all tissues and body fluids where, at physiological conditions, plays a role of a very potent inhibitor of cysteine proteases. Biologically active hCC is a monomeric protein but during cellular trafficking it forms dimers, transiently losing its inhibitory activity. In vitro, dimerization of cystatin C was observed for the mature protein during crystallization trials, revealing that the mechanism of this process is based on the three dimensional swapping of the protein domains. In our work we have focused on the impact of two proposed "hot spots" in cystatin C structure on its conformational stability. Encouraged by promising results of the theoretical calculations, we designed and produced several hCC hinge region point mutation variants that display a variety of conformational stability and propensity for dimerization and aggregation. A similar approach, i.e., rational mutagenesis, has been also applied to study the amyloidogenic L68Q variant to determine the contribution of hydrophobic interactions and steric effect on the stability of monomeric cystatin C. In this overview we would like to summarize the results of our studies. The impact of a particular mutation on the properties of the studied proteins will be presented in the context of their thermal and mechanical stability, in vitro dimerization tendency as well as the outcome of crystallization. Better understanding of the mechanism and, especially, factors affecting conformational stability of cystatin C and access to stable monomeric and dimeric versions of the protein opens new perspectives in explaining the role of dimers and the domain swapping process in hCC oligomerization, as well as designing potential inhibitors of this process.
人胱抑素 C（hCC）是一种体积虽小但非常有趣的蛋白质。所有有核细胞均可产生 hCC，它几乎存在于所有组织和体液中，在生理条件下，作为半胱氨酸蛋白酶的一种非常有效的抑制剂发挥作用。生物活性 hCC 是一种单体蛋白，但在细胞运输过程中会形成二聚体，暂时失去其抑制活性。在体外，在结晶试验中观察到成熟蛋白的胱抑素 C 二聚化，这表明该过程的机制基于蛋白质结构域的三维交换。在我们的工作中，我们专注于 hCC 结构中两个提出的“热点”对其构象稳定性的影响。受理论计算有希望结果的鼓舞，我们设计并产生了几个 hCC 铰链区点突变变体，这些变体显示出多种构象稳定性以及二聚化和聚集的倾向。类似的方法，即合理的诱变，也已应用于研究淀粉样变性 L68Q 变体，以确定疏水性相互作用和空间位阻对单体胱抑素 C 稳定性的贡献。在这篇综述中，我们将总结我们的研究结果。将特定突变对研究蛋白性质的影响置于其热和机械稳定性、体外二聚化倾向以及结晶结果的背景下进行介绍。更好地了解胱抑素 C 构象稳定性的机制，特别是影响构象稳定性的因素，并获得稳定的单体和二聚体形式的蛋白，为解释二聚体的作用和 hCC 寡聚化过程中的结构域交换提供了新的视角，以及设计该过程的潜在抑制剂。
