Abstract:
αB-Crystallin (αB-Cry) is expressed in many tissues, and mutations in this protein are linked to various diseases, including cataracts, Alzheimer\'s disease, Parkinson\'s disease, and several types of myopathies and cardiomyopathies. The p.D109G mutation, which substitutes a conserved aspartate residue involved in the interchain salt bridges, with glycine leads to the development of both restrictive cardiomyopathy (RCM) and skeletal myopathy. In this study, we generated this mutation in the α-Cry domain (ACD) which is crucial for forming the active chaperone dimeric state, using site-directed mutagenesis. After inducing expression in the bacterial host, we purified the mutant and wild-type recombinant proteins using anion exchange chromatography. Various spectroscopic evaluations revealed significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry caused by this mutation. Furthermore, this pathogenic mutation led to the formation of protein oligomers with larger sizes than those of the wild-type protein counterpart. The mutant protein also exhibited increased chaperone activity and decreased chemical, thermal, and proteolytic stability. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and fluorescence microscopy (FM) demonstrated that p.D109G mutant protein is more prone to forming amyloid aggregates. The misfolding associated with the p.D109G mutation may result in abnormal interactions of human αB-Cry with its natural partners (e.g., desmin), leading to the formation of protein aggregates. These aggregates can interfere with normal cellular processes and may contribute to muscle cell dysfunction and damage, resulting in the pathogenic involvement of the p.D109G mutant protein in restrictive cardiomyopathy and skeletal myopathy.
摘要:
αB-晶体蛋白(αB-Cry)在许多组织中表达,这种蛋白质的突变与各种疾病有关,包括白内障,老年痴呆症,帕金森病,以及几种类型的肌病和心肌病。p.D109G突变,它取代了链间盐桥中保守的天冬氨酸残基,与甘氨酸一起导致限制性心肌病(RCM)和骨骼肌病的发展。在这项研究中,我们在α-Cry结构域(ACD)中产生了这种突变,这对于形成活性伴侣二聚体状态至关重要,使用定点诱变。在细菌宿主中诱导表达后,我们使用阴离子交换色谱纯化了突变和野生型重组蛋白。各种光谱评估揭示了次要的显着变化,第三级,和由这种突变引起的人αB-Cry的四级结构。此外,这种致病性突变导致形成比野生型蛋白质对应物更大的蛋白质寡聚物。突变蛋白还表现出增加的伴侣活性和降低的化学,热,和蛋白水解稳定性。原子力显微镜(AFM),透射电子显微镜(TEM),和荧光显微镜(FM)表明,p.D109G突变蛋白更容易形成淀粉样蛋白聚集体。与p.D109G突变相关的错误折叠可能导致人αB-Cry与其天然伴侣的异常相互作用(例如,desmin),导致蛋白质聚集体的形成。这些聚集体可以干扰正常的细胞过程,并可能导致肌肉细胞功能障碍和损伤,导致p.D109G突变蛋白在限制性心肌病和骨骼肌病中的致病参与。