Biophysical Characterisation of Calumenin as a Charged F508del-CFTR Folding Modulator

The cystic fibrosis transmembrane regulator (CFTR) is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF) patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs). We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with IDPs and is hypothesized to regulate F508del-CFTR folding by electrostatic effects. This work provides useful insights for designing optimized synthetic structural correctors of CFTR mutant proteins in the future.囊性纤维化跨膜传导调节因子（cystic fibrosis transmembrane regulator, CFTR）是一种环腺苷单磷酸（cyclic-AMP, cAMP）依赖型氯离子通道，表达于呼吸道等多种器官的上皮细胞顶膜表面。由CFTR基因突变引发的该蛋白加工与功能缺陷，会导致离子平衡丧失、黏液清除障碍、生物膜增殖增加以及人类气道炎症，这也是囊性纤维化（cystic fibrosis, CF）患者的典型病理表现。目前，CFTR在分泌通路中多种分子伴侣作用下折叠与成熟的完整机制仍未完全阐明。近期研究发现，分泌蛋白钙连蛋白（calumenin）属于CREC家族低亲和力钙结合蛋白，被认定为一种潜在的CFTR分子伴侣，但其生物物理特性与功能尚未得到系统表征。本研究对比了钙连蛋白及其他CREC家族成员与CFTR相关分子伴侣、钙结合蛋白、野生型及突变型CFTR蛋白、内在无序蛋白（intrinsically disordered proteins, IDPs）的亲水性、不稳定性、电荷性、解折叠特性、无序性与聚集倾向。结果显示，与其他CREC家族蛋白类似，钙连蛋白的电荷水平显著更高，而折叠程度相较于CFTR相关分子伴侣更低。此外，与内在无序蛋白一致，钙连蛋白及其他CREC家族蛋白均表现出较低的疏水性与聚集倾向。系统发育分析表明，钙连蛋白与其他CREC家族蛋白存在紧密的进化关联，提示进化过程如何塑造了二者相似的生物物理特性。实验观测显示，钙连蛋白可显著降低F508del-CFTR的聚集，其作用模式与已被充分表征的内在无序蛋白AavLEA1相似。基于荧光显微镜的成像分析还揭示，在表达F508del-CFTR的支气管细胞中，钙连蛋白的运输过程发生异常，这表明其在囊性纤维化的病理生理过程中发挥直接作用。综上，钙连蛋白是一种高电荷蛋白，与内在无序蛋白具有高度相似性，研究推测其可通过静电效应调控F508del-CFTR的折叠。本研究为未来设计优化的CFTR突变蛋白合成结构矫正剂提供了有价值的理论参考。