PDF(Pubmed)
Abstract：
Variations in genes coding for calcium and integrin binding protein 2 (CIB2) and whirlin cause deafness both in humans and mice. We previously reported that CIB2 binds to whirlin, and is essential for normal staircase architecture of auditory hair cells stereocilia. Here, we refine the interacting domains between these proteins and provide evidence that both proteins have distinct role in the development and organization of stereocilia bundles required for auditory transduction. Using a series of CIB2 and whirlin deletion constructs and nanoscale pulldown (NanoSPD) assays, we localized the regions of CIB2 that are critical for interaction with whirlin. AlphaFold 2 multimer, independently identified the same interacting regions between CIB2 and whirlin proteins, providing a detailed structural model of the interaction between the CIB2 EF2 domain and whirlin HHD2 domain. Next, we investigated genetic interaction between murine Cib2 and Whrn using genetic approaches. Hearing in mice double heterozygous for functionally null alleles (Cib2 KO/+ ;Whrn wi/+ ) was similar to age-matched wild type mice, indicating that partial deficiency for both Cib2 and Whrn does not impair hearing. Double homozygous mutant mice (Cib2 KO/KO ;Whrn wi/wi ) had profound hearing loss and cochlear stereocilia exhibited a predominant phenotype seen in single Whrn wi/wi mutants. Furthermore, over-expression of Whrn in Cib2 KO/KO mice did not rescue the stereocilia morphology. These data suggest that, CIB2 is multifunctional, with key independent functions in development and/or maintenance of stereocilia staircase pattern in auditory hair cells.
摘要：
编码钙和整联蛋白结合蛋白2（CIB2）和呼耳素的基因变异会导致人和小鼠耳聋。我们之前报道了CIB2与Whirlin结合，对于听觉毛细胞立体纤毛的正常楼梯结构至关重要。这里,我们完善了这些蛋白质之间的相互作用域，并提供了证据表明这两种蛋白质在听觉转导所需的立体纤毛束的发育和组织中具有不同的作用。使用一系列CIB2和Whirlin缺失构建体和纳米级下拉（NanoSPD）测定，我们定位了CIB2的区域，这些区域对于与惠林相互作用至关重要。AlphaFold2多聚体,独立鉴定了CIB2和Whirlin蛋白之间的相同相互作用区域，提供CIB2EF2结构域和WhirlinHHD2结构域之间相互作用的详细结构模型。接下来,我们使用遗传学方法研究了鼠Cib2和Whrn之间的遗传相互作用。功能性无效等位基因（Cibb2KO/+;Whrnwi/+）双杂合子小鼠的听力与年龄匹配的野生型小鼠相似，表明Cib2和Whrn的部分缺陷不会损害听力。双纯合突变小鼠（Cib2KO/KO；Whrnwi/wi）具有严重的听力损失，耳蜗立体纤毛表现出在单个Whrnwi/wi突变体中可见的主要表型。此外，Whrn在Cib2KO/KO小鼠中的过表达不能挽救立体纤毛的形态。这些数据表明,CIB2是多功能的，在听觉毛细胞中发育和/或维持立体纤毛楼梯模式方面具有关键的独立功能。
