Differential requirements for the EF-hand domains of human centrin 2 in primary ciliogenesis and nucleotide excision repair
Differential requirements for the EF-hand domains of human centrin 2 in primary ciliogenesis and nucleotide excision repair
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DOI：
10.1242/jcs.228486
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发表时间：
2019-10-01
影响因子：
4
通讯作者：
Morrison, Ciaran G.
中科院分区：
文献类型：
--
作者：
Khouj, Ebtissal M.;Prosser, Suzanna L.;Morrison, Ciaran G.
中心蛋白2是一种小的保守的钙结合蛋白，定位于人类细胞的中心粒远端腔。它是有效的初级纤毛发生和核苷酸切除修复（NER）所必需的。中心蛋白2形成着色性干皮病C组蛋白复合物的一部分。为了探索中心蛋白2如何促进这些不同的过程，我们突变了人类中心蛋白2的四个钙结合EF-手结构域。其中所有四个EF-手已经突变以消融钙结合（4DA突变体）的中心蛋白2能够支持体外NER，并且在拯救中心蛋白2缺失细胞的UV敏感性方面与野生型蛋白一样有效。然而，我们发现任何EF-手结构域的突变损害了人类TERT-RPE 1细胞中的初级纤毛发生，其程度与中心蛋白2的缺失相同。4DA突变体的表型分析显示，缺陷的中心体定位，中心粒卫星组装，纤毛组装和功能，并在与POC 5和SFI 1的相互作用。这些观察结果表明，中心蛋白2需要钙结合能力，其主要纤毛功能，但不是NER，并建议这些功能需要中心蛋白2能够形成复合物与伴侣蛋白。
Centrin 2 is a small conserved calcium-binding protein that localizes to the centriolar distal lumen in human cells. It is required for efficient primary ciliogenesis and nucleotide excision repair (NER). Centrin 2 forms part of the xeroderma pigmentosum group C protein complex. To explore how centrin 2 contributes to these distinct processes, we mutated the four calcium-binding EF-hand domains of human centrin 2. Centrin 2 in which all four EF-hands had been mutated to ablate calcium binding (4DA mutant) was capable of supporting in vitro NER and was as effective as the wild-type protein in rescuing the UV sensitivity of centrin 2-null cells. However, we found that mutation of any of the EF-hand domains impaired primary ciliogenesis in human TERT-RPE1 cells to the same extent as deletion of centrin 2. Phenotypic analysis of the 4DA mutant revealed defects in centrosome localization, centriole satellite assembly, ciliary assembly and function and in interactions with POC5 and SFI1. These observations indicate that centrin 2 requires calcium-binding capacity for its primary ciliogenesis functions, but not for NER, and suggest that these functions require centrin 2 to be capable of forming complexes with partner proteins.