Cockayne syndrome (CS), which was discovered by Alfred Cockayne nearly 75 years ago, is a rare autosomal recessive disorder characterized by growth failure, neurological dysfunction, premature aging, and other clinical features including microcephaly, ophthalmologic abnormalities, dental caries, and cutaneous photosensitivity. These alterations are caused by mutations in the CSA or CSB genes, both of which are involved in transcription-coupled nucleotide excision repair (TC-NER), the sub-pathway of NER that rapidly removes UV-induced DNA lesions which block the progression of the transcription machinery in the transcribed strand of active genes. Several studies assumed that CSA and CSB genes can play additional roles outside TC-NER, due to the wide variations in type and severity of the CS phenotype and the lack of a clear relationship between genotype and phenotype. To address this issue, our lab generated isogenic cell lines expressing wild type as well as different versions of mutated CSA proteins, fused at the C-terminus with the Flag and HA epitope tags (CSAFlag-HA). In unpublished data, the identity of the CSA-interacting proteins was determined by mass spectrometry. Among which three subunits (namely, CCT3, CCT8, and TCP1) of the TRiC/CCT complex appeared as novel interactors. TRiC is a chaperonin involved in the folding of newly synthesized or unfolded proteins. The aim of this study is directed to investigate by immunofluorescence analysis the impact of the selected CSA mutations on the subcellular localization of the CSA protein itself as well as on its novel interactors CCT3, CCT8, and TCP1. We showed that specific CSA mutations impair the proper cellular localization of the protein, but have no impact on the cellular distribution of the TRiC subunits or CSA/TRiC co-localization. We suggested that the activity of the TRiC complex does not rely on the functionality of CSA.

中文翻译：

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免疫荧光研究剖析 Cockayne 综合征 A 改变对蛋白质相互作用和细胞定位的影响

Cockayne 综合征 (CS) 由 Alfred Cockayne 于近 75 年前发现，是一种罕见的常染色体隐性遗传病，其特征是生长障碍、神经功能障碍、过早衰老和其他临床特征，包括小头畸形、眼科异常、龋齿和皮肤光敏性. 这些改变是由 CSA 或 CSB 基因的突变引起的，这两种基因都涉及转录偶联核苷酸切除修复 (TC-NER)，这是 NER 的子途径，可快速去除紫外线诱导的 DNA 损伤，从而阻止疾病的进展活性基因转录链中的转录机制。一些研究假设 CSA 和 CSB 基因可以在 TC-NER 之外发挥其他作用，由于 CS 表型的类型和严重程度差异很大，并且基因型和表型之间缺乏明确的关系。为了解决这个问题，我们的实验室生成了表达野生型以及不同版本的突变 CSA 蛋白的同基因细胞系，在 C 端与 Flag 和 HA 表位标签 (CSAFlag-HA) 融合。在未发表的数据中，CSA 相互作用蛋白的身份是通过质谱法确定的。其中，TRiC/CCT 复合体的三个亚基（即 CCT3、CCT8 和 TCP1）作为新型相互作用物出现。TRiC 是一种伴侣蛋白，参与新合成或未折叠蛋白质的折叠。本研究的目的是通过免疫荧光分析研究选定的 CSA 突变对 CSA 蛋白本身的亚细胞定位及其新型相互作用因子 CCT3、CCT8 和 TCP1 的影响。我们发现特定的 CSA 突变会损害蛋白质的正确细胞定位，但对 TRiC 亚基或 CSA/TRiC 共定位的细胞分布没有影响。我们建议 TRiC 复合物的活性不依赖于 CSA 的功能。