Immunofluorescence studies to dissect the impact of Cockayne syndrome A alterations on the protein interaction and cellular localization

Amr Ghit

doi:10.1186/s43141-021-00190-7

Immunofluorescence studies to dissect the impact of Cockayne syndrome A alterations on the protein interaction and cellular localization

J Genet Eng Biotechnol. 2021 Jun 16;19(1):88. doi: 10.1186/s43141-021-00190-7.

Author

Amr Ghit^{1

2}

Affiliations

¹ Department of Biology and Biotechnology, University of Pavia, Pavia, Italy. amr.saadshehatariz01@universitadipavia.it.
² Department of Biotechnology, Institute of Graduate Studies and Research (IGSR), Alexandria University, Alexandria, Egypt. amr.saadshehatariz01@universitadipavia.it.

Abstract

Background: 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 (CSA^Flag-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 In unpublished data, the identity of the CSA-interacting proteins was determined by mass spectrometry.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.

Results: 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.

Conclusion: We suggested that the activity of the TRiC complex does not rely on the functionality of CSA.

Keywords: CSA; CSB; Cockayne syndrome; Immunofluorescence; Nucleotide excision repair; TRiC/CCT complex.