The role of charged amino acids in the nuclear transport activity of triple C2H2 zinc finger domains

• Main Authors: Lin, Chih-Ying, 林志穎
• Other Authors: Lin, Lih-Yuan
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/6d9x58

博士 === 國立清華大學 === 分子與細胞生物研究所 === 106 === Zinc finger (ZF) motifs on proteins are frequently recognized as a structure for DNA binding. Accumulated reports indicate that ZF motifs contain nuclear localization signal (NLS) to facilitate the transport of ZF proteins into nucleus. We investigated the critical factors that are involved in the nuclear transport of triple C2H2 ZF proteins. Three conserved basic residues (hot spots) were identified among the ZF sequences of triple C2H2 ZF proteins that reportedly have NLS function. Additional basic residues can be found on the α-helix of the ZFs. Using the ZF domain (ZFD) of Egr-1 as a template, various mutants were constructed and expressed in cells. The nuclear transport activity of various mutants was estimated by analyzing the proportion of protein localized in the nucleus. Mutation at any hot spot of the Egr-1 ZFs reduced the nuclear transport activity. Changes of the basic residues at the α-helical region of the second ZF (ZF2) of the Egr-1 ZFD abolished the NLS activity. However, this activity can be restored by substituting the acidic residues at the homologous positions of ZF1 or ZF3 with basic residues. The restored activity dropped again when the hot spots at ZF1 or the basic residues in the α-helix of ZF3 were mutated. This study was extended to other triple C2H2 ZF proteins. SP1 and KLF families, similar to Egr-1, have charged amino acid residues at the second (α2) and the third (α3) positions of the α-helix. Replacing the amino acids at α2 and α3 with acidic residues reduced the NLS activity of the SP1 and KLF families including KLF5, KLF6, KLF8 and KLF11. The reduced activity can be restored by substituting the α3 with histidine at any ZFs of SP1 and KLF family members. The variations in nuclear transport activity of Egr-1, SP1 and KLF6 are directly linked to the binding activity of the ZFDs with importins. Besides, basic residues at α6 of ZF2 and ZF3 enhance the NLS activity of SP1, KLF6 and KLF11. However, the mechanism may not apply to multiple C2H2 ZF proteins, such as Wilms' tumor 1 and metal responsive transcription factor 1. The results show the interchangeable role of ZFs and charge residues in the α-helix in regulating the NLS activity of triple C2H2 ZF proteins.