C2H2鋅指 (zinc finger, ZF) 區域為一種常見的DNA辨識結構。近來許多研究都指出C2H2鋅指區域含有核定位訊號 (nuclear localization signal, NLS) 可將蛋白帶入細胞核中。我們的研究主要探討影響三指C2H2鋅指區域進核能力的因素。我們將已知具有核定位訊號能力的三指C2H2鋅指區域進行序列比對，找出了三個高度保留性的鹼性胺基酸 (hot spots)，另外在α-helix區域也發現有若干鹼性胺基酸的分布。Egr-1首先被作為研究的對象，將其帶有鋅指區域的片段與綠色螢光蛋白連結，並在該區域進行各種突變後，將它們表現在細胞中，分析綠色螢光在細胞核與質的分布並加以量化。結果顯示任意hot spot的突變都會影響Egr-1的進核。另外，將第二個鋅指 (ZF2) 上的鹼性胺基酸突變也會抑制Egr-1的進核能力。然而，進一步將ZF1與ZF3相對位置的酸性胺基酸突變成鹼性胺基酸之後回復了Egr-1的進核能力，但此回復又會進一步分別被ZF1的hot spot突變以及ZF3 α-helix區域的鹼性胺基酸突變所抑制。我們將此結果也拓展到其他的三指 C2H2鋅指家族。類似於Egr-1，SP1和KLF家族在α-helix區域的第二 (α2) 以及第三 (α3) 位置的胺基酸也有帶有電性，將這些位置置換為酸性胺基酸會使得SP1以及KLF家族成員的進核活性下降，而將任意ZF的α3置換成組胺酸之後即可回復進核能力。我們同樣證明Egr-1、SP1與KLF6的進核能力變化是由於與importin的親和力改變所造成的。此外，位於SP1、KLF6與KLF11 ZF2和ZF3 α6的鹼性胺基酸也會強化鋅指的NLS活性。然而三指C2H2鋅指區域的進核機制卻無法適用於多指C2H2鋅指蛋白如WT1與MTF-1。我們的結果證明了α-helix區域的帶電性胺基酸調控了三指 C2H2鋅指蛋白的進核能力，而且其角色具有可互換性。

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.

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