探究Znf179的分子功能

腦指蛋白Znf179 在神經發育及分化的過程中扮演重要的角色，由於Znf179 的結構包含C3HC4 環指區域，此區域為E3 泛素連接酶重要的結構特徵，並且在我們的研究結果發現，在293T 細胞過度表達Znf179，會增加內生性蛋白的泛素化，顯示Znf179 可能具有E3 泛素連接酶的活性。在本研究中我們發現，藉由細胞內以及細胞外實驗觀察到Znf179 具有自我泛素化的現象；並且我們也發現會與Znf179 結合的E2，包含 UbcH2、UbcH3、UbcH5b、UbcH6 以及UbcH8，顯示Znf179 可能有E3 泛素連接酶的活性。先前的研究利用yeast two-hybrid 篩選結果發現，PLZF (promyelocytic leukemia zinc finger 早幼粒細胞白血病鋅指蛋白，a kruppel-like zinc finger gene) 會與Znf179 結合，我們亦利用免疫沉澱實驗進一步確認兩者交互作用的關係，且進一步發現，表現 PLZF 會促進Znf179 以及內生性蛋白的泛素化。此外，在先前研究發現一些E3 連接酶，像是parkin，能夠調控蛋白酶體(proteasome)的活性。因此，我們利用一個泛素融合螢光蛋白GFP 報導性載體（UbG76V-GFP）探討Znf179 是否能夠調控蛋白酶體(proteasome)的活性。實驗結果發現，Znf179 會促使UbG76V-GFP 表現量下降，顯示Znf179 會促進26S蛋白酶體的活性；我們亦發現Znf179 剔除小鼠與正常小鼠相較，26S 蛋白酶體相關的蛋白(20,19 次單元) PSMA1、PSMC6 以及PSMD10 表現量明顯減少，而在293T 細胞中過度表現Znf179 蛋白，26S 蛋白酶體中的20S 次單元PSMA1 與PSMB4 以及19S 次單元的PSMC6 表現量上升，這些結果顯示Znf179 可以藉由透過調節蛋白酶體次單元的表現量來調控其活性。綜觀上述結果，我們初步發現Znf179 具有E3 泛素連接酶活性，而PLZF 會促進Znf179 及內生性蛋白的泛素化，扮演一載體蛋白的角色。而Znf179 能透過調節26S 蛋白酶體的次單元蛋白表現量進而影響到26S 蛋白酶體的活性。

關鍵字

腦指蛋白Znf179 ；泛素蛋白酶體代謝路徑； E3泛素連接酶早幼粒細胞白血病鋅指蛋白PLZF

並列摘要

Brain finger protein, Znf179, is critical for neuronal differentiation. Due to Znf179 contains the classic C3HC4 RING finger domain and our results revealed that overexpression of Znf179 enhanced the ubiquitylation of endogenous proteins in 293T cells, suggesting that Znf179 may have E3 ubiquitin ligase activity. In this study, we found Znf179 have E3 ligase activity since we observed the autoubiquitylation -like patterns of Znf179 in vivo and in vitro. We found that UbcH2, UbcH3, UbcH5b, UbcH6 and UbcH8 interact with Znf179, suggested they are candidate E2 conjugating enzymes of Znf179. On the other hand, Dr. Lee's groups and our results revealed that Znf179 interacted with promyelocytic leukemia zinc finger (PLZF), a Kruppel-like zinc finger gene, in yeast two-hybrid screening and immunoprecipitation. Overexpression of PLZF enhanced the ubiquitylation levels of Znf179 and Znf179-mediated ubiquitylation of endogenous proteins. In addition, some E3 ligase like Parkin can regulate the proteasome activity. So we further investigated whether Znf179 plays a role in regulating the ubiquitin proteasome activity by using a plasmid, UbG76V-GFP, as the reporter of the proteasome activity. These results show that the level of UbG76V-GFP protein is decreased in 293T cells co-expressing Znf179 than those expressing UbG76V-GFP only, suggested that Znf179 could increase the proteasome activity. We also noted the level of a series proteins relative to 26S proteasome such as 20S/19S proteasome subunits PSMA1,PSMC6 and PSMD10 were decreased in Znf179 knock out mice, whereas the 20S proteasome subunits PSMA1 and PSMB4 and 19S proteasome subunits PSMC6 were increased in Znf179 overexpressed 293T cells.Here, we proof Znf179 could regulate the proteasome activity by modulating the level of 26S proteasome subunits. Together, these results suggested that Znf179 has E3 ubiquitin ligase activity, which is enhanced by adaptor/substrate protein of Znf179, PLZF. Moreover, Znf179 regulates the activity of 26S proteasome through modulating the protein levels of 19S and 20S proteasome subunits.