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標題: | 探討人類Cav2.1鈣離子通道蛋白之降解機制 Regulation of human P/Q-type calcium (Cav2.1) channel degradation. |
作者: | 傅斯如 Ssu-Ju Fu |
指導教授: | 湯志永 |
關鍵字: | 鈣離子,泛素,E3連接?, Calcium channel,ubiquitin,E3 ligase, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 電壓控制型鈣離子通道CaV2.1,由α1A次單元以及α2δ、β4 附屬單元組成功能性的鈣離子通道,其在調控神經傳導物質釋放與調節神經突觸訊號傳遞上扮演重要角色。當人類CaV2.1基因(CACNA1A)帶有突變時,可能會導致第一型家族性偏頭痛(familial hemiplegic migraine-1; FHM-1)、第二型遺傳性共濟失調(episodic ataxia type 2 (EA2)、以及第6型脊髓小腦萎縮症(spinocerebellar ataxia type 6; SCA6)。文獻指出,當CaV2.1帶有EA2疾病突變時,會使得突變蛋白質的穩定度下降,甚至引起顯性抑制作用(dominant-negative suppression)而使得正常型蛋白質經proteasome進行降解。因此,我們試圖找出調控CaV2.1蛋白質降解之途徑。
我們確立了E3泛素連接酶RNF138與CaV2.1間的交互作用,RNF138與CaV2.1共同表現在神經細胞之突觸前後。共同表現RNF138會促進CaV2.1之泛素化,並加速CaV2.1蛋白質降解速度。相反的,降低細胞內生性RNF138表現,會顯著增加CaV2.1蛋白質表現量並提升CaV2.1蛋白質穩定度,也會有效的增加EA2突變型蛋白質,並且改善由EA2突變型引起的蛋白質表現量顯性抑制作用。然而利用電生理進行CaV2.1離子通道功能測試時卻發現,降低內生性RNF138表現量僅能部分回復由EA2突變型所引起的電流抑制作用。利用biotinylation實驗觀察,降低內生性RNF138表現,雖然改善了EA2突變型對CaV2.1 WT的蛋白質抑制作用,但仍然無法改變由EA2突變型所導致的CaV2.1 WT trafficking降低的問題。 除了RNF138之外,我們還確立了Pin1與CaV2.1間的交互作用。與RNF138類似,Pin1與CaV2.1也共同表現在神經細胞之突觸前後。我們發現,CaV2.1蛋白質表現量會受到其磷酸化程度所調控。Pin1會促進CaV2.1之泛素化,並加速CaV2.1蛋白質降解速度。利用Pin1阻斷劑,抑制細胞內生性Pin1活性,會顯著增加CaV2.1蛋白質表現量,以及增加CaV2.1蛋白質穩定度。有趣的是,相較於CaV2.1正常型,Pin1與EA2突變型有較高的親合力。統合實驗結果我們推論,Pin1扮演上游調控分子,並參與在由RNF138媒介的CaV2.1蛋白質降解途徑中。 CACNA1A mRNA具有除了promotor以外的內生性ribosome結合位(internal ribosomal entry site; IRES) 。CACNA1A基因bicistronic的特性,會使得mRNA轉譯出全長的CaV2.1鈣離子通道與僅具有CaV2.1部分羧基端片段的carboxyl-terminal fragment (CTF)。當CTF含有過度延長的polyglutamine時,會導致遺傳性第6型脊髓小腦萎縮症。實驗發現,CTF較全長CaV2.1次單元蛋白質表現量低,然而卻有較穩定的蛋白質半衰期。與CaV2.1次單元相同,CTF蛋白質也是經由proteasome進行降解,並依然受到Pin1與RNF138調控。CTF會受到SUMOylation的修飾作用,或許與CTF在細胞內分布的位置有關,然而這是未來我們需要進一步釐清的問題。 總結此篇論文發現,我們認為Pin1與RNF138在調控CaV2.1蛋白質表現與離子通道功能上扮演著重要的角色。利用調整內生性Pin1與RNF138之功能,進一步調控CaV2.1的蛋白質表現量,或許可以提供未來治療EA2與SCA6疾病的可能契機。 Voltage-gated Ca2+ (CaV) channels comprise a pore-forming α1A subunit with auxiliary α2δ and β subunits. CaV2.1 (P/Q-type) channels play an essential role in regulating synaptic signaling. Mutations in CACNA1A, the human gene encoding the CaV2.1 subunit, are associated with the neurological disease familial hemiplegic migraine-1 (FHM-1), episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 (SCA6). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of CaV2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human CaV2.1. We identified RNF138, an E3 ubiquitin ligase, as a novel CaV2.1-binding partner. In neurons, RNF138 and CaV2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of CaV2.1. Disrupting endogenous RNF138 function significantly upregulates the CaV2.1 protein level, enhances CaV2.1 protein stability, and effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of CaV2.1 WT subunits. However, only partially restores the dominant-negative effect of EA2 mutants on CaV2.1 WT functional expression, which can be attributed to defective membrane trafficking of CaV2.1 WT in the presence of EA2 mutants. We also identified Pin1, a peptidyl-prolyl cis/trans isomerase, as another novel CaV2.1-binding partner. Like RNF138, Pin1 colocalizes with CaV2.1 in neurons at both presynaptic and postsynaptic region. We demonstrated a phosphorylation-dependent change in CaV2.1 protein level. Pin1 promotes polyubiquitination and accelerates protein turnover of CaV2.1, and application of a specific Pin1 blocker leads to enhanced CaV2.1 protein level. Interestingly, Pin1 exhibits a significantly higher affinity to an EA2 mutant than CaV2.1 WT. Take together Pin1 appears to act as an upstream regulator for the RNF138-mediated CaV2.1 degradation pathway. The CACNA1A gene has a cryptic internal ribosomal entry site (IRES). The bicistronic mRNA of CACNA1A gene encodes the full-length CaV2.1 protein, as well as the CaV2.1 carboxyl-terminal fragment (CTF) that harbors polyglutamine repeats implicated in the pathogenesis of SCA6. We demonstrate that the CaV2.1 CTF has lower expression protein level, but longer protein half-life, than full-length CaV2.1 subunit. The protein degradation of CaV2.1 CTF is also via proteasome and is regulated by Pin1 and RNF138. The posttranslational modification of CaV2.1 CTF may also involve SUMOylation, which may regulate its subcellular localization. Overall, we conclude that Pin1 and RNF138 play a critical role in the homeostatic regulation of CaV2.1 protein level and functional expression. Regulation CaV2.1 protein level via specific modulator of endogenous Pin1 and RNF138 function may potentially contribute to future development of novel therapeutic strategies for CaV2.1-related disease such as EA2 and SCA6. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70081 |
DOI: | 10.6342/NTU201800380 |
全文授權: | 未授權 |
顯示於系所單位: | 生理學科所 |
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