E3泛素連接酶與NSF蛋白對第二型氯離子通道的調節機制

Modulation of CLC-2 chloride channel by E3 ubiquitin ligase and NSF

方心妤(Hsin-Yu Fang)

指導教授 ： 湯志永

國立臺灣大學/醫學院/生理學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.01000

摘要

CLC-2為一種廣泛存在於各種組織的氯離子通道，主要受到膜電位過極化、細胞體積增加、胞內氯離子濃度增加與胞外酸性微量增加等因素調控而活化；但是，CLC-2在細胞中確切的分子調控機轉則仍有待釐清。先前研究指出，cereblon (CRBN)是CLC-2的結合蛋白。近來的研究亦發現CRBN可能是cullin RING E3泛素連接酶複合體 (E3 ubiquitin ligase complex) (CRL)中的受體蛋白受器 (substrate receptor)。然而CRBN是否的確參與CLC-2的蛋白質降解作用，則尚不清楚。另一方面，本實驗室過去利用酵母菌雙雜交技術 (yeast-two hybrid screening)發現NSF是能與CLC-2交互作用的蛋白。本論文的研究方向分為二個主軸，其一是針對CLC-2的降解途徑進行探討，其二則是觀察抑制NSF對於CLC-2蛋白表現量的影響。
我們先以MG132和MLN4924這二種藥物作用的結果推論CLC-2可能是經由CRL進行蛋白酶體降解，我們接著以生化實驗發現CLC-2會與Cullin 4 (CUL4)、DDB1和CRBN存在於同一蛋白質複合體 (protein complex)。我們也證明CUL4和CRBN都會參與CLC-2的降解作用。此外，我們還以只能進行單一泛素 (monoubiquitination)作用的突變種泛素Ub-K0證明CLC-2的降解機制應該是以聚泛素鏈的形式進行泛素標定。另一方面，我們發現若對NSF進行shRNA抑制，雖然不會影響CLC-2的mRNA表現量，但卻造成CLC-2蛋白總量的增加。

關鍵字

CLC-2氯離子孔道 ； 蛋白質降解 ； E3泛素連接酶 ； NSF

並列摘要

CLC-2 is a ubiquitously expressed chloride channel, which is mainly activated by membrane potential hyperpolarization, increased cell volume, elevated intracellular chloride concentration and mild extracellular acidification; however, the precise cellular regulatory properties of CLC-2 channel still remain elusive. In recent studies, Cereblon (CRBN) has been considered to be a binding partner of CLC-2. CRBN has also been suggested to play a role as a substrate receptor of the cullin RING E3 ubiquitin ligase complex (CRL). Nevertheless, it is unclear whether CRBN is involved in the protein degradation of CLC-2. In addition, previous studies in our lab utilizing yeast-two hybrid screening of rat brain cDNA library has identified NSF as a CLC-2 interacting protein. Therefore, there are two aims in this study. One aim is to understand the degradation pathway of CLC-2, and the other aim is to observe the effect on CLC-2 protein expression level caused by NSF shRNA knockdown.
Firstly, we reason that the proteasomal degradation of CLC-2 can be mediated by CRL according to effects caused by MG132 and MLN4924 treatments. We then find out the fact that CLC-2 can coexist in the same protein complex with Cullin 4 (CUL4), DDB1 and CRBN based on our biochemical experimental results; moreover, we also prove that both CUL4 and CRBN do play a role in the degradation of CLC-2. By utilizing an ubiquitin mutant, Ub-K0, which can only undergo monoubiquitination, we further prove that CLC-2 shall be ubiquitinated through polyubiquitin chains in its degradation mechanism. Besides, we have also noted that NSF shRNA knockdown results in an increased CLC-2 total protein level. In contrast, the mRNA level of CLC-2 does not be affected by NSF shRNA knockdown.

並列關鍵字

CLC-2 chloride channel ； protein degradation ； E3 ubiquitin ligase ； NSF

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