人類第一型氯離子通道之蛋白酶體降解途徑

Abstract

先天性肌肉強直症 (myotonia congenita) 是一種影響骨骼肌舒張的神經肌肉遺傳疾病，這是由於負責轉錄骨骼肌上人類第一型氯離子通道的CLCN1基因突變所造成的結果。人類第一型氯離子通道 (human CLC-1 Chloride channels, CLC-1) ，其功能主要為調節骨骼肌細胞膜的興奮性。先前研究已知，先天性肌肉強直症之相關突變會改變CLC-1的電位依賴性進而影響離子通道的門閥開關比例；此外，某些相關突變也會影響CLC-1表現總量與分布型態。近來亦有研究指出，先天性肌肉強直症相關突變會促進CLC-1之降解 (degradation)，但現今對於CLC-1之降解調控途徑及機制尚未釐清。本篇論文旨在利用正常型以及突變型A531V探討CLC-1之降解途徑。 我們的主要目標是要找出負責CLC-1降解之E3連接酶 (E3 ligase)。首先，我們利用MLN4924藥物處理辨別出CLC-1之E3連接酶屬於cullin-RING ubiquitin ligases (CRLs)次家族。為了進一步找出參與CLC-1泛素化作用之E3連接酶，我們選用了具有dominant negative (DN)效果的cullin (CUL)構築質體，利用西方墨點法分析比較共同轉染 (co-expression)後CLC-1之蛋白質表現量的差異，發現CUL4A與CUL4B組別中CLC-1之表現量顯著增加。我們也以泛素化標定 (ubiquitination)實驗觀察到DN-CUL4A組別明顯減少了CLC-1被泛素化的程度。而後我們利用共同免疫沉澱法 (Co-IP)發現CLC-1會與CUL4A之adaptor蛋白DDB1以及CUL4A之substrate receptor蛋白cereblon (CRBN)共同存在相同的protein complex。此外，DN-CRBN也會造成CLC-1蛋白總量的增加，並使CLC-1之泛素作用程度減少。我們還證明DN-CUL4A與DN-CRBN皆可延長CLC-1之蛋白質代謝半衰期。細胞表面生物素標定 (surface biotinylation)的實驗結果顯示在與DN-CUL4A共同轉染的情況下，CLC-1蛋白質運送至細胞膜效率並不會有明顯的改變。當我們以肌強直相關突變型A531V重複以上之實驗結果發現其蛋白質總量之增加、泛素化作程度之下降以及蛋白質代謝半衰期之延長的效果都比正常型的反應更為明顯，代表A531V突變型CLC-1蛋白質確實是受到顯著的降解調控機制。由以上實驗結果我們證明了調控CLC-1的E3連接酶應當為CUL4-DDB1-CRBN所組成的多單元複合體。 為了進一步探討CLC-1泛素化之鍵結方式，我們發現利用泛素 (Ubiquitin, Ub)所有位置之離胺酸 (K)皆定點突變為精胺酸 (R)而只能形成單一泛素化作用的構築質體 (Ub-K0)可以使CLC-1蛋白總量明顯增加。而泛素不同位置之離胺酸定點突變為精胺酸的構築質體雖然在正常型CLC-1組別不具有顯著差異，但是Ub-K6R以及Ub-K48R可以使A531V CLC-1蛋白質表現總量增加。綜合以上結果，我們推測CLC-1是以聚泛素鏈形式進行降解機制之標定，並且其聚泛素鏈是由泛素K48位置鍵結所形成。

Myotonia congenita, a hereditary neuromuscular disorder that disrupts skeletal muscle relaxation, is caused by mutations in the human CLCN1 gene on chromosome 7. The function of human CLC-1 chloride channels (CLC-1) is to regulate the membrane excitability of skeletal muscles. Myotonia congenita-related mutations are known to alter the voltage dependence and open probability of CLC-1 channels, as well as the biogenesis and the subcellular distribution of the Cl- channel. Recent evidence further indicates that myotonia congenita mutation may enhance the degradation of CLC-1 channels. In this thesis, we aim to decipher the proteasomal degradation mechanism of CLC-1 channels by studying the wild type (WT) and the mutant A531V. The substrate specificity of the ubiquitin-conjugation system is mainly mediated by E3 ligases. By treating with MLN4924, we examine the E3 ligase of CLC-1 belongs to CRLs. To find E3 ligases of CLC-1, we choose CUL constructs that has dominant-negative effects to co-express with CLC-1. We find that DN-CUL4A and DN-CUL4B increase the total protein expression level of CLC-1. DN-CUL4A also reduced the ubiquitination level of CLC-1. We further find the adaptor, DDB1, and the substrate receptor, CRBN, of CRL4s by using co-immunoprecipitation. Furthermore, we find that DN-CRBN could increase the total protein expression level of CLC-1 as well as reduce the ubiquitination level of CLC-1by, prolong the protein half-life via cycloheximide treatment. Moreover, we discover that co-expression CLC-1 with DN-CUL4A or DN-CUL4B can enhance CLC-1 protein trafficking to cell membrane by biotinylation technique. We also proved that the mutation related to myotonia congenita, A531V, has more significant effect to DN-RING domain E3 ligase comparing to wild type CLC-1. These results are consistent with the mutant type were more significant than wild-type. In addition, in order to understand how ubiquitin is covalently linked to CLC-1 protein via specific residues, can use different sites of lysine to form different types of ubiquitin chain, co-expressed CLC-1 WT and A531V proteins with various ubiquitin constructs harboring different lysine mutations. In this result, we find that ubiquitin covalently linked to CLC-1 by using K48 residues, which would be recognized by proteasome.