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  • 學位論文

NRIP 需要藉由 WD40 蛋白序列與 AChR 結合以達到穩固 NMJ 的結構

NRIP WD40 domain is essential to interact with AChR for stabilization of neuromuscular junction

指導教授 : 陳小梨

摘要


WD40 domain 廣泛存在於真核細胞的基因組中。包含 WD40 domain 的蛋白質具有 β-螺旋結構,可為提供蛋白質相互作用的平台。先前的研究表明,含有 WD40 domain 的蛋白質參與在各種細胞功能,如細胞骨架組織,信號傳遞等。大部分含有 WD40 domain 的蛋白質都充當了蛋白質-蛋白質相互作用或蛋白質-DNA 相互作用的支架 蛋白。核受體相互作用蛋白(NRIP)由 860 個氨基酸組成,其中包括 7 個 WD40 domain(大約 40 個氨基酸大小的短片段,並終止於色氨酸-天冬氨酸二肽 )和一 個 IQ motif。NRIP 是與雄激素受體(AR)相互作用以及 Ca2 +依賴的鈣調蛋白結 合的蛋白。神經肌肉接合處(NMJ)是運動神經元和骨骼肌纖維之間的化學突觸, 被 Schwann 細胞(SCs)包裹。 NMJs 包括突觸前末梢,棘突裂和突觸後膜或細胞, 突觸之間的神經傳遞物質交換且會導致肌肉收縮。根據我們以前的研究,肌肉 NRIP 基因剔除(cKO)小鼠在 16 週齡時表現出肌肉萎縮,運動神經元退化和 NMJ 異常 的表現型,這說明肌肉中的 NRIP 可能影響運動神經元的生長。此外,NRIP 被發 現與 NMJ 組成(例如乙酰膽鹼受體(AChR),rapsyn 和 α-actinin(ACTN2)共定 位。 Rapsyn 是一種 45 kDa 的膜結合蛋白,可直接與 AChR 結合併增強 AChR clusters 的形成。因此,rapsyn 被定義為 NMJ 突觸後膜的 AChR 複合物的支架蛋 白。因此,NRIP 與 AChR 和 rapsyn 的共定位顯示 NRIP 可能是一種支架蛋白,有 助於維持 AChR,rapsyn 和 α-actinin 的相互作用,從而穩定了 NMJ 的形成。NRIP 可以結合 AChR, 共同表現 NRIP 和 AChR 可以在 HEK293T 細胞中形成 clusters。 對 NRIP cKO 小鼠進行肌內 AAV-NRIP 基因治療可以恢復 NMJ 的形成並恢復運動 神經元的生長。NRIP-C(包含一個 IQ motif 和兩個 WD40 domain)可以與 AChR 結合,誘導 AChR clusters 形成,並通過 NMJ 形成來復原運動神經元的生長。相 反,NRIP-C-ΔWD7(包含一個 IQ motif 和一個 WD40 domain)失去了與 AChR 結 合,AChR clustering 的能力以及 NRIP cKO 治療效果。因此,我們假設位於 NRIP- C 片段的 WD40 domain6/7 可能對於 NRIP 與 AChR 結合並誘導 clusters 形成,進 III 一步穩定 NMJ 形成是至關重要的。在這項研究中,我們將重點放在 WD 域 6/7 與 AChR 的相互作用上,並確定它是否與 AChR cluster 形成相關。因此,我們通過進 行免疫沉澱法研究了 NRIP-WD6 / 7 和 NRIP-WD7 的 AChR 結合能力。結果表明, 均含有 WD40 domain7(WD7)的 NRIP-WD6 / 7 和 NRIP-WD7 能夠與細胞中的 AChR-α 結合。為了研究 NRIP-WD6 / 7 和 NRIP-WD7 誘導 AChR cluster 形成的能 力,我們將 EGFP-NRIP 突變體和 mCherry-AChR-α 共轉染到 HEK293T 細胞中, 並通過共聚焦顯微鏡觀察 cluster 的形成。結果表明,可以與 AChR-α 結合的 NRIP- WD6 / 7 和 NRIP-WD7 也可以誘導 AChR cluster 的形成。通過與 NRIP 全長進行比 較,我們發現 NRIP-WD6 / 7 和 NRIP-WD7 都達到了 NRIP 全長 AChR 聚類能力的 70%,這表示,AChR 與 WD40 domain 7(WD7)之間的結合,提供了 NRIP 的大 多數聚類能力。此外,我們還對 NRIP cKO 小鼠進行了肌肉注射 AAV-NRIP-WD7 基因治療,以研究其對 NMJ 的影響。我們發現 AAV-NRIP-WD7 處理可以通過增 加突觸后區域的 NMJ 面積來恢復 NMJ 的形成。但是,AAV-NRIP-WD7 無法恢復 端板的神經支配。簡而言之,具有 AChR 結合能力的 NRIP-WD7 可以誘導細胞中 AChR clusters 的形成,也可以增加 NRIP cKO 小鼠的 NMJ 面積,但不能影響運動 神經元。接下來,為了探討 NRIP 的作用機制,我們研究了 NRIP 是否可以恢復 NMJ complex 之間 AChR 與 rapsyn 或 ACTN2 之間的結合親和力。我們利用肌肉 注射 AAV-NRIP 的 NRIP cKO 小鼠的肌肉蛋白進行 BTX 下拉試驗和免疫沉澱試 驗,以研究 AChR 聚合物之間的結合親和力。綜合來說,位於 C 端的 NRIP WD40 domain7(WD7)負責 NRIP 和 AChR 之間的結合,並且其結合與 NRIP 誘導 AChR cluster 形成的能力相關。 但是,僅用 WD7domain 無法實現 NRIP 從突觸後肌肉向 突觸前運動神經元的逆行作用。

並列摘要


WD40 domain is one of the most abundant domain in eukaryotic genome. WD40 domain- containing protein exhibits a β-propeller architecture which provide a platform for protein-protein interaction and involved in various cellular function, such as cytoskeleton organization, signal transduction and served as a scaffold protein. Nuclear receptor interaction protein (NRIP) consists of 860 amino acids which includes seven WD40 domains (short ~40 amino acids terminating in a tryptophan-aspartic acid dipeptide) and one IQ motif. NRIP was identified as an androgen receptor (AR)-interacting protein and Ca2+ dependent calmodulin-binding protein. Neuromuscular junction (NMJ) is a chemical synapse between motor neurons and skeletal muscle fiber which is shielded by Schwann cells (SCs). NMJs include a presynaptic terminal, snaptic cleft and postsynaptic membrane or cell, the transmission of neurotransmitter occur between synapse resulting in the contraction of muscle. Muscle-restricted NRIP knockout (cKO) mice showed the phenotypes with muscle atrophy, motor neuron degeneration and abnormal NMJ at the age of 16-weeks which revealed that NRIP in muscle could affect the motor neuron growth. Moreover, NRIP found to co-localize with NMJ components, such as acetylcholine receptor (AChR), rapsyn and α-actinin (ACTN2). Rapsyn, a 45 kDa membrane-bound protein, which is directly bind with AChR, enhance AChR clusrtering and defined as a scaffold protein of AChR complex. Therefore, NRIP also defined as a V scaffold protein, since it co-expressed with rapsyn. NRIP could bind AChR and co- expressed with AChR which induce cluster formation in cells. The intramuscular AAV- NRIP gene therapy on NRIP cKO mice can restore the NMJ formation and retrogradely rescue motor neuron growth. NRIP-C (containing one IQ motif and two WD40 domains) can bind with AChR, induce AChR cluster formation, and rescue motor neuron growth with NMJ formation. On the contrast, NRIP-C-ΔWD7 (containing one IQ motif and one WD40 domain) losses the AChR binding, clustering ability in cells, and the rescue effects of NRIP cKO. Thus, we hypothesized that the WD40 domain 6/7 located at NRIP-C fragment might be crucial for the NRIP to bind with AChR and induce cluster formation, and further stabilize the NMJ formation. In this study, we would focus on the interaction between WD40 domain 6/7 and AChR, and determine whether it is corelated with AChR cluster formation. Therefore, we investigate the AChR binding ability of NRIP-WD6/7 and NRIP-WD7 by performing immunoprecipitation assay. The results showed that NRIP-WD6/7 and NRIP-WD7 which both containing WD40 domain 7 (WD7) are able to bind with AChR-α in cells. We co-transfected EGFP-NRIP mutants and mCherry- AChR-α into HEK293T cells and examined the clusters formation by confocal microscopy. The resulted showed that NRIP-WD6/7 and NRIP-WD7 which could bind with AChR-α can also induce the AChR clusters formation. We found that NRIP-WD6/7 and NRIP-WD7 both reached about 70% of AChR clustering ability of NRIP full length, VI which indicate that WD7 is responsible for AChR binding also contributed most of the clustering ability of NRIP. Besides, we also applied intramuscular AAV-NRIP-WD7 gene therapy on NRIP cKO mice. Strikingly, we found that AAV-NRIP-WD7 treatment could rescue the NMJ formation by increasing the NMJ area at postsynaptic region. However, AAV-NRIP-WD7 fail to restore the endplate innervation. In short, NRIP-WD7 which have AChR binding ability could induce AChR clusters formation in cells, also able to increase the NMJ area of NRIP cKO mice, but could not retrogradely retore the endplate innervation. Next, we investigate whether NRIP could restore the binding affinity between AChR and rapsyn or ACTN2 among NMJ complex components. NRIP cKO mice treated with AAV-NRIP were used for performing BTX pull down assay and immunoprecipitation assay to investigate the binding affinity between AChR complex. However, the results showed that no effects were observed on the binding affinity between AChR-rapsyn and rapsyn-ACTN2 after AAV-NRIP treatment. Collectively, NRIP WD40 domain 7 (WD7) located at C-terminal is responsible for the binding between NRIP and AChR and its binding is co-related to the ability of NRIP in inducing AChR clusters formation. However, the retrograde effect of NRIP from postsynaptic muscle to presynaptic motor neuron could not achieved with only WD7 domain.

並列關鍵字

NRIP WD40 domain AChR ACTN2 binding affinity gene therapy

參考文獻


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