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標題: | NRIP在神經肌肉接合處可作為乙醯膽鹼受體複合物的結構性蛋白 NRIP is a structural component of AChR cluster complex at neuromuscular junction |
作者: | Tzu-Yun Lai 賴姿云 |
指導教授: | 陳小梨 |
關鍵字: | 核受體交互作用蛋白,神經肌肉接合處,乙醯膽鹼受體,基因治療,WD40 domain, NRIP,neuromuscular junction,acetylcholine receptor,gene therapy,WD40 domain, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 在神經肌肉接合處(neuromuscular junction, NMJ)的發展上,agrin-Lrp4-MuSK 信號會促使乙醯膽鹼受體(acetylcholine receptors, AChRs)在肌肉細胞膜上形成緊密的聚集,以此形成功能良好,能有效率接收訊號的神經肌肉接合處。乙醯膽鹼受體由α, β, γ, δ四個子單位構成,在內質網形成α2βγδ的聚合體並以此型態表現在肌肉細胞膜上。Rapsyn為近細胞膜蛋白,可以和乙醯膽鹼受體有直接交互作用並促使乙醯膽鹼受體在神經肌肉接合處形成聚集。缺乏rapsyn的小鼠神經肌肉接合處上的乙醯膽鹼受體無法形成聚集,導致老鼠無法存活。另外,輔肌動蛋白異構體 (α-actinin 2, ACTN2) 在神經肌肉接合處與rapsyn有直接交互作用,在agrin的調控下ACTN2-rapsyn也會影響乙醯膽鹼受體聚集。因此,rapsyn, ACTN2 與乙醯膽鹼受體三者為目前已知的乙醯膽鹼受體複合物 (AChR complex)。核受體交互作用蛋白(Nuclear receptor interaction protein, NRIP)是一個鈣離子依賴性的攜鈣素(calmodulin)結合蛋白,其中涵蓋7個WD domain與1個IQ domain。在本實驗室先前的研究中,全身性NRIP基因剃除小鼠有肌肉失養及受損的運動能力。另外 在16周大的肌肉NRIP基因剃除小鼠中觀察到神經肌肉接合處的異常,包括神經肌肉接合處的面積減少和神經支配比例(軸突神經支配/去神經支配的比例 (axonal innervtion/denervation)),和運動神經元(α-motor neuron)退化。這代表NRIP可能參與在神經肌肉接合處的行程與維持之中。在先前研究我們發現NRIP可以透過自身的IQ domain與 ACTN2的EF-hand有交互作用。鑑於ACTN2為乙醯膽鹼受體複合物的一員,加上我們所觀察到的NRIP對神經肌肉接合處的影響,我們推測NRIP可能也是組成乙醯膽鹼受體複合物的結構性蛋白。透過免疫螢光染色我們觀察到NRIP是一個近膜蛋白,且與rapsyn、ACTN2與乙醯膽鹼受體共定位(co-localize)在肌肉細胞膜上。使用免疫沉澱法(Immunoprecipitation) 也看到NRIP與rapsyn、ACTN2可以共同被乙醯膽鹼受體沉澱下來,因此代表NRIP是乙醯膽鹼受體複合物之中的一個結構性蛋白。
我們進一步想探討NRIP與乙醯膽鹼受體是如何進行交互作用的。我們使用免疫沉澱法觀察到NRIP可與乙醯膽鹼受體的α, β和δ子單位有交互作用,並使用不同片段的NRIP觀察到在把NRIP的WD7 domain去除後,NRIP便無法和AChR-α 結合。因此可以得知NRIP是透過WD7 domain來與AChR-α有交互作用。為了瞭解NRIP與乙醯膽鹼受體的交互作用與促進乙醯膽鹼受體聚集產生是否有關連,我們共轉染EGFP-NRIP不同片段和mCherry-α再以共軛焦顯微鏡觀察乙醯膽鹼受體聚集產生的情形。結果顯示和AChR-α有交互作用的片段,包括NRIP-FL與NRIP-C皆可使AChR-α在細胞內產生許多的聚集。而C-ΔWD7在先前沒有看到與AChR-α有交互作用,在此實驗也觀察到較少的AChR-α聚集。這顯示NRIP透過WD7 domain和AChR-α的交互作用與AChR-α形成聚集是有相關的。 最後,我們想知道在細胞觀察到的AChR-α聚集形成與生物體內神經肌肉接合處的行成是否有相關性。我們使用肌肉注射給予帶有NRIP-C與C-ΔWD7基因的相關腺病毒(AAV)進行基因治療,觀察肌肉NRIP基因剃除小鼠的神經肌肉接合處的形成是否會受到影響。先前實驗室研究結果顯示給予AAV-NRIP基因治療可以顯著改善使神經肌肉接合處型態正常與運動神經元的存活。我們使用肌肉注射給予AAV-NRIP-C與AAV-C-ΔWD7基因治療,並觀察到AAV-NRIP-C可以改善肌肉NRIP基因剃除小鼠的神經肌肉接合處的面積減少和神經支配比例,也改善與運動神經元的存活率,而AAV-C-ΔWD7則無法。因此我們知道NRIP透過WD7 domain和AChR-α的交互作用與神經肌肉接合處的完整和運動神經元的退化死亡有相關。綜合上述所發現的,NRIP是乙醯膽鹼受體複合物之中的一個結構性蛋白,透過WD7 domain與乙醯膽鹼受體有交互作用並參與神經肌肉接合處的乙醯膽鹼受體聚集形成,藉此影響神經肌肉接合處的形成與穩定。 Synapses formed between motor neurons and skeletal muscle fibers are named neuromuscular junction (NMJ). During development of neuromuscular junction (NMJ), agrin-Lrp4-MusK signaling pathway regulates acetylcholine receptors (AChRs) to form dense clusters at postsynaptic muscle membrane, which are essential for well function NMJ. AChR are consist of α, β, γ, δ four subunits and assembles to pentamer as α2βγδ at ER, then transport and localized on membrane. Rapsyn, a 43kDa peripheral membrane protein, is known to bind directly to AChR subunits and participate in AChR cluster formation through its binding ability. Mice with absence of rapsyn would cause failure AChR clustering at NMJ and mice died perinatally. Moreover, ACTN2 interacts with rapsyn directly at NMJ. With agrin’s regulation, rapsyn-ACTN2 also take part in AChR clustering in NMJ. Taken together, AChR-rapsyn-ACTN2 form a ternary complex on muscle membrane, components of AChR complex are associate with AChR clustering and NMJ formation. Nuclear receptor interaction protein (NRIP) is a Ca2+-dependent calmodulin-binding protein, consists of 860 amino acids and containing seven WD-40 repeats and one IQ motif. In our previous study, NRIP global knockout (gKO) mice show muscle dystrophy and impaired motor function. Furthermore, muscle specific NRIP knockout (cKO) mice demonstrate NMJ abnormality with decreased NMJ area and axonal denervation, loss of motor neuron in spinal cord and motor function defects at adult age (16week). These indicates that NRIP may play a role in NMJ formation and maintenance. In previous study we also investigated that NRIP can interact with ACTN-2 EF-hand through its IQ domain. Since ACTN2 is a component of AChR complex, combined NRIP’s influence at NMJ, we hypothesis that NRIP is a structural component of AChR and participate in AChR clustering at NMJ. By immunofluorescence assay, we examined that NRIP is a membrane bound protein and colocalized with rapsyn, ACTN2 and AChR at cell membrane. NRIP can also be pulled down with ACTN2 and rapsyn by AChR. These indicates that NRIP is a novel structural component of AChR complex. To further discuss how NRIP bind with AChR, we performed immunoprecipitation and examined NRIP can bind to AChR α, β and δ with different affinity. On the other hand, NRIP loss AChR-α binding ability when WD7 domain were truncated, indicating that NRIP and AChR-α is reciprocally interaction through WD7 domain. To investigate whether NRIP’s binding ability to AChR is associated with AChR cluster formation, we co-transfected EGFP-NRIP mutants and mCherry-AChR-α into HEK293T cells, examined cluster formation in cells by confocal microscopy. The result shows that NRIP mutants that have binding ability to AChR-α can form AChR clusters in cells, while C-ΔWD7, which loss binding ability to AChR-α, cannot form AChR clusters. Taken together, WD7 domain of NRIP is responsible for AChR-α binding, and this interaction is essential for AChR cluster formation. Last, to see whether the ability of AChR cluster formation in cells can represent AChR clustering in vivo, we examine NMJ formation by given gene therapy of AAV-NRIP-C and AAV-C-ΔWD7 to 6weeks-old NRIP cKO mice. Our previous study showed AAV-NRIP treated cKO mice had improved NMJ integrity and motor neuron survival. Here we performed gene therapy of AAV-NRIP-C and AAV-C-ΔWD7 by intramuscular injection and the results show that AAV-NRIP-C can increase NMJ area size, axonal denervation, and enhance motor neuron survival in NRIP cKO mice, while AAV-C-ΔWD7 cannot. Collectively, NRIP WD7 domain’s AChR binding ability is responsible for NMJ formation, as well as motor neuron degeneration. To sum up, NRIP is a novel structural component of AChR complex, participates in AChR clustering at NMJ formation and stabilization through its binding with AChR by WD7 domain. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7244 |
DOI: | 10.6342/NTU201902676 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 微生物學科所 |
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