體染色體隱性遺傳中央核肌肉病變的致病機制

Pathogenic Mechanism of Autosomal Recessive Centronuclear Myopathy

駱怡君(Julie Loh)

指導教授 ： 劉雅雯

國立臺灣大學/醫學院/分子醫學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201800973

摘要

中央核肌肉病變 (Centronuclear myopathy, CNM)是一種罕見的遺傳疾病，病徵是骨骼肌的衰弱與退化。CNM分為三種類型，其中的體染色體隱性遺傳型是由一個骨骼肌中負責橫小管(T-tubule)發育的蛋白Bin1突變所造成。T-tubule是骨骼肌細胞膜內凹陷入細胞內的結構，對肌肉細胞的興奮收縮連結作用(E-C coupling)非常重要。許多造成CNM的Bin1突變體是發生在產生膜彎曲的區域進而產生T-tubule發生的缺陷。然而，有兩個造成Bin1 SH3區域截斷的無義突變(nonsense mutations)體， Q434X與K436X，目前仍不清楚其致病機制。在這篇論文中，我發現這兩個Bin1突變體喪失了對Dynamin 2(Dyn2)的調控而造成T-tubule無法維持。Dyn2是一個全身性表達的細胞膜切斷酵素。從體外實驗的結果，我發現Bin1 Q434X與K436X 對Dyn2的結合力下降因此造成Dyn2切斷膜的能力上升。相同的影響也在肌纖維母細胞中利用共同表達Bin1 Q434X-GFP 或K436X-GFP 與 Dyn2-mCherry 觀察到。因此，我們發現Bin1是Dyn2活性的調控蛋白，而Bin1 Q434X與K436X突變造成了Dyn2的過度活躍而造成T-tubule斷裂而產生了骨骼肌的病變。我的實驗結果除了提供CNM的致病機制，並指出降低Dyn2的活性將是治療Bin1-CNM的一個方法。

關鍵字

肌肉病 ； 致病機制 ； 肌肉細胞 ； 膜張力 ； 膜販運 ； 體染色體隱性遺傳中央核肌肉病變

並列摘要

Centronuclear myopathy (CNM) is a rare genetic disorder characterized by muscle weakness that is debilitating from an early age. Autosomal recessive CNM is caused by mutations in Amphiphysin-2 (Bin1), a protein important for membrane curvature generation and critical for the formation of highly curved plasma membrane invaginations in muscle cells, the T-tubules, which are crucial for excitation-contraction coupling. There have been several CNM-related Bin1 mutations found to be located at its membrane curvature generating domain or membrane binding domain resulting in defects in Bin1’s tubulation ability. Two nonsense mutations, Q434X and K436X, which result in truncated Src homology 3 (SH3) domains, have not been well-characterized for their pathogenic mechanism. The phenotypes of these Bin1 mutants are illustrated in relation to their effects on dynamin-2 (Dyn2) activity in this study. Dyn2 is a GTPase that is ubiquitously expressed that is well-known for its scission of vesicles from the membrane. I discovered that Bin1 mutants, Q434X and K436X, have an enhancing effect on Dyn2 fission activity because Bin1 Q434X and K436X are diminished in their binding affinity with Dyn2 compared to full-length Bin1. Consistent results were also observed in vivo by co-transfection of the Q434X-GFP or K436X-GFP with Dyn2-mCherry in C2C12 myoblast cells. In conclusion, wild-type Bin1 was found to negatively regulate dynamin fission activity and the hyperactivity quality of Dyn2 membrane fission underpins the autosomal recessive CNM caused by SH3 domain truncated Bin1. Together, my study provides a mechanistic explanation for the characteristics of T-tubule fragmentation displayed by CNM and implicates downregulation of dynamin to be a therapeutic strategy in mitigating muscle atrophy by CNM.

並列關鍵字

centronuclear myopathy ； dynamin ； Amphiphysin-2 ； Bin1 ； membrane trafficking ； membrane remodeling ； membrane fission

參考文獻

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