肌間線蛋白突變導致潛在細胞凋亡路徑與其和aB-水晶體的交互作用

Desmin mutations cause potential apoptosis pathway and its interaction with aB-crystallin

江政任(Cheng-Jen Chiang)

指導教授 ： 彭明德

國立清華大學/生命科學院/分子醫學研究所/碩士(2013年)

摘要

肌間線蛋白是一分子量53 kDa，表現在肌肉的中間絲蛋白，它形成外肌節的細胞骨架，將肌纖維串連起來且把收縮裝置連接至粒線體、細胞核和細胞間接合。當肌間線蛋白突變將導致肌間線蛋白肌病變，這是一種會影響骨骼肌和心肌的嚴重罕見疾病。肌間線蛋白肌病變的特徵是肌節間的大量含肌間線蛋白粒絲狀蛋白堆積物。且經由先前的發現，一小型熱休克蛋白B-水晶體的突變亦可造成與肌間線蛋白肌病變相同的病理特徵，支持B-水晶體和肌間線蛋白功能性上交互作用的首次證據。在本次研究中，我們使用許多生化方法驗證B-水晶體與肌間線蛋白的交互作用關係。此外，我們也設計出可專一性辨認被凋亡蛋白酶6處理的N端肌間線蛋白片段的抗體。當細胞表現疾病特有的肌間線蛋白突變時，透過這種抗體也可偵測到N端肌間線蛋白的產生。在體外環境中，N端肌間線蛋白無法自行產生十奈米寬的中間絲構造，反而形成高密度的蛋白堆積物。Co-assembly的研究也顯示N端肌間線蛋白會干擾正常肌間線蛋白的結合，促使其形成蛋白堆積物。綜合以上研究結果，可知藉由凋亡蛋白酶處理所產生的N端肌間線蛋白有傾向形成蛋白堆積物的能力，且建立了N端肌間線蛋白促進細胞凋亡能力的基礎，以及藉由影響粒線體功能以致危害細胞存活，這都是肌間線蛋白肌病變的重要病理生理學特徵。

關鍵字

肌間線蛋白 ； aB-水晶體 ； 粒線體 ； 細胞凋亡

並列摘要

Desmin is a 53 kDa muscle-specific intermediate filament protein, which forms extrasarcomeric cytoskeleton that interconnects myofibrils and links the contractile apparatus to mitochondria, nuclei and intercellular junctions. Mutations in desmin cause desmin-related myopathy (DRM), a rare and devastating genetic disorder affecting both skeletal and cardiac muscles. DRM is characterized by intrasarcolemmal aggregates of granulofilamentous material containing predominantly desmin. Since that initial discovery, mutations in the small heat shock protein B-crystallin was found to phenocopy DRM caused by desmin mutation, providing the first genetic evidence in support of a functional interaction of B-crystallin and desmin. In this study, we used a variety of biochemical assay to show that B-crystallin can interact with desmin. In addition, we generated polyclonal antibodies specific to the N-terminal fragment of desmin (N-desmin) generated by caspase 6 cleavage. Using this antibody, N-desmin was detected in cells undergoing apoptosis. In vitro assembly studies had demonstrated that the N-desmin was unable to form 10-nm intermediate filaments, but instead form electron-dense aggregates. Coassembly studies showed that N-desmin interfered with the normal assembly of intact desmin in a way that promoted filament aggregation. Taken together, these data suggest caspase-mediated cleavage of desmin produces N-terminal proteolytic fragment that is prone to aggregation. Furthermore, N-desmin and other disease-causing mutations interfere intermediate filaments networks and mitochondrial phenotypes in cells. This study provides a firm and testable foundation to the hypothesis that the N-terminal desmin is pro-apoptotic, affecting mitochondria function that compromises cell viability as key part of the pathophysiology of DRM.

並列關鍵字

Desmin ； aB-crystallin ； Mitochondria ； Apoptosis

參考文獻

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