摘要
我們實驗室在2005年發現了一個基因，命名為nuclear receptor interaction protein (縮寫：NRIP，又名DCAF6或IQWD1)。NRIP的蛋白質結構由七個WD-40 repeats以及一個IQ motif所組成。且實驗室先前的研究證明在有鈣離子的情況下，NRIP會利用IQ motif與調鈣素（Calmodulin）下進行交互作用。利用實驗室的NRIP基因剔除小鼠進行行為測試，其結果暗示NRIP可能參與心臟功能的調控。因此，我們利用心臟超音波檢測，針對NRIP基因剔除小鼠進行長期性的心臟功能監測。根據監測的結果，我們發現剔除NRIP基因確實會影響到小鼠的心臟功能，並且伴隨著年紀增長，心肌有趨於肥厚的現象（cardiac hypertrophy）。為了進一步驗證這項發現，我們將小鼠的心臟取出進行一連串的組織分析，得到的結果與心臟超音波的結果一致。在年紀大的基因剔除小鼠中，我們不但找到心肌肥厚的現象，還發現這些老鼠有心肌纖維化的情形發生。 所以，為了找出NRIP調節心臟功能的分子機制，我們進一步利用yeast two-hybrid進行大量篩選，發現到一群屬於α-actinin家族的蛋白質會與NRIP產生交互作用。α-actinin有四種異構型，其中特定表現在肌肉細胞的異構型ACTN2 是組成肌節上Z-disc的主要成分。Z-disc可以與肌動蛋白絲連結，是維持肌節構形以及穩定肌肉收縮的重要結構。因此，我們利用in vitro及in vivo binding assay進一步確認了NRIP與ACTN2的交互作用，之後又分析兩者間交互作用的區塊並且利用免疫螢光染色法，證明兩者在組織中共同存在於Z-disc上，也意外發現了NRIP是一個新的Z-disc protein。然而，許多論文指出Z-disc protein一旦有缺失便會造成肌節排列錯亂，影響心臟功能而導致心肌症（cardiomyopathy）。因此，我們利用穿透視電子顯微鏡來觀察NRIP基因剔除小鼠的肌節構造。結果顯示缺少NRIP的小鼠其肌節的結構受到影響，特別的是肌節的I-band變窄以及Z-disc變寬。另外，實驗室先前的研究證實NRIP會在有鈣離子的情況下與調鈣素(calmodulin)交互作用，且許多研究指出調鈣素在心肌細胞裡，可以與許多鈣離子通道或者與其他蛋白質交互作用，直接或間接的影響心肌細胞內鈣離子濃度的變化。因此，我們純化並檢測老鼠心肌細胞收縮時鈣離子的變化，發現缺少NRIP會影響心肌細胞收縮時的鈣離子變化量。根據目前的證據我們作出以下的推論：NRIP是一個能夠與ACTN2進行交互作用的Z-disc protein，且缺少NRIP會影響肌節的結構，以及心肌細胞收縮時鈣離的變化量，進而導致心臟收縮功能受損，而引發最終我們看到的心肌肥大的結果。
並列摘要
Previously, we demonstrated a novel gene, nuclear receptor interaction protein (NRIP, also named as DCAF6 or IQWD1), which could cooperate with nuclear receptors such as androgen and glucocorticoid receptors and its gene expression was regulated by androgen via androgen receptor. We also identified NRIP as a Ca2+- dependent calmodulin binding protein that activates calcineurin phosphatase activity. To investigate insights into in vivo function of NRIP, we generated NRIP-null mice and found that loss of NRIP impairs cardiac function and lead to cardiac hypertrophy progressively. Furthermore, NRIP-/- mice display weaker muscle strength, reduced cardiac function, and cardiac fibrosis at elder stage compared with WT. To verify the regulatory mechanism, we found that α-actinin-2 (ACTN2), which is a biomarker of muscular Z-disc complex is one of NRIP-interacting proteins from the yeast two-hybrid system. ACTN2 cross-links with actin filament to stabilize sarcomeric structure and muscle contraction, which is an essential constituent of sarcomere. Through the in vitro and in vivo binding assays, we further confirmed the interaction and defined the interacting domains between NRIP and ACTN2. Plus co-localization of NRIP and ACTN2 was discovered in cardiac tissue by immunofluorescence assays, we firstly defined NRIP as a Z-disc protein. Although the Z-disc has been viewed as a passive constituent of the sarcomere traditionally, increasing numbers of mutations in Z-disc proteins leading to disruption and malfunction of the contractile apparatus have been shown to cause cardiomyopathies and/or muscular dystrophies. Hence, we analyzed the sarcomeric structure of NRIP-/- cardiomyocytes and found reduction of I-band width and extension of Z-disc. Besides, we know that NRIP is a Ca2+- dependent calmodulin binding protein. In cardiomyocytes, calmodulin interacts with multiple calcium ion channels or proteins to directly or indirectly regulate the variation of calcium concentration during muscle contraction. Therefore, we isolated and measured the calcium transient of cardiomyocytes. Then, we found that deficiency of NRIP decreases the amplitude of calcium transient. In a conclusion, we speculated that loss of NRIP impairs the structure of sarcomere, the amplitude of calcium transient during muscle contraction and the function of muscle contraction resulting in cardiomyopathy.
參考文獻
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