透過與MyoD和NFATc1的結合研究探討NRIP在骨骼肌調控中的角色

Abstract

核受體相互作用蛋白(NRIP)，又稱IQWD1或DCAF6 (DDB1及CUL4相關因子6)，由860個胺基酸組成，目前已知其包含7個WD40 domain與1個IQ motif。我們先前的研究發現，在全身性NRIP基因剔除(NRIP-gKO)小鼠中，受傷後第六天Myogenin (MyoG) mRNA與蛋白質的表現量皆顯著低於野生型，說明MyoG為NRIP的下游調控目標。此外，實驗室進一步發現NRIP可與MyoD協同作用以共同活化MyoG啟動子(MyoG promoter)，並透過純化蛋白質的pull-down實驗以及共免疫沉澱(co-IP)實驗，分別在體外與C2C12肌母細胞中確認了NRIP與MyoD之間的交互作用，顯示NRIP可作為MyoD的轉錄共激活因子以促進MyoG表現。在我的研究中，我進一步確認了在 HEK293T 細胞中過度表現下，NRIP與MyoD之間的物理性互作，支持先前關於內源性NRIP與MyoD結合的結果。總結來說NRIP作為MyoD的轉錄輔助因子，以促進MyoG的表現。 另一方面，NRIP亦被發現參與於calcineurin/NFAT訊息傳導路徑中。其IQ motif能與calmodulin結合並進一步活化calcineurin，使NFATc1去磷酸化。在NRIP-gKO小鼠中，去磷酸化的NFATc1表現量下降，顯示NRIP能於細胞質層級調控NFATc1。Pull-down實驗顯示，NRIP可與來自C2C12細胞的NFATc1發生交互作用。鑒於MyoG啟動子上含有NFATc1的結合位點，本研究旨在探討NRIP是否透過與NFATc1的互作來調控 MyoG 的表現。 首先，為驗證NRIP與NFATc1之間的交互關係，我建構了EGFP-NFATc1質體，並與Flag-NRIP共轉染至HEK293T細胞中；同時也從小鼠肌肉組織中萃取蛋白質以進行co-IP實驗。無論是過表達還是內源性蛋白的實驗結果皆證實NRIP與NFATc1之間存在交互作用。 為進一步探討NRIP與NFATc1是否能協同活化MyoG啟動子，我們在HEK293T細胞中共轉染Flag-NRIP與EGFP-NFATc1並進行luciferase assay。實驗結果顯示，兩者在促進MyoG_P-luc2活性上具有協同效應。 接著，為釐清NRIP中哪一結構區域對此轉錄共激活功能至關重要，我們進行了luciferase assay，測試多個NRIP截短構建體與NFATc1共轉染對MyoG_P-luc2的影響。結果顯示，僅缺乏WD40區域的NRIP-ΔWD67無法與NFATc1共同活化MyoG表現，而其餘含WD40區域之構建體則仍具共激活能力。 我們也使用同一組NRIP突變構型進行了共免疫沉澱實驗，進一步鑑定與NFATc1結合所需的NRIP結構區域。結果與luciferase assay的實驗結果一致：所有能與NFATc1結合的構型（包括NRIP-FL、NRIP-ΔIQ、NRIP-N、NRIP-C和NRIP-WD67）皆可顯著增強MyoG啟動子的活性。相反地，NRIP-ΔWD67既無法與NFATc1結合，也無法活化啟動子，進一步支持WD40結構域介導的NFATc1結合對於這種協同轉錄活化作用至關重要的結論。此結果獲得AlphaFold 3結構預測的支持，該預測指出NFATc1與NRIP的WD40區域頂表面相互作用。 總結而言，我們確認NRIP為一種新型的NFATc1結合蛋白，並發現NRIP可作為NFATc1的轉錄共激活因子，兩者協同調控MyoG表現，提供了一個新的NRIP調控MyoG轉錄的機制。

Nuclear receptor interaction protein (NRIP), also known as IQWD1 or DCAF6 (DDB1 and CUL4 associated factor 6), is composed of 860 amino acids and contains seven WD40 domains and one IQ motif. Our previous research demonstrated that, in global NRIP knockout (NRIP-gKO) mice, both mRNA and protein levels of Myogenin (MyoG) were significantly reduced on day six post-injury compared to wild-type mice, indicating that MyoG is a downstream regulatory target of NRIP. Furthermore, we found that NRIP synergistically activates the MyoG promoter in cooperation with MyoD. The interaction between NRIP and MyoD was confirmed both in vitro via pull-down assays using purified proteins, and in C2C12 myoblasts via co-immunoprecipitation (co-IP), suggesting that NRIP acts as a transcriptional coactivator of MyoD to enhance MyoG expression. In my study, I further confirmed that physical interaction between NRIP and MyoD upon overexpression in HEK293T cells supporting the previous results of endogenous NRIP and MyoD binding. In sum, NRIP as a transcription cofactor for MyoD to activate MyoG expression. On the other hand, NRIP has also been implicated in the calcineurin/NFAT signaling pathway. Its IQ motif can bind calmodulin, thereby activating calcineurin and promoting dephosphorylation of NFATc1. In NRIP-gKO mice, the levels of dephosphorylated NFATc1 were reduced, indicating that NRIP regulates NFATc1 at the cytoplasmic level. The pull-down assays indicated that NRIP interacts with NFATc1 from C2C12 cells. Given that the MyoG promoter contains NFATc1 binding sites, this study aims to investigate whether NRIP regulates MyoG expression through its interaction with NFATc1. Firstly, the constructed an EGFP-NFATc1 plasmid was performed; and co-transfected it with Flag-NRIP into HEK293T cells; in parallel, we also performed co-IP experiments using proteins extracted from mouse skeletal muscle tissue. Both overexpression and endogenous co-IP results confirmed the interaction between NRIP and NFATc1. To further explore whether NRIP and NFATc1 cooperate to activate the MyoG promoter, we co-transfected Flag-NRIP and EGFP-NFATc1 into HEK293T cells and performed a luciferase assay. The results demonstrated a synergistic effect between the two in activating MyoG_P-luc2 activity. To clarify which structural region of NRIP is essential for this transcriptional co‑activating function, we performed luciferase reporter assays using a panel of NRIP truncation constructs co‑transfected with NFATc1, and assessed their effects on MyoG_P‑luc2 activity. The results showed that only NRIP‑ΔWD67—lacking all WD40 domains—failed to synergize with NFATc1 and activate MyoG expression. All other constructs retaining the WD40 region maintained co‑activating ability. Next, using the same set of NRIP variants, we carried out co IP assays to identify which NRIP domain mediates binding to NFATc1. The results were consistent with those from the luciferase assays: all constructs that bound NFATc1 (NRIP FL, NRIP ΔIQ, NRIP N, NRIP C, and NRIP WD67) significantly enhanced MyoG promoter activity. In contrast, NRIP ΔWD67 neither interacted with NFATc1 nor activated the promoter, reinforcing the conclusion that NFATc1 binding—mediated by the WD40 domain—is essential for the observed synergistic transcriptional activation. These findings are further supported by AlphaFold 3 structural predictions, which indicate that NFATc1 interacts with the top surface of NRIP’s WD40 region. In summary, we identify NRIP as a novel NFATc1-binding protein and demonstrate that NRIP functions as a transcriptional coactivator of NFATc1, synergistically regulating MyoG expression. These findings reveal a new mechanism by which NRIP regulates MyoG transcription.