研究ACTN2和calmodulin結合NRIP競爭關係

Abstract

核受體結合蛋白(Nuclear receptor interaction protein, NRIP)又稱為DDB1 and CUL4 associated factor 6 (DCAF6)以及IQ motif and WD repeats 1 (IQWD1)，其結構由七個WD-40 repeats和一個IQ motif所組成。NRIP當作雄激素受體(androgen receptor, AR)，糖皮質激素受體(glucocorticoid receptor, GR)和人類乳突病毒(human papillomavirus ,HPV) E2的共同激活劑，來增加它們的轉錄活性，或是預防AR和HPV E2被蛋白質降解。以前的研究已經證明，NRIP的IQ motif在鈣離子(Ca2+)存在下與鈣調蛋白(CaM)相連，以激活調節肌肉功能和運動性能的鈣調神經磷酸酶(CaN)和Ca2+/鈣調蛋白依賴性蛋白激酶II (CaMKII)。 除了NRIP在骨骼肌功能的角色下，我們已發表的結果證明NRIP肌肉特異性剔除鼠的心髒顯示收縮能力減少和心臟有肥大的現象。此外，NRIP和心臟特異性肌動蛋白異構體α-actinin-2 (ACTN2)之間的相互作用也在體外和體內測定。NRIP IQ motif和CaM結合，NRIP IQ motif與CaM和α-actinin-2 (ACTN2)相互作用。根據Michael等人的研究，含有IQ motif 的N-甲基-D-天冬氨酸受體 (N-methyl-D-aspartate receptor, NMDAR)同時與CaM和α-輔肌動蛋白結合 (α-actinin)，並且透過NMDAR的IQ motif，然而在Ca2+依賴條件下，CaM使NMDAR和ACTN2的連結分開，造成CaMKII和NMDAR-CaM綜合體結合，用於激酶信號傳導。目前已經鑑定出可以與CaM和ACTN2相互作用的蛋白質，例如NMDAR、網格蛋白重鏈(clathrin heavy chain, CHC)、內皮一氧化氮合酶（endothelial nitric oxide synthase, eNOS）、L-type鈣離子通道(L-type calcium channel, LTCC)和腺苷2A受體(adenosine 2A receptor, A2AR)。其中一些研究發現，蛋白質與ACTN和CaM的結合親和力在不同的Ca2+存在下改變而導致不同的生物性結果。因此，我們假設CaM和ACTN2可以相互競爭結合NRIP IQ motif，並在與NRIP結合的同時發揮不同的生物功能。我們首先將His-MBP-NRIP，GST-ACTN2和CaM蛋白進行組合，並進行體外His-tag pull down assay。除了體外實驗，我們也將NRIP-GFP，ACTN2-V5和CaM-GFP表現在293T細胞中，並進行NRIP的體內免疫沈澱實驗。我們發現，在Ca2+存在下，NRIP與ACTN2和CaM的結合無法達到一致性的結果，因此，根據歷年來的研究，我們進行這兩項實驗的檢討。

Nuclear receptor interaction protein (NRIP) also named DDB1 and CUL4 associated factor 6 (DCAF6) and IQ motif and WD repeats 1 (IQWD1), is composed of seven WD-40 domains and an IQ motif. NRIP acts as a coactivator to enhance the transcriptional activity of androgen receptor (AR), glucocorticoid receptor (GR) and human papillomavirus (HPV) E2 or prevent AR and HPV E2 from proteasomal degradation. The physiological role of NRIP also determined by using of NRIP global and muscle-specific knockout mice. Previous studies have demonstrated that IQ motif of NRIP associates with calmodulin (CaM) in the presence of calcium (Ca2+) to activate calcineurin (CaN) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) that regulates muscle functions and motor performance. Except NRIP role in skeletal muscle function, our unpublished results demonstrated that NRIP muscle-specific conditional knockout (cKO) mice revealed decrease of heart contraction and performed dilated cardiac hypertrophy. Furthermore, the interaction between NRIP and α-actinin-2 (ACTN2), a heart specific α-actinin isoforms, was also determined in vitro and in vivo. NRIP interacts either ACTN2 or CaM through its IQ motif. According to the study of Michael et al., N-methyl-D-aspartate receptor (NMDAR) containing IQ domain can bind to CaM and ACTN2 in the presence of Ca2+ resulting in recruiting CaMKII to NMDAR-CaM complex for kinase signaling. Like NMDAR, clathrin heavy chain (CHC), endothelial nitric oxide synthase (eNOS), L-type calcium channel (LTCC) and adenosine 2A receptor (A2AR), can interact both with CaM and α-actinin (ACTN). Some of these studies found the binding affinity of proteins with CaM and ACTN would be changed in the presence of Ca2+ that influences different biological consequences. Hence, we hypothesized that CaM and ACTN2 could compete for each other for binding with NRIP through NRIP IQ domain and exerted different biological functions. We firstly combined the His-MBP-NRIP, GST-ACTN2 and CaM proteins and performed in vitro His-tag pull down assay. In addition to in vitro experiments, we also expressed NRIP-GFP, ACTN2-V5 and CaM in 293T cells and performed immunoprecipitation experiments in vivo. We found that the combination of NRIP with ACTN2 and CaM did not achieve consistent results in the presence of Ca2+. Therefore we follow researches over the years and review our experiments.