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標題: | NRIP的結構及功能分析 Structural and functional analysis of NRIP |
作者: | Wen-Bin Ke 柯文斌 |
指導教授: | 陳小梨(Show-Li Chen) |
關鍵字: | 核受體交互作用蛋白,IQ domain,顯性抑制突變,鈣調蛋白依賴性蛋白質激?第二型,DxR motif, NRIP,IQ domain,calmodulin,dominant-negative mutant,calcium/calmodulin-dependent protein kinase type II,DxR motif, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 核受體交互作用蛋白 (Nuclear receptor interaction protein, NRIP),又被稱為DCAF6或IQWD1,是由七個WD40 repeat和一個IQ domain組成的。之前我們發現它可以活化AR標定基因並穩固AR。最近我們發現它在鈣離子存在的情況下,可以透過IQ domain和攜鈣素 (calmodulin)結合,然後活化鈣調神經磷酸酶-活化T細胞核因子 (CaN-NFATc1)和鈣調蛋白依賴性蛋白質激酶第二型 (CaMKII)訊息途徑來調控肌發生 (myogenesis)、鈣離子動態平衡及肌肉再生。此外,我們發現NRIP在前列腺癌上扮演著致癌蛋白的角色。
最近,它被其他研究團隊發現在同一位肢帶型肌肉失養症病患的肌萎縮組織中,其mRNA的表現量和其他組織的相比有較低的表現量。另外有報導指出在患有心肌症的病患中,和NRIP一樣同為DCAF家族蛋白一員的DCAF8的高度保留在WD40 repeat的DxR motif有突變發生,而我們也發現缺乏NRIP會造成心室肥大的現象。此外,另有報導指出在患有腦視力障礙的病患中,也有發現變異發生在DCAF6。因此,我們想探討NRIP的functional domain的結構和功能在臨床病症中扮演著何種角色。基於上述所提的結構domain (或motif),例如IQ domain、DxR motif、及第670號胺基酸;因此,我想藉由NRIP在這幾個突變位置來探討這幾個位置的結構和功能的關聯性。 首先,我們探討IQ domain在肌發生及肌管形成所扮演的角色,我們用帶有在IQ domain上四個高度保留蛋白被突變的NRIP的Ad-NRIP4A來感染骨骼肌成肌細胞C2C12,並讓其分化。我們發現其有較少的肌管形成伴隨著較低的分化、融合及成肌素(myogenin)的表現量。這代表在IQ domain上的突變會阻斷肌生成及肌管形成,代表著顯性抑制的效用。由於NRIP會和攜鈣素直接作用造成肌生成的發生,為了想證實在IQ domain上突變是否會造成NRIP和攜鈣素結合的顯性抑制的效用,我們用不帶有IQ domain的NRIP的Ad-NRIPΔIQ來感染C2C12細胞,並讓其分化。我們發現NRIP和攜鈣素結合會隨著逐漸增加NRIPΔIQ的量而有比例上下降的趨勢。 我們已有NRIP在IQ domain和DxR motif上突變的突變體,因此我們想藉由定點突變來建構在患有腦視力障礙的病患中發現在DCAF6上的變異體(c.2240G>A)到NRIP cDNA上。因此我們有了3個NRIP的突變體,例如NRIP 670突變 (R670Q)、NRIPΔIQ、和NRIP-DM。另外,我們也想探討心臟疾病患者的NRIP的functional domain的基因是否會有突變發生。然而,可能是由於我們心肌症患者的檢體太少的緣故,我們並沒有發現任何突變。 Nuclear receptor interaction protein (NRIP), also known as DCAF6 or IQWD1, is composed of seven WD40 repeats and one IQ domain. Previously, we found it can activate AR-targeted genes and stabilize AR. Recently, we found that it can bind to calmodulin (CaM) through its IQ domain in the presence of calcium, and then activate calcineurin-nuclear factor of activated T-cells, cytoplasmic 1 (CaN-NFATc1) and calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling pathways to regulate myogenesis, calcium homeostasis, and muscle regeneration. Furthermore, NRIP is also found as an oncoprotein in prostate cancer recently. NRIP related clinical significance; it was discovered by other group that lower expression of IQWD1 (NRIP) mRNA in dystrophy muscle tissue compared to other tissue in patients with limb-girdle muscular dystrophy (LGMD). Another report demonstrated that mutation on DxR motif, conserved within WD40 repeat, of DCAF8, like NRIP as one of DCAF family proteins, was identified in patients with cardiomyopathy, while we also found that NRIP deficiency would cause cardiac hypertrophy in NRIP-knockout mice. In addition, there was a report illustrating a variant on DCAF6 in a patient with cerebral visual impairment. Based on above-mentioned structural domain (or motif) such as IQ domain, DxR motif, and 670th amino acid; therefore, I would examine the structural and functional relationship based on these three mutation sites of NRIP. Firstly to examine role of IQ domain in myogenesis and myotube formation, we infected Ad-NRIP4A, which carried NRIP with four conserved amino acid mutated on IQ domain, to skeletal myoblast, C2C12 cells, then we found reduced myotube formation and less differentiation and fusion with decreased myogenin expression during differentiation. It implied that mutation on IQ domain may disrupt myogenesis and myotube formation that seems dominant-negative effect. Due to NRIP directly interacting with CaM resulting in myogenesis; to confirm whether mutation on IQ domain caused a dominant-negative effect for disrupting NRIP/CaM binding, we infected Ad-NRIPΔIQ (equivalent Ad-NRIP4A), which carried NRIP with deletion of IQ domain, to C2C12 cells. The results showed that the level of declination of NRIP/CaM binding was proportional to increasing NRIPΔIQ dose. We already had truncated IQ domain and DxR mutant of NRIP, hence here we constructed variant identified on DCAF6 (c.2240G>A) related cerebral visual impairment with site-directed mutagenesis. We then had these three mutants, such as NRIP 670 mutant (R670Q), NRIPΔIQ, and NRIP-DM. On the other hand, we investigated whether there was mutation on functional domains of genomic NRIP in heart clinical patients. However, we didn’t find any mutation sequences in heart clinical patients, which might result from low sample size of cardiomyopathy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67610 |
DOI: | 10.6342/NTU201702197 |
全文授權: | 有償授權 |
顯示於系所單位: | 微生物學科所 |
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