| 摘要註: |
本研究利用分子動態模擬探討Keap1-Nrf2之間及Nedd4-ENaC之間的作用模式。Keap1藉由與Nrf2結合以抑制生物體內抗氧化機制，在氧化壓力下，Keap1被刺激進而改變構型而失去與Nrf2之結合能力，致使Nrf2進入細胞核啟動抗氧化基因以保護細胞；當氧化還原恢復穩定，細胞質的ProTα因帶有核定位序列，可幫助Keap1進入細胞核中，將Nrf2帶至細胞質降解，關閉抗氧化基因。在此進行兩個子題:一、 比較Keap1與Nrf2ETGE、Nrf2DLG及ProTαENGE結合模式以驗證Keap1、Nrf2和ProTα三者之間的調控機制。由模擬結果得知Keap1與三組peptide均以靜電交互作用的方式結合，Keap1與Nrf2ETGE、ProTαENGE結合模式非常相似，然而與Nrf2DLG結合時的親和力較低，且結合模式也不同，其主要原因為帶電胺基酸的分布方式不同。二、探討Keap1上的七個單點突變所造成對於辨認Nrf2ETGE之影響，推論G364C及G430C單點突變造成Keap1的Nrf2ETGE結合位附近的帶負電之胺基酸活動度大增，進而屏蔽Keap1和Nrf2ETGE之間的遠距離靜電作用力；相較之下，S363A、N387A、 S508A、S555A和S602A雖在Nrf2ETGE結合位附近卻因為保有與Keap1WT相仿的靜電表面，故不影響Keap1辨識Nrf2ETGE。Nedd4之WW domain可藉由辨認ENaC之βP2片段所攜帶的PPxY motif，再通過胞吞作用降解而抑制ENaC的活性，ENaC突變會致使Nedd4無法辨識而造成鈉離子過度累積進而引發高血壓相關疾病。在此進行兩個子題：一、探討何以Nedd4具有序列相似度極高WW I~III domain與ENaC之攜有PPxY motif 的αP2、βP2及γP2 peptide在兩兩結合時親和性差異甚大。由模擬結果知形成WW-P2複合物的關鍵在於由WW domain上的XP groove提供親合力並以Tyrosine口袋輔助，及P2 peptide之C端胺基酸與WW domain表面靜電的交互作用。二、前人研究指出經磷酸化修飾的βP2與WW domain有較高的親和力，但原因卻未被探究。因為磷酸化後βP2 peptide同時攜有PPxY及phospho-TP兩種可被WW domain辨識的motif，在此建構分子模型、分析ΔG值數據及探討結合方向性來闡述親和力提升與WW III domain依然辨識PPxY motif的關聯性。 Molecular dynamics simulations are applied to investigate the protein-protein interaction of two systems including Keap1-Nrf2 and Nedd4-ENaC. Keap1 is a BTB-Kelch substrate adaptor protein that regulates steady-state levels of Nrf2, a bZIP transcription factor, in response to oxidative stress. In basal condition, Nrf2 is negatively regulated through ubiquitination by Keap1. However, upon exposure to oxidative stress, ubiquitination of Nrf2 is inhibited, resulting increased steady-state level of Nrf2 in nucleus and increased transcription of cytoprotective genes. As the oxidative stress ceases, ProTα mediated nuclear import of Keap1 for ubiquitination and degradation of Nrf2 regulates nuclear level of Nrf2. Both ProTα (with an ENGE motif) and Nrf2 (with one ETGE and one DLG motif in different degrees of affinity) bind to the same pocket of Keap1. Two issues are studied in Keap1-Nrf2 system. (i) Binding free energy ΔG calculation based on MD simulation is applied to shed light on the interplay among ProTα, Nrf2, and Keap1. Our ΔG result shows the binding affinities are in the order of Keap1-Nrf2ETGE > Keap1-ProTαENGE > Keap1-Nrf2DLG which suggest that Keap1-ProTαENGE is replaced by Keap1-Nrf2ETGE in the first place and subsequently the association of Keap1-Nrf2DLG fulfills the competition over ProTα, where the Nrf2ETGE and Nrf2DLG work as hinge and latch, respectively, in Keap1-Nrf2 complex formation. (ii) Point mutations such as G364C and G430C on Keap1 greatly impair Keap1-Nrf2ETGE interaction whereas S363A, N387A, S508A, S555A, and S602A do not affect, regardless that G364 and G430 are not near by the Nrf2ETGE binding site whereas the rest five residues are involved to the Nrf2ETGE binding. Principal component analysis (PCA) on the collected MD trajectories of the above seven mutated systems is utilized to provide dynamic observation. It is found that G364C and G430C cause the negatively charged amino acid residues surrounding the Nrf2ETGE binding site more mobile and consequently hamper the long-range electrostatic attraction R380-E82’ and R483-E79’ between Keap1 and Nrf2ETGE. In contrast, the other five mutated systems maintain an electrostatic surface almost the same as that of Keap1WT.The protein-protein interaction between Nedd4 and ENaC plays a key role in regulating salt and fluid homeostasis. Reduced Nedd4-ENaC affinity caused by mutation of ENaC leads to Liddle syndrome, a hereditary form of hypertension. Nedd4 contains three WW domains and ENaC has three distinct PPxY motif-containing subunits recognizable by the Nedd4 WW domains. Two issues are addressed in this system. (i) In spite of the sequence similarity among the WWI, WWII and WWIII domains, earlier experimental observation indicated that the αP2, βP2, and γP2 peptides derived from ENaC and carrying PPAY, PPNY, and PPRY, respectively, show various affinities when forming WW-P2 complexes. In this part, we compare two series of WW-P2 complexes: one series includes WWI-βP2, WWII-βP2, and WWIII-βP2, and the other series includes WWIII-αP2, WWIII-βP2, and WWIII-γP2. Our simulation data suggests WW domain’s XP groove and a Tyrosine pocket play essential roles in PPxY motif recognition by the corresponding WW domains. (ii) Previous experimental finding shows phosphorylation on βP2’s T613’ enhances the binding affinity toward the Nedd4 WW domains. Interestingly, the phosphorylated βP2 carries two WW binding motif preferences, PPxY and phospho-TP, which adopt opposite orientations when associating with a WW domain. In this part, we apply MD simulation and ΔG calculations to compare WWIII-βP2 (in negative orientation), WWIII-phosphorylated βP2 (in negative orientation), and WWIII-phosphorylated βP2 (in positive orientation) and conclude that the increase in binding affinity is attributed by the electrostatic interaction from the phosphorous group on the footing of the PPxY motif, instead of phospho-TP motif, recognition. |