發(fā)布時間：2018-08-27 15:26

【摘要】：胱抑素的淀粉樣突變體在人體內(nèi)發(fā)生淀粉樣聚集后，會引起腦淀粉樣血管病和阿爾茨海默病。其聚集的分子機制與胱抑素單體通過結(jié)構(gòu)域交換形成穩(wěn)定的二聚體密切相關(guān)。但是，迄今為止，有關(guān)胱抑素淀粉樣突變體的單體如何通過構(gòu)象轉(zhuǎn)換形成二聚體的機制一直缺乏足夠的實驗論證。 本研究基于前期的生化和細胞水平的研究成果，利用分子動力學模擬的方法，研究點突變對胱抑素結(jié)構(gòu)及淀粉樣聚集特性的影響。對突變體I66Q的模擬結(jié)果表明，與野生型胱抑素相比，其構(gòu)象發(fā)生顯著變化，穩(wěn)定性明顯降低。其中AS、loop區(qū)域以及α-β界面的變化最明顯，其結(jié)構(gòu)更加靈活。這些變化使得胱抑素整體結(jié)構(gòu)變得松散，AS區(qū)域與β區(qū)域的張口變大，處于伸展狀態(tài)，作用力減弱，因此更易誘導參與結(jié)構(gòu)域交換的兩個亞結(jié)構(gòu)域分開。對I66Q/I108T的模擬結(jié)果表明，I108T對I66Q起著促進作用，其中AS、loop、helix1變化更加明顯，結(jié)構(gòu)中疏水核心面積增大，整體結(jié)構(gòu)疏散，AS區(qū)域的helix2基本全部解螺旋為coil結(jié)構(gòu)，AS區(qū)域和β區(qū)域的張口幾乎全部打開，相互作用力嚴重減弱，極易發(fā)生結(jié)構(gòu)域交換。隨后的研究發(fā)現(xiàn)，Val55位點突變V55D、V55N對I66Q突變體都有一定的穩(wěn)定作用，但V55N突變體的穩(wěn)定作用更為顯著。這種穩(wěn)定作用的分子機制主要表現(xiàn)在V55D、V55N點突變增強了cC的55位殘基的穩(wěn)定性，從而進一步穩(wěn)定了Loop1、β2-β3以及α2-helix。而55位殘基穩(wěn)定性的不同是由不同突變體形成的氫鍵數(shù)量來決定的，其中55位殘基與Gln53形成的氫鍵可能發(fā)揮著最重要的作用。另外我們也發(fā)現(xiàn)，V55D、V55N兩種突變體對I66Q疏水核心都有顯著的穩(wěn)定作用，并伴隨著疏水核心氫鍵的輕微增加，進而增強了I66Q單體的穩(wěn)定性。 上述研究結(jié)果不僅有助于在理論上解釋點突變影響cC單體穩(wěn)定性和分子間聚集的分子機制，，對于深入研究胱抑素結(jié)構(gòu)域交換的分子機制也具有重要的促進意義。另外，本研究中所采用的研究方法，為其它蛋白質(zhì)錯誤折疊引起的蛋白質(zhì)淀粉樣聚集疾病的分子機制研究提供了一個新的思路。
[Abstract]:Amyloid mutants of cystatin cause cerebral amyloid angiopathy and Alzheimer's disease after amyloid aggregation occurs in the human body. The molecular mechanism of its aggregation is closely related to the exchange of cystatin monomers through domain to form stable dimers. However, the mechanism of conformational conversion of cystatin amyloid mutant monomers to dimer has not been sufficiently demonstrated. In this study, the effects of point mutation on cystatin structure and amyloid aggregation were studied by molecular dynamics simulation based on previous biochemical and cellular studies. The simulation results of the mutant I66Q showed that the conformation and stability of the mutant I66Q were significantly different from those of wild type cystatin. The change of AS,loop region and 偽-尾 interface is the most obvious, and its structure is more flexible. These changes make the whole structure of cystatin become loose, the opening of as region and 尾 region becomes larger, and the interaction force is weakened, so it is easier to induce the separation of two subdomains involved in domain exchange. The simulation results of I66Q/I108T show that I108T promotes I66Q, and the change of AS,loop,helix1 is more obvious, and the hydrophobic core area in the structure increases. The helix2 unspirals in the as region of the whole structure are almost completely opened in the as and 尾 region of the coil structure, and the interaction forces are weakened seriously, so the domain exchange is easy to occur. Subsequently, it was found that the V55D- V55N mutation had a certain stabilizing effect on the I66Q mutants, but the V55N mutants had a more significant stabilizing effect on the I66Q mutants. The molecular mechanism of this stabilization is that the point mutation of V55DV 55N enhances the stability of 55 residues of cC, thus further stabilizing Loop1, 尾 2- 尾 3 and 偽 2-helix. The stability of 55 residues is determined by the number of hydrogen bonds formed by different mutants, and the hydrogen bonds formed between 55 residues and Gln53 may play the most important role. These results not only help to explain the molecular mechanism of point mutation affecting the stability and intermolecular aggregation of cC monomers, but also contribute to the further study of the molecular mechanism of cystatin domain exchange. In addition, the methods used in this study provide a new idea for the study of molecular mechanism of protein amyloid aggregation disease caused by other protein misfolding.
【學位授予單位】：遼寧大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R341

【參考文獻】

相關(guān)期刊論文 前1條

1 宋有濤;張慧麗;宮雅楠;;淀粉樣蛋白沉積疾病在細胞水平的研究進展[J];細胞生物學雜志;2008年05期

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