人胰岛淀粉样多肽11-20片段聚集行为和肽抑制剂的研究

发布时间：2018-06-06 22:56

本文选题：胰岛淀粉样多肽 + 淀粉样纤维　；参考：《郑州大学》2017年博士论文

【摘要】：构象病是由于蛋白质错误折叠引起的一种疾病,其典型特征为具有正常功能的蛋白质构型发生非正常变化,引起错误折叠,导致蛋白质从正常形态逐渐变为富含大量β折叠结构的淀粉样斑块,沉积在组织器官中,从而使细胞功能衰退直至凋亡。2型糖尿病作为一种常见的构象病,由于它患病人数逐年增加,已经成为一种严重危害人类生活质量和身体健康的流行性疾病。胰岛淀粉样多肽的纤维化沉积被认为是2型糖尿病的主要病理特征之一。虽然口服降糖药以及直接注射胰岛素均可用于治疗2型糖尿病,但是此种干预性治疗只能初步改善和维持正常血糖值,在治疗后期,随着并发症逐渐发生,此种治疗方法具有一定局限性。胰岛淀粉样多肽聚集形成的淀粉样纤维能够破坏胰岛β细胞膜,导致β细胞逐渐凋亡,加剧了2型糖尿病的病症。抑制胰岛淀粉样多肽的聚集能够减缓2型糖尿病的病发,因此研发抑制胰岛淀粉样多肽聚集抑制剂为2型糖尿病的治疗提供了一种新方法。人胰岛淀粉样多肽(hIAPP)的不同片段具有不同性质,阻断hIAPP自身π-π堆积作用,可以有效减弱hIAPP成淀粉样纤维的能力。因此本论文针对人胰岛淀粉样多肽自聚集行为进行了研究并设计合成了多种多肽类抑制剂。首先,本研究以牛血清白蛋白(BSA)为模版蛋白,运用微量热泳动技术(MST)建立了一种能够研究蛋白聚集行为的方法,考察了人胰岛淀粉样多肽片段_(11-20)(hIAPP_(11-20))在不同溶液中聚集行为,最终以hIAPP_(11-20)为目标物,设计并合成了一系列具有不同构型的多肽,考察了它们对hIAPP_(11-20)淀粉样纤维形成的抑制效果,探讨了影响hIAPP_(11-20)淀粉样纤维形成的主要因素。具体研究内容如下:1.运用微量热泳动技术建立研究蛋白聚集行为的方法在药物研发过程中,探究蛋白与其目标分子的结合行为为药物分子设计提供了大量的基本信息。本实验以牛血清白蛋白(BSA)为模板蛋白,分别运用微量热泳动技术(MST)和普通荧光猝灭方法,考察了BSA与一种常见小分子异硫氰酸钠(FITC)之间的相互作用。MST实验表明,在短时间内,BSA与FITC发生了两种形式的相互作用。在MST竞争实验中,以华法林和布洛芬分别为BSA不同位点的特异性结合标记物,结果表明,FITC主要与BSA的位点II位结合,但也与位点I位有少量结合,同时也证明了FITC并不以标记的形式与BSA发生相互作用,而是与BSA聚集体发生结合,从而表现出2种结合方式。MST实验所得的结果与荧光实验结果相一致,进一步表明MST在研究生物分子间相互作用中的可靠性。因此,MST不仅可以为生物分子间相互作用提供大量有用的信息,也可以研究蛋白聚集行为,获得其它方法不能得到的信息。2.hIAPP_(11-20)在不同溶液中的聚集行为研究hIAPP_(11-20)能够在体外形成与全长类似的富含β折叠的淀粉样纤维,因此选择hIAPP_(11-20)作为探究淀粉样多肽聚集行为的目标物。本实验运用MST技术和传统的Th T荧光法,考察了hIAPP_(11-20)在不同溶液(Tris-HCl,HEPES,PBS和超纯水)中的淀粉样纤维的形成过程。实验结果表明,hIAPP_(11-20)在不同溶液中表现出不同的聚集行为,hIAPP_(11-20)不仅与自身发生相互作用,也可能与溶质分子发生结合,说明缓冲溶液不仅只是起到维持溶液环境稳定的作用,还有可能参与目标分子的相互作用。hIAPP_(11-20)与Tris的亲和力远大于hIAPP_(11-20)与其它溶质分子的亲和力,说明hIAPP_(11-20)在Tris-HCl缓冲溶液中自聚能力要远小于hIAPP_(11-20)在其它溶液中的自聚能力。在HEPES和超纯水中,hIAPP_(11-20)表现出相似的自聚集行为。PBS缓冲溶液中带负电的磷酸根离子可能会“中和”一部分hIAPP_(11-20)自身的正电,从而导致hIAPP_(11-20)在PBS中快速形成淀粉样纤维聚集。因此,在研究hIAPP_(11-20)淀粉样纤维化过程时,缓冲溶液的影响需考虑在内。同时,MST技术也为研究多肽或蛋白的早期聚集行为提供了一种简单有效的方法。3.新型阻断肽抑制hIAPP_(11-20)聚集效果的研究hIAPP错误折叠所产生的淀粉样纤维是2型糖尿病的一种重要病理特征。本实验设计并合成了17条具有不同长度,构型以及组成的多肽类抑制剂,以hIAPP_(11-20)为模板,考察了它们抑制hIAPP_(11-20)淀粉样纤维形成的效果。通过MST实验以及Th T荧光实验深入探究了这些多肽抑制hIAPP_(11-20)淀粉样纤维形成的机理,并用TEM表征了hIAPP_(11-20)最终形成淀粉样纤维的形貌。结果表明短肽AT、SA、RF、KS、KT和KN,以及环肽cyclic-KS、cyclic-KT和cyclic-KN能够有效抑制hIAPP_(11-20)淀粉样纤维的形成。其中,芳香型氨基酸的种类,多肽氨基酸的特定组成以及多肽的构型在抑制淀粉样纤维形成中起重要作用。MST实验量化了hIAPP_(11-20)与多肽之间的亲和力,表明具有较高亲和力的多肽在抑制聚集中表现出良好的抑制效果,该结果与Th T荧光实验以及分子对接结果相一致。因此,MST是一种简单快速的方法,可用于初步筛选聚集抑制剂。本研究也为以后设计多肽抑制剂提供了一些实验基础,同时也为探究其他蛋白构象病的抑制剂扩展了思路。4.脂肽抑制hIAPP_(11-20)聚集效果的研究根据有些脂肽能够保护细胞免受淀粉样纤维破环,而且脂肽与报道的抑制剂均含有较多的疏水基团等特点,本实验设计了一系列新型脂肽,以hIAPP_(11-20)为探究淀粉样多肽聚集的目标物,考察了这些脂肽抑制hIAPP_(11-20)淀粉样纤维形成的效果。我们通过MST、Th T荧光实验深入讨论了这些脂肽抑制聚集的机理,同时利用TEM表征了hIAPP_(11-20)最终形成淀粉样纤维的形貌及hIAPP_(11-20)与脂肽共同孵育后的hIAPP_(11-20)聚集形貌。结果表明,随着脂肽链长的逐渐增加,其抑制hIAPP_(11-20)聚集的效果逐渐增强。另外,含正电氨基酸的数量也在抑制hIAPP_(11-20)淀粉样纤维形成中起到一定作用。因此,本实验拓宽了hIAPP淀粉样纤维化抑制剂的研究范围。本论文通过运用MST建立了一种能够研究蛋白聚集行为的方法,并采用该方法联合使用其他技术考察了hIAPP_(11-20)在不同溶液中的自聚集行为,探讨了不同类型抑制剂的抑制hIAPP_(11-20)聚集的效果,总结出影响hIAPP_(11-20)淀粉样纤维化的因素,
[Abstract]:Conformational disease is a disease caused by the wrong folding of proteins, typically characterized by abnormal changes in the structure of proteins with normal functions, causing false folds, causing proteins to gradually change from normal form to amyloid plaques rich in beta folded structures and accumulate in tissues and organs so that cell function decline straight. As a common conformation disease, apoptotic type.2 diabetes has become a popular disease that seriously endangers the quality of human life and health. The fibrotic deposition of islet amyloid peptide is considered to be one of the main pathological features of type 2 diabetes. Insulin can be used in the treatment of type 2 diabetes, but this intervention therapy can only improve and maintain normal blood glucose. In the later period of treatment, with the gradual occurrence of complications, this method has some limitations. The amyloid Fibrinosis formed by amyloid polypeptide of islet amyloid peptide can destroy the islet beta cell membrane and lead to beta cells gradually. Apoptosis, exacerbates the disease of type 2 diabetes. Inhibition of the aggregation of islet amyloid peptide can slow the onset of type 2 diabetes. Therefore, the development of the inhibition of islet amyloid peptide aggregation inhibitors provides a new method for the treatment of type 2 diabetes. The different fragments of the human islet amyloid polypeptide (hIAPP) have different properties and block the hIAPP itself - pion. Pion accumulation can effectively weaken the ability of hIAPP to form amyloid fibers. Therefore, this paper studies the self aggregation behavior of human islet amyloid peptide and designs a variety of polypeptide inhibitors. First, a kind of MST was established by using BSA as a template and a micro thermo swimming technique (MST). In the method of protein aggregation behavior, the aggregation behavior of human islet amyloid polypeptide fragment (11-20) (11-20) (hIAPP_ (11-20)) was investigated in different solutions. A series of polypeptides with different configurations were designed and synthesized with hIAPP_ (11-20) as the target, and their inhibition effect on the formation of hIAPP_ (11-20) amyloid fibers was investigated and the influence of hI was discussed. The main factors for the formation of APP_ (11-20) amyloid fiber are as follows: 1. the method of using microcalorimetry to establish the behavior of protein aggregation in the process of drug development, the combination of protein and its target molecules provides a large amount of basic information for the design of drug molecules. This experiment is based on bovine serum albumin (BSA). For template protein, the interaction between BSA and a common sodium isothiocyanate (FITC) was investigated by microcalorimetry (MST) and common fluorescence quenching. The interaction between BSA and FITC in a short time showed that there were two forms of interaction between BSA and FITC. In the MST competition experiment, Hua Falin and Bloven were respectively BSA The specific binding markers of different loci show that FITC is mainly associated with the II site of the site of BSA, but also has a small combination with the site I position. It also shows that FITC does not interact with BSA in the form of labeling, but is a combination with BSA aggregates, thus showing the results obtained from the 2 binding modes.MST experiment and fluorescence reality. The results are consistent, which further indicates the reliability of MST in the study of intermolecular interaction. Therefore, MST can not only provide a large number of useful information for intermolecular interaction, but also study the aggregation behavior of protein, and obtain the aggregation behavior of.2.hIAPP_ (11-20) in different solutions (hI), which are not obtained by other methods. APP_ (11-20) can form amyloid like fiber rich in beta fold in vitro, so hIAPP_ (11-20) is selected as a target to explore the aggregation behavior of amyloid polypeptide. The experiment used MST technology and traditional Th T fluorescence method to investigate the starch like fiber of hIAPP_ (11-20) in different solutions (Tris-HCl, HEPES, PBS, and ultra pure water). The experimental results show that hIAPP_ (11-20) shows different aggregation behavior in different solutions. HIAPP_ (11-20) not only interact with itself, but also combine with solute molecules, indicating that the buffer solution not only plays a role in maintaining the stability of the solution, but may also be involved in the interaction of the target molecules. The affinity between.HIAPP_ (11-20) and Tris is far greater than the affinity of hIAPP_ (11-20) to other solute molecules, indicating that the self polymerization of hIAPP_ (11-20) in Tris-HCl buffer solution is much smaller than that of hIAPP_ (11-20) in other solutions. In HEPES and super pure water, hIAPP_ (11-20) shows a similar self aggregation behavior in.PBS buffer solution. Negatively charged phosphate ions may "neutralize" a portion of hIAPP_ (11-20) itself, leading to the rapid formation of amyloid fibrils in hIAPP_ (11-20) in PBS. Therefore, the impact of the buffer solution needs to be taken into account in the study of hIAPP_ (11-20) amyloid fibrosis. At the same time, MST technology is also the early study of the peptide or protein. Aggregation behavior provides a simple and effective method of inhibition of hIAPP_ (11-20) aggregation effect of.3. new blocking peptide. The amyloid fiber produced by hIAPP error folding is an important pathological feature of type 2 diabetes. This experiment designed and synthesized 17 polypeptide inhibitors with different length, structure and composition, with hIAPP_ (11-2). 0) the effect of their inhibition on the formation of hIAPP_ (11-20) amyloid fibers was investigated for the template. The mechanism of inhibiting the formation of hIAPP_ (11-20) amyloid fibers by these peptides was investigated by MST and Th T fluorescence. The morphology of hIAPP_ (11-20) in the final formation of starch like fibers was characterized by TEM. The results showed that the short peptide AT, SA, RF, KS, KT and minerals were used. And cyclic peptide cyclic-KS, cyclic-KT and cyclic-KN can effectively inhibit the formation of hIAPP_ (11-20) amyloid fibers. Among them, the species of aromatic amino acids, the specific composition of polypeptide amino acids and the configuration of peptides play an important role in inhibiting the formation of amyloid fibers..MST experiments have quantified the affinity between hIAPP_ (11-20) and polypeptide. Polypeptides with high affinity exhibit good inhibitory effects in inhibition of aggregation. This result is consistent with the results of Th T fluorescence experiments and molecular docking. Therefore, MST is a simple and rapid method for preliminary screening of aggregation inhibitors. This study also provides some experimental basis for the future design of polypeptides. In order to explore other inhibitors of protein conformation disease, a study on the inhibition of.4. lipopeptides to inhibit the aggregation of hIAPP_ (11-20) is based on some lipopeptides that protect cells from amyloid fibrils, and both lipopeptides and reported inhibitors contain many hydrophobic groups. A series of new lipopeptides were designed in this experiment, with hIAPP_ (11-20). In order to explore the target of amyloid polypeptide aggregation, the effects of these lipopeptides on the formation of hIAPP_ (11-20) amyloid fibers were investigated. The mechanism of inhibiting aggregation of these lipopeptides was discussed through MST and Th T fluorescence experiments. At the same time, the morphology of hIAPP_ (11-20) amyloid fibers was characterized by TEM and hIAPP_ (11-20) was shared with lipopeptides. The aggregation morphology of hIAPP_ (11-20) after incubation showed that with the gradual increase of the length of the lipoprotein chain, the effect of its inhibition of hIAPP_ (11-20) aggregation increased gradually. In addition, the number of positive amino acids also played a role in inhibiting the formation of hIAPP_ (11-20) amyloid fibers. Therefore, this experiment broadened the hIAPP amyloid fibrosis inhibitor. In this paper, a method to study the aggregation behavior of protein was established by using MST, and the self aggregation behavior of hIAPP_ (11-20) in different solutions was investigated by using the method combined with other techniques. The effect of different inhibitors on the inhibition of hIAPP_ (11-20) aggregation was discussed, and the effect of hIAPP_ (11-20) starch samples was summarized. Factors of fibrosis,
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ460.1

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