人胰岛淀粉样多肽与磷脂膜相互作用的研究
发布时间：2018-08-02 20:39
【摘要】：正常可溶性的蛋白质错误折叠形成高度有序的聚集体后可能引发一系列的疾病,II型糖尿病(T2DM)、阿尔兹海默症(AD)、帕金森病(PD)等神经退行性疾病都与这类蛋白质的错折叠相关。虽然这些蛋白质有各自的氨基酸序列和空间结构,但它们均可以由可溶性的蛋白质错折叠成不溶性的淀粉样沉积,由这类蛋白导致的淀粉样疾病很可能有相似的致病机理。其中,T2DM患者的显著特征是人胰岛淀粉样多肽(h IAPP)错折叠形成β-折叠结构的淀粉样纤维并沉积在胰岛β-细胞表面,导致β-细胞的死亡。h IAPP由37个氨基酸残基组成,和胰岛素共同由胰岛β-细胞分泌。在h IAPP对细胞产生毒性进而导致细胞死亡的过程中,其与膜的相互作用是关键的因素。因此了解h IAPP与磷脂膜是如何相互作用的,对抑制淀粉样蛋白的聚集和开发治疗此类疾病的药物都有重要的意义。由于细胞环境的复杂性,h IAPP与膜的结合受到许多环境因素的影响。此外,胆固醇是细胞膜中的重要成分,对淀粉样蛋白的聚集行为及毒性机理可能有重要的影响。本论文中,我们主要研究了h IAPP在磷脂膜上的聚集以及硫酸软骨素和血清白蛋白对h IAPP在磷脂膜上聚集的影响;我们还研究了胆固醇对h IAPP的N端区域与磷脂膜相互作用的影响。我们对h IAPP与磷脂膜相互作用的研究工作可以归纳为以下几点:1、我们用两性离子磷脂膜POPC、阴离子磷脂膜POPG以及它们以POPG:POPC为3:7的比例组成的混膜作为模型膜,通过Th T荧光实验、圆二色谱(CD)、原子力显微镜、31P-NMR等实验方法研究了硫酸软骨素(CSA)和牛血清白蛋白(BSA)对全长h IAPP在磷脂膜环境中聚集的影响。CSA和BSA属于细胞外基质的成分,它们的引入为h IAPP的聚集创造了一个多相异质环境。我们发现h IAPP在多相异质环境和在均相环境中的聚集行为是不一样的。在均相水环境中,CSA对h IAPP的聚集有促进作用,BSA则很好地抑制了h IAPP的聚集。而在POPC膜中,由于CSA和POPC的特异性结合,导致CSA对h IAPP聚集的促进作用有一个额外的增强效果。在含POPG的阴离子膜(POPG和POPG/POPC)中,BSA因与POPG磷脂成分的结合而减弱了对h IAPP的抑制作用。与单一环境相比,多相环境可能更接近h IAPP所处的真实环境,所以在含有磷脂膜和胞外基质成分的复杂体系中进行研究有可能为了解h IAPP的聚集机理提供更加有意义的信息。2、h IAPP的N端区域是其与膜结合的关键区域,研究发现,虽然h IAPP有很强的聚集倾向,但h IAPP1-19与全长的h IAPP不同,h IAPP1-19不管是在水溶液还是磷脂膜中都不会形成长纤维,有很弱的聚集倾向,但是有与全长h IAPP类似的细胞毒性。1-19肽段中包含的R11、F15和V17三个残基组成反向胆固醇识别氨基酸共有序列(CARC),可能参与肽-胆固醇的结合,其中第15位的苯丙氨酸是CARC序列的关键残基。因此,我们选取1-19肽段(h IAPP1-19)作为h IAPP与膜相互作用的结构模型,并将第15位的苯丙氨酸(Phe或F)突变成亮氨酸(Leu或L)得到h IAPP1-19/F15L,通过差示扫描量热法(DSC)、31P-NMR、1H-NMR、CD和染料泄漏实验等实验方法研究了h IAPP1-19与DPPC膜的相互作用以及CARC序列对h IAPP1-19与DPPC膜相互作用的影响。我们发现,胆固醇可以在DPPC膜上形成脂筏区,并促进h IAPP1-19与DPPC的结合,诱导多肽的二级结构由无规卷曲转变为α-螺旋,产生更多的毒性寡聚体,对膜造成更大的破坏,在这个过程中,F15参与了肽与胆固醇的结合。3、我们在第二部分工作的基础上用具有柔性链的DOPC磷脂膜代替刚性链的DPPC膜,同样以野生型肽h IAPP1-19和突变体肽h IAPP1-19/F15L作为研究对象,用31P-NMR、1H-NMR滴定实验、CD和染料泄漏实验等实验方法研究了h IAPP1-19与DOPC膜的相互作用以及在这个过程中胆固醇的影响。我们发现,胆固醇也能增强h IAPP1-19与具有柔性链的DOPC膜的结合,并增加磷脂膜的透性,对磷脂膜造成破坏。但与DPPC膜的结果相比,h IAPP1-19与DOPC膜的结合较弱,这可能归因于胆固醇没能在柔性的DOPC膜上产生脂筏区。
[Abstract]:Normal soluble protein folds to form highly ordered aggregates may lead to a series of diseases. Neurodegenerative diseases such as type II diabetes (T2DM), Alzheimer's disease (AD), and Parkinson's disease (PD) are all related to the misfolding of this protein. Although these proteins have their own amino acid sequence and spatial structure, they have their own amino acid sequence and space structure. All of them can be misfolded from soluble proteins to insoluble amyloidosis. The amyloid disease caused by this type of protein is likely to have similar pathogenicity. Among them, the T2DM patients are characterized by the misfolding of the H IAPP of human islet amyloid (IAPP) to form amyloid fibrils of beta folded structure and deposited on the islet beta cell surface, .h IAPP, which leads to the death of beta cells, is composed of 37 amino acid residues, and insulin is secreted by islet beta cells. In the process of H IAPP toxicity to cells and cell death, the interaction with the membrane is the key factor. Therefore, it is understood how the H IAPP and the phospholipid membrane interact and inhibit the accumulation of amyloid protein. The collection and development of drugs for the treatment of such diseases are of great significance. Due to the complexity of the cell environment, the combination of H IAPP with the membrane is affected by many environmental factors. In addition, cholesterol is an important component of the cell membrane and may have important effects on the aggregation behavior and toxic mechanism of amyloid. In this paper, we mainly study The effect of H IAPP aggregation on the phospholipid membrane and the effect of chondroitin sulfate and serum albumin on the aggregation of H IAPP on the phospholipid membrane. We also studied the effect of cholesterol on the interaction between the N end region of H IAPP and the phospholipid membrane. Our research on the interaction of H IAPP and phospholipid membrane can be summed up as follows: 1, we use sex separation. The subphospholipid membrane POPC, the anion phospholipid membrane POPG and their mixed membrane with the proportion of POPG:POPC as 3:7 were used as model membranes. The effects of CSA and bovine serum white egg white (BSA) on the accumulation of full h IAPP in the phospholipid membrane environment were investigated by Th T fluorescence experiments, circular two chromatography (CD), atomic force microscopy, 31P-NMR and other experimental methods. A and BSA are components of extracellular matrix, and their introduction of H IAPP creates a heterogeneous heterogeneous environment. We found that h IAPP has different aggregation behaviors in heterogeneous and homogeneous environments. In homogeneous water environment, CSA promotes the aggregation of H IAPP, while BSA inhibits h IAPP. In the POPC membrane, due to the specific binding of CSA and POPC, CSA has an additional enhancement effect on the promotion of H IAPP aggregation. In the anion membrane containing POPG (POPG and POPG/POPC), BSA is weakened by the binding of the POPG phospholipid component, and the multiphase environment may be more close to the single environment than in the single environment. The real environment, so the study in the complex system containing the phospholipid membrane and the extracellular matrix may provide more meaningful information for understanding the aggregation mechanism of H IAPP. The N terminal region of H IAPP is the key area of its binding with the membrane. It is found that although h IAPP has a strong tendency to gather, H IAPP1-19 and H IAPP. H IAPP1-19, in both water and phospholipid membranes, will not form a fiber and has a weak tendency to aggregation, but there are R11, F15 and V17 three residues, which are composed of R11, F15 and V17, which are similar to the full-length h IAPP, and may be involved in the binding of peptide cholesterol, of which first The 5 site phenylalanine is the key residue of the CARC sequence. Therefore, we select the 1-19 peptide segment (H IAPP1-19) as the structural model of the interaction between H IAPP and the membrane, and change the fifteenth bits of phenylalanine (Phe or F) into leucine (Leu or L) to obtain h IAPP1-19/F15L, and through differential scanning calorimetry (DSC) The interaction between H IAPP1-19 and DPPC membrane and the effect of CARC sequence on the interaction between H IAPP1-19 and DPPC membrane. We found that cholesterol can form lipid rafts on DPPC membrane, and promote the combination of H IAPP1-19 and DPPC, inducing the two structure of peptide to be transformed from random curling to alpha helix, producing more toxic oligomers, In this process, F15 participates in the binding of.3 to the peptide and cholesterol. On the basis of the second part, we use the DOPC phospholipid membrane with a flexible chain to replace the rigid chain DPPC membrane, and the wild peptide h IAPP1-19 and the mutant peptide h IAPP1-19/F15L as the research object, 31P-NMR, 1H-NMR titrated experiments, CD. The interaction between H IAPP1-19 and DOPC membrane and the effect of cholesterol in this process are studied by the experimental method of dye leakage test. We found that cholesterol can also enhance the binding of H IAPP1-19 to the DOPC membrane with a flexible chain, increase the permeability of the phospholipid membrane and destroy the phospholipid membrane. But compared with the results of the DPPC membrane, H IAPP1-19 The binding to DOPC membrane is rather weak, which may be attributed to the failure of cholesterol to produce lipid rafts on flexible DOPC films.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R363
本文编号：2160666
[Abstract]:Normal soluble protein folds to form highly ordered aggregates may lead to a series of diseases. Neurodegenerative diseases such as type II diabetes (T2DM), Alzheimer's disease (AD), and Parkinson's disease (PD) are all related to the misfolding of this protein. Although these proteins have their own amino acid sequence and spatial structure, they have their own amino acid sequence and space structure. All of them can be misfolded from soluble proteins to insoluble amyloidosis. The amyloid disease caused by this type of protein is likely to have similar pathogenicity. Among them, the T2DM patients are characterized by the misfolding of the H IAPP of human islet amyloid (IAPP) to form amyloid fibrils of beta folded structure and deposited on the islet beta cell surface, .h IAPP, which leads to the death of beta cells, is composed of 37 amino acid residues, and insulin is secreted by islet beta cells. In the process of H IAPP toxicity to cells and cell death, the interaction with the membrane is the key factor. Therefore, it is understood how the H IAPP and the phospholipid membrane interact and inhibit the accumulation of amyloid protein. The collection and development of drugs for the treatment of such diseases are of great significance. Due to the complexity of the cell environment, the combination of H IAPP with the membrane is affected by many environmental factors. In addition, cholesterol is an important component of the cell membrane and may have important effects on the aggregation behavior and toxic mechanism of amyloid. In this paper, we mainly study The effect of H IAPP aggregation on the phospholipid membrane and the effect of chondroitin sulfate and serum albumin on the aggregation of H IAPP on the phospholipid membrane. We also studied the effect of cholesterol on the interaction between the N end region of H IAPP and the phospholipid membrane. Our research on the interaction of H IAPP and phospholipid membrane can be summed up as follows: 1, we use sex separation. The subphospholipid membrane POPC, the anion phospholipid membrane POPG and their mixed membrane with the proportion of POPG:POPC as 3:7 were used as model membranes. The effects of CSA and bovine serum white egg white (BSA) on the accumulation of full h IAPP in the phospholipid membrane environment were investigated by Th T fluorescence experiments, circular two chromatography (CD), atomic force microscopy, 31P-NMR and other experimental methods. A and BSA are components of extracellular matrix, and their introduction of H IAPP creates a heterogeneous heterogeneous environment. We found that h IAPP has different aggregation behaviors in heterogeneous and homogeneous environments. In homogeneous water environment, CSA promotes the aggregation of H IAPP, while BSA inhibits h IAPP. In the POPC membrane, due to the specific binding of CSA and POPC, CSA has an additional enhancement effect on the promotion of H IAPP aggregation. In the anion membrane containing POPG (POPG and POPG/POPC), BSA is weakened by the binding of the POPG phospholipid component, and the multiphase environment may be more close to the single environment than in the single environment. The real environment, so the study in the complex system containing the phospholipid membrane and the extracellular matrix may provide more meaningful information for understanding the aggregation mechanism of H IAPP. The N terminal region of H IAPP is the key area of its binding with the membrane. It is found that although h IAPP has a strong tendency to gather, H IAPP1-19 and H IAPP. H IAPP1-19, in both water and phospholipid membranes, will not form a fiber and has a weak tendency to aggregation, but there are R11, F15 and V17 three residues, which are composed of R11, F15 and V17, which are similar to the full-length h IAPP, and may be involved in the binding of peptide cholesterol, of which first The 5 site phenylalanine is the key residue of the CARC sequence. Therefore, we select the 1-19 peptide segment (H IAPP1-19) as the structural model of the interaction between H IAPP and the membrane, and change the fifteenth bits of phenylalanine (Phe or F) into leucine (Leu or L) to obtain h IAPP1-19/F15L, and through differential scanning calorimetry (DSC) The interaction between H IAPP1-19 and DPPC membrane and the effect of CARC sequence on the interaction between H IAPP1-19 and DPPC membrane. We found that cholesterol can form lipid rafts on DPPC membrane, and promote the combination of H IAPP1-19 and DPPC, inducing the two structure of peptide to be transformed from random curling to alpha helix, producing more toxic oligomers, In this process, F15 participates in the binding of.3 to the peptide and cholesterol. On the basis of the second part, we use the DOPC phospholipid membrane with a flexible chain to replace the rigid chain DPPC membrane, and the wild peptide h IAPP1-19 and the mutant peptide h IAPP1-19/F15L as the research object, 31P-NMR, 1H-NMR titrated experiments, CD. The interaction between H IAPP1-19 and DOPC membrane and the effect of cholesterol in this process are studied by the experimental method of dye leakage test. We found that cholesterol can also enhance the binding of H IAPP1-19 to the DOPC membrane with a flexible chain, increase the permeability of the phospholipid membrane and destroy the phospholipid membrane. But compared with the results of the DPPC membrane, H IAPP1-19 The binding to DOPC membrane is rather weak, which may be attributed to the failure of cholesterol to produce lipid rafts on flexible DOPC films.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R363
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