人胰島淀粉樣多肽與磷脂膜相互作用的研究
發(fā)布時間：2018-08-02 20:39
【摘要】：正�？扇苄缘牡鞍踪|(zhì)錯誤折疊形成高度有序的聚集體后可能引發(fā)一系列的疾病,II型糖尿病(T2DM)、阿爾茲海默癥(AD)、帕金森病(PD)等神經(jīng)退行性疾病都與這類蛋白質(zhì)的錯折疊相關(guān)。雖然這些蛋白質(zhì)有各自的氨基酸序列和空間結(jié)構(gòu),但它們均可以由可溶性的蛋白質(zhì)錯折疊成不溶性的淀粉樣沉積,由這類蛋白導(dǎo)致的淀粉樣疾病很可能有相似的致病機(jī)理。其中,T2DM患者的顯著特征是人胰島淀粉樣多肽(h IAPP)錯折疊形成β-折疊結(jié)構(gòu)的淀粉樣纖維并沉積在胰島β-細(xì)胞表面,導(dǎo)致β-細(xì)胞的死亡。h IAPP由37個氨基酸殘基組成,和胰島素共同由胰島β-細(xì)胞分泌。在h IAPP對細(xì)胞產(chǎn)生毒性進(jìn)而導(dǎo)致細(xì)胞死亡的過程中,其與膜的相互作用是關(guān)鍵的因素。因此了解h IAPP與磷脂膜是如何相互作用的,對抑制淀粉樣蛋白的聚集和開發(fā)治療此類疾病的藥物都有重要的意義。由于細(xì)胞環(huán)境的復(fù)雜性,h IAPP與膜的結(jié)合受到許多環(huán)境因素的影響。此外,膽固醇是細(xì)胞膜中的重要成分,對淀粉樣蛋白的聚集行為及毒性機(jī)理可能有重要的影響。本論文中,我們主要研究了h IAPP在磷脂膜上的聚集以及硫酸軟骨素和血清白蛋白對h IAPP在磷脂膜上聚集的影響;我們還研究了膽固醇對h IAPP的N端區(qū)域與磷脂膜相互作用的影響。我們對h IAPP與磷脂膜相互作用的研究工作可以歸納為以下幾點(diǎn):1、我們用兩性離子磷脂膜POPC、陰離子磷脂膜POPG以及它們以POPG:POPC為3:7的比例組成的混膜作為模型膜,通過Th T熒光實(shí)驗(yàn)、圓二色譜(CD)、原子力顯微鏡、31P-NMR等實(shí)驗(yàn)方法研究了硫酸軟骨素(CSA)和牛血清白蛋白(BSA)對全長h IAPP在磷脂膜環(huán)境中聚集的影響。CSA和BSA屬于細(xì)胞外基質(zhì)的成分,它們的引入為h IAPP的聚集創(chuàng)造了一個多相異質(zhì)環(huán)境。我們發(fā)現(xiàn)h IAPP在多相異質(zhì)環(huán)境和在均相環(huán)境中的聚集行為是不一樣的。在均相水環(huán)境中,CSA對h IAPP的聚集有促進(jìn)作用,BSA則很好地抑制了h IAPP的聚集。而在POPC膜中,由于CSA和POPC的特異性結(jié)合,導(dǎo)致CSA對h IAPP聚集的促進(jìn)作用有一個額外的增強(qiáng)效果。在含POPG的陰離子膜(POPG和POPG/POPC)中,BSA因與POPG磷脂成分的結(jié)合而減弱了對h IAPP的抑制作用。與單一環(huán)境相比,多相環(huán)境可能更接近h IAPP所處的真實(shí)環(huán)境,所以在含有磷脂膜和胞外基質(zhì)成分的復(fù)雜體系中進(jìn)行研究有可能為了解h IAPP的聚集機(jī)理提供更加有意義的信息。2、h IAPP的N端區(qū)域是其與膜結(jié)合的關(guān)鍵區(qū)域,研究發(fā)現(xiàn),雖然h IAPP有很強(qiáng)的聚集傾向,但h IAPP1-19與全長的h IAPP不同,h IAPP1-19不管是在水溶液還是磷脂膜中都不會形成長纖維,有很弱的聚集傾向,但是有與全長h IAPP類似的細(xì)胞毒性。1-19肽段中包含的R11、F15和V17三個殘基組成反向膽固醇識別氨基酸共有序列(CARC),可能參與肽-膽固醇的結(jié)合,其中第15位的苯丙氨酸是CARC序列的關(guān)鍵殘基。因此,我們選取1-19肽段(h IAPP1-19)作為h IAPP與膜相互作用的結(jié)構(gòu)模型,并將第15位的苯丙氨酸(Phe或F)突變成亮氨酸(Leu或L)得到h IAPP1-19/F15L,通過差示掃描量熱法(DSC)、31P-NMR、1H-NMR、CD和染料泄漏實(shí)驗(yàn)等實(shí)驗(yàn)方法研究了h IAPP1-19與DPPC膜的相互作用以及CARC序列對h IAPP1-19與DPPC膜相互作用的影響。我們發(fā)現(xiàn),膽固醇可以在DPPC膜上形成脂筏區(qū),并促進(jìn)h IAPP1-19與DPPC的結(jié)合,誘導(dǎo)多肽的二級結(jié)構(gòu)由無規(guī)卷曲轉(zhuǎn)變?yōu)棣?螺旋,產(chǎn)生更多的毒性寡聚體,對膜造成更大的破壞,在這個過程中,F15參與了肽與膽固醇的結(jié)合。3、我們在第二部分工作的基礎(chǔ)上用具有柔性鏈的DOPC磷脂膜代替剛性鏈的DPPC膜,同樣以野生型肽h IAPP1-19和突變體肽h IAPP1-19/F15L作為研究對象,用31P-NMR、1H-NMR滴定實(shí)驗(yàn)、CD和染料泄漏實(shí)驗(yàn)等實(shí)驗(yàn)方法研究了h IAPP1-19與DOPC膜的相互作用以及在這個過程中膽固醇的影響。我們發(fā)現(xiàn),膽固醇也能增強(qiáng)h IAPP1-19與具有柔性鏈的DOPC膜的結(jié)合,并增加磷脂膜的透性,對磷脂膜造成破壞。但與DPPC膜的結(jié)果相比,h IAPP1-19與DOPC膜的結(jié)合較弱,這可能歸因于膽固醇沒能在柔性的DOPC膜上產(chǎn)生脂筏區(qū)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R363
本文編號：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R363
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