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The role of cystic fibrosis transmembrane conductance regulator in insulin secretion in pancreatic islet β-cells. / Role of cystic fibrosis transmembrane conductance regulator in insulin secretion in pancreatic islet beta-cells / CUHK electronic theses & dissertations collectionJanuary 2013 (has links)
囊性纖維化(CF)是由囊性纖維化跨膜電導調節器(CFTR)的突變引起的一種隱性遺傳病。CF病人的肺、肝、胰腺、腸道與生殖道受到嚴重影響,其中有50%的成年病人患有糖尿病。由CF引起的糖尿病被稱為CF相關糖尿病(CFRD), 关于它的病因至今仍然存有爭議。2007年,人們發現CFTR在分泌胰島素的胰島β細胞上有表達。儘管如此,β細胞上的CFTR与糖尿病发病的关系却一直被忽略。我們的研究目標是闡述β細胞上的CFTR在胰島素分泌中的作用。 / 在β細胞上,葡萄糖刺激的胰島素分泌伴隨著複雜的電活動,這種電活動被描述為細胞膜電位去极化疊加的動作電位的爆發。葡萄糖引起的ATP敏感的鉀離子通道(K[subscript Asubscript Tsubscript P])的關閉被普遍認為是β細胞去極化的初始事件,初始的去極化啟動了電壓依賴的鈣離子通道,由此產生的鈣離子內流成為構成動作電位的去極化電流,引起了細胞內鈣離子的震盪,從而引起胰島素的釋放。雖然氯離子電流被認為參與了β細胞去極化電流,但是,人們仍然不能確定是哪一種氯離子通道介導了這個去極化電流。在我們研究的第一部分,CFTR被證明功能性的表達在β細胞上,並且可以被葡萄糖激活。CFTR可以被葡萄糖激活这一性质,在CFTR超表達的CHO 细胞上被進一步驗證。在原代培養的β細胞與β細胞株RIN-5F细胞中的葡萄糖引起的全細胞電流、膜電位的去極化、動作電位的幅度與頻率、鈣震盪和胰島素的分泌可以被CFTR的抑制劑或缺陷所降低。與野生型小鼠相比,CFTR基因敲除的小鼠,禁食之後,具有更高的血糖濃度,然而其胰島素的濃度低。 / 我們研究中的第二部分,利用了數學模型去闡明CFTR 在胰島素分泌的電活動中的角色。結果顯示, CFTR電導的減低可以使細胞的細胞膜去極化,從而導致需要更高的電刺激去引發動作電位,这些結果證明了CFTR對於维持細胞膜電位的貢獻。同時增加細胞內氯離子濃度和CFTR的電導可以引起更大頻率的膜電位的震盪,這一點證明了氯離子對於細胞膜電位震盪有著重要的作用。在数学模型中,CFTR電導的降低可以消除通過改變ATP/ADP值所引起的電火花, 這與我們在試驗中發現的CFTR參與了葡萄糖引起的動作電位是一致的。總而言之,我們的数学模型證明了CFTR對於胰島素的分泌是非常重要的,它通過介導氯離子外流對細胞膜電位的產生貢獻並且參與了電火花的產生,所有這些都進一步驗證了我們在實驗部分的發現。 / 综上所述,現有的研究揭示了CFTR,通過對β細胞膜電位作用與参与了動作電位的產生,在葡萄糖刺激胰島素分泌过程中的鮮為人知的重要角色。這個發現為揭示CFRD的病理機制提供了全新的視角,並且可能為開發治療CFRD的方法帶来了曙光。 / Cystic fibrosis (CF) is a recessive autosomal genetic disease resulted from mutations of cystic fibrosis transmembrane conductance regulator (CFTR). CF affects critically the lung, liver, pancreas, intestine and reproductive tract. CF patients also exhibit a high percentage of diabetes, which almost reach 50% in adult. The pathological cause of diabetes in CF patients, also called CF related diabetes (CFRD), is still controversial. It has been reported that CFTR expressed in the islet β cells, which is responsible for insulin secretion. However, the exact role of CFTR in islet β-cell and its relation to diabetes have been ignored. The present study aims to elucidate the role of CFTR in the process of insulin secretion by pancreatic islet β cells. / Glucose-stimulated insulin secretion is associated with a complex electrical activity in the pancreatic islet β-cell, which is characterized by a slow membrane depolarization superimposed with bursts of action potentials. Closing ATP-sensitive K⁺ channels (K[subscript Asubscript Tsubscript P]) in response to glucose increase is generally considered the initial event that depolarizes the β-cell membrane and activates the voltage-dependent Ca²⁺ channels, which constitutes the major depolarizing component of the bursting action potentials giving rise to the cytosolic calcium oscillations that trigger insulin release. While Cl⁻ has been implicated in an unknown depolarization current of the β-cell, the responsible Cl⁻ channel remains unidentified. In the first part of our study, we show functional expression of CFTR and its activation by glucose in the β-cell. Activation of CFTR by glucose was also demonstrated in CHO cell over-expression system. The glucose-elicited whole-cell currents, membrane depolarization, electrical bursts (both magnitude and frequency), Ca²⁺ oscillations and insulin secretion could be abolished or reduced by inhibitors/knockdown of CFTR in primary mouse β-cells or RIN-5F β-cell line, or significantly attenuated in isolated mouse islet β-cells from CFTR mutant mice compared to that of wildtype. Significantly increased blood glucose level accompanied with reduced level of insulin is found in CFTR mutant mice compared to the wildtype. The results strongly indicate a role of CFTR in the process of insulin secretion. / In the second part of our study, mathematical model is built up to clarify the role of CFTR in the electrical activity during insulin secretion. It is shown that reduction of CFTR conductance hyperpolarizes the membrane of the β-cell, for which it requires a larger electrical stimulus to evoke an action potential, indicating the contribution of CFTR to the membrane potential as demonstrated by our experimental results. Increase in intracellular Cl⁻ concentration and the conductance of CFTR result in higher frequency of membrane potential oscillations, demonstrating that Cl⁻ is crucial for the membrane potential oscillations. The electrical spikes induced by increase of ATP/ADP in the model are abolished by decreasing CFTR conductance, which is consistent with our findings that CFTR is involved in the generation of action potentials induced by glucose. In other word, our model demonstrates that CFTR is crucial for insulin secretion by its contribution to membrane potential and participating in the generation of electrical spikes via conducting Cl⁻ efflux, which confirms our findings in the experimental study. / Taken together, the present study reveals a previously unrecognized important role of CFTR in glucose-stimulated insulin secretion via contributing to the membrane potential and the participating in the generation of action potential in islet β cells. This finding sheds new light into the understanding of the pathogenesis of CFRD and may provide grounds for the development of new therapeutic approaches for CFRD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Guo, Jinghui. / "December 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 156-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要: --- p.iii / Acknowledgement: --- p.v / LIST OF PUBLICATIONS --- p.vi / Declaration --- p.viii / ABBREVIATIONS --- p.xi / LIST OF FIGURES --- p.xiii / Chapter Chapter 1: --- General introduction --- p.1 / Chapter 1.1 --- The function of islet β cells and diabetes --- p.1 / Chapter 1.1.1 --- The introduction of the pancreas --- p.1 / Chapter 1.1.2. --- Glucose metabolism and blood glucose regulation --- p.6 / Chapter 1.1.2.2 --- Blood glucose regulation --- p.7 / Chapter 1.1.3 --- Insulin secretion by the islet β cell --- p.10 / Chapter 1.1.4 --- Diabetes --- p.14 / Chapter 1.2 --- Cystic fibrosis-related diabetes --- p.17 / Chapter 1.2.1 --- Cystic fibrosis --- p.17 / Chapter 1.2.2 --- CFTR --- p.19 / Chapter 1.3 --- Mathematical model for insulin secretion --- p.25 / Chapter 1.4 --- Aim and hypothesis --- p.27 / Chapter 1.4.1 --- CFTR may be activated by glucose --- p.27 / Chapter 1.4.2 --- Activation of CFTR may depolarize the membrane potential --- p.28 / Chapter 1.4.3 --- CFTR-mediating Cl-efflux may be involved in the generation of electrical spikes --- p.28 / Chapter 1.4.4 --- Calcium oscillation depends on CFTR --- p.28 / Chapter 1.4.5 --- Insulin secretion --- p.29 / Chapter 1.5 --- Approaches to test the hypothesis --- p.29 / Chapter Chapter 2: --- Materials and Methods --- p.31 / Chapter 2.1 --- Cell culture --- p.31 / Chapter 2.1.1 --- RIN-5F cell --- p.31 / Chapter 2.1.2 --- CHO cell --- p.31 / Chapter 2.2 --- Islet isolation and culture --- p.32 / Chapter 2.3 --- CFTR knockdown --- p.33 / Chapter 2.4 --- Western blot --- p.35 / Chapter 2.5 --- Immunofluorescence --- p.37 / Chapter 2.6 --- Membrane potential (Vm) measurement --- p.38 / Chapter 2.7 --- Intracellular chloride imaging --- p.39 / Chapter 2.8 --- Intracellular calcium imaging --- p.40 / Chapter 2.9 --- Patch-clamp --- p.40 / Chapter 2.10 --- Blood glucose measurement --- p.42 / Chapter 2.11 --- Insulin ELISA --- p.42 / Chapter 2.12 --- Statistics --- p.42 / Chapter Chapter 3: --- Contribution of CFTR on the eletrophysiological properties in insulin secretion --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Results --- p.45 / Chapter 3.2.1 --- Functional expression of CFTR in mouse islet β cells --- p.45 / Chapter 3.2.2 --- CFTR activation by glucose --- p.46 / Chapter 3.2.3 --- Involvement of CFTR in the maintenance of membrane potential of islet β cells --- p.47 / Chapter 3.2.4 --- CFTR is involved in the generation of spikes induced by glucose --- p.50 / Chapter 3.2.5 --- Generation of spikes burst in the β cell depends on intracellular chloride. --- p.52 / Chapter 3.2.6 --- Inhibition/mutation of CFTR attenuates calcium oscillation induced by glucose --- p.53 / Chapter 3.2.7 --- Inhibition/mutation of CFTR impairs insulin secretion --- p.53 / Chapter 3.3 --- Discussion --- p.71 / Chapter Chapter 4: --- Mathematical model for the role of CFTR in the process of insulin secretion in islet β cell --- p.74 / Chapter 4.1 --- Introduction to the mathematical modeling in the process of insulin secretion --- p.74 / Chapter 4.2 --- Methods --- p.77 / Chapter 4.2.1 --- Components in the model --- p.77 / Chapter 4.2.2 --- Assumptions and approaches in modeling --- p.78 / Chapter 4.2.3 --- Modeling ion channels and transporters --- p.79 / Chapter 4.2.3.1 --- KATP channel --- p.79 / Chapter 4.2.3.2 --- Sodium channel --- p.82 / Chapter 4.2.3.3 --- Voltage Dependent calcium channel --- p.83 / Chapter 4.2.3.4 --- NCX --- p.84 / Chapter 4.2.3.5 --- Na-K pump --- p.85 / Chapter 4.2.3.6 --- Kv channel --- p.87 / Chapter 4.2.3.7 --- Ca pump --- p.88 / Chapter 4.2.3.9 --- CFTR --- p.90 / Chapter 4.2.3.10 --- NKCC --- p.91 / Chapter 4.3 --- Results --- p.93 / Chapter 4.3.1 --- Role CFTR in regulation of the basal membrane potential in β cells --- p.93 / Chapter 4.3.2 --- Role of intracellular chloride concentration in the burst spikes induced by glucose --- p.95 / Chapter 4.3.3 --- Role of CFTR in the burst spikes induced by glucose --- p.96 / Chapter 4.4 --- Discussion --- p.105 / Chapter Chapter 5: --- General discussion and conclusion --- p.109 / Chapter 5.1 --- General discussion --- p.109 / Chapter 5.1.1 --- Role of CFTR in endocrine pancreas and diabetes --- p.109 / Chapter 5.1.2 --- Role of CFTR as a cell metabolic sensor --- p.111 / Chapter 5.1.3 --- Role of CFTR in excitable cells --- p.113 / Chapter 5.2 --- Conclusion --- p.114 / Appendix A --- p.115 / Appendix B --- p.118 / Reference: --- p.156
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Immunoregulatory role of human islet amyloid polypeptide through FoxP3+CD4+CD25+ T regulatory cells. / 人類淀粉樣蛋白通過CD4+CD25+調節性T細胞的免疫調節作用 / CUHK electronic theses & dissertations collection / Ren lei dian fen yang dan bai tong guo CD4+CD25+ diao jie xing T xi bao de mian yi tiao jie zuo yongJanuary 2010 (has links)
Background. Islet amyloid polypeptide (IAPP, also known as amylin) is a 37-amino acid peptide principally co-secreted with insulin from the beta-cells of the pancreatic islets. Some of the physiological actions of human amylin (hIAPP) include glucose regulation, suppression of appetite and stimulation of renal sodium and water reabsorption. Amylin deficiency and diminished post-prandial amylin response have been reported in advanced stages of type 1 and type 2 diabetes. In autopsy specimens of type 2 diabetes, amyloid is found in 40--90% of cases. During the characterization of islet morphology of aged hIAPP transgenic mice, I observed pathological features suggestive of immune dysregulation. Review of literature also suggested possible immuno-modulating functions of human amylin in in vitro experiments. Since autoimmunity and innate immunity are implicated in aging and diabetes, I explored the immunological role of amylin with particular focus on CD4+CD25+ T regulatory cells and toll-like receptors (TLR) which are known mediators of autoimmunity and innate immunity respectively. / Conclusions. Human amylin may play an important role in modulating immunity mainly through stimulating CD4+CD25+ Treg cells, decreasing PLN and altering expression of TLR-4 and cytokines. If these findings are confirmed in in vivo model, human amylin has the potential to become a novel and promising therapy to prevent and reverse autoimmune disease such as autoimmune type 1 diabetes. / Hypothesis. Human amylin may have immunomodulating effects which may have implications on pathogenesis of autoimmune type 1 diabetes. / Materials and methods. Male hemizygous hIAPP transgenic mice (n=32) and their nontransgenic littermates (n=20) were fed with normal chow and studied longitudinally up to 18 months of age with measurement of plasma insulin, glucose and amylin at regular intervals. Detailed oral glucose tolerance test, intra-peritoneal insulin tolerance test, insulin and amylin protein expression were examined at 3, 7, 12 and 18 months of age. Histological changes of pancreas and spleen including changes in CD4+CD25+ T regulatory cells and cytokines were examined at 12 and 18 months. / Objectives. (1) I systemically characterized the morphological, functional and immune regulatory role of human amylin in aged hIAPP transgenic mice which include metabolic profiles, plasma levels of amylin and insulin as well as morphological changes of pancreatic lymph nodes (PLN). (2) I then examined splenic expression of TLR-4 associated changes in cytokines (TNF-alpha, TGF-beta, and IL-6). (3) I also examined the expression level of receptor activity modifying proteins (RAMPs) in pancreas and spleen. (4) I finished by investigating the role of human amylin on stimulating CD4+CD25+ T regulatory (Treg) cells in hIAPP transgenic mice and peripheral blood monocytes (PBMC) from healthy subjects. / Results. (1) With aging, the hIAPP transgenic mice demonstrated increased plasma amylin, decreased plasma insulin, reduced insulin to amylin ratio and improved insulin sensitivity (p<0.05). (2) The aged hIAPP transgenic mice showed changes in immune function as indicated by: (a) Reduced number and size of PLN (p<0.05). (b) Decreased expression level of TLR-4 in splenocytes (p<0.05). (c) Increased expression of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) protein but decreased level of IL-6 in splenocytes (p<0.05). (3) The changes in the levels of immune cytokines such as IL-1, IL-2, IL-4, IL-10, IL-17, interferon-gamma and GM-CSF were similar between hIAPP transgenic and nontransgenic mice (p>0.05). (4) The levels of RAMP1, RAMP2, and RAMP3 were higher in the spleen of hIAPP transgenic mice than nontransgenic mice (p<0.05). (5) The hIAPP transgenic mice showed higher percentage of CD4+CD25+ Treg cells compared with nontransgenic littermates. Treatment with human amylin, but not rat amylin, increased the percentage of FoxP3+CD4+CD25+ Treg cells in both splenic T lymphocytes of hIAPP transgenic mice and PBMCs of healthy subjects ex vivo (p<0.05). / He, Lan / Adviser: Juliana C.N. Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 176-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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Studies of neuropeptides in pancreatic beta cell function with special emphasis on islet amyloid polypeptide (IAPP)Karlsson, Ella January 2000 (has links)
<p>The presence of protein amyloid in pancreas and its association to diabetes was first described 100 years ago in 1901, but was not identified as Islet Amyloid Polypeptide (IAPP) until 1986. The aim of the present work was to determine the role of the beta cell hormone, IAPP, in normal pancreatic islet physiology and during early disturbances of islet function.</p><p>Intra-islet peptides, i.e. chromogranin peptides and an extra-islet peptide, i.e. leptin, were studied to identify possible endogenous regulators of IAPP and insulin secretion. Chromogranin-B, but not chromogranin-A or pancreastatin, had the ability to inhibit islet IAPP and insulin release, suggesting that chromogranin-B may serve as an autocrine regulator of IAPP and insulin secretion. </p><p>Leptin had a more potent effect on IAPP secretion than on insulin secretion, which was dissociated from effects on islet glucose metabolism. Glucose oxidation rates were increased at physiological leptin concentrations, whereas higher leptin concentrations showed an inhibitory effect and chronically high leptin concentrations had no effect.</p><p>Female NOD mice were studied to investigate the release of IAPP in the progression to type 1 diabetes. The release of IAPP was lower than that of insulin from immune cell infiltrated islets, indicating preferential insulin release during the early course of the disease. </p><p>IAPP is expressed at an early embryonic stage. The effect of IAPP on cell proliferation in neonatal rat islets was studied in the search for a physiological role of IAPP. IAPP concentrations of (1-1000) nM stimulated neonatal islet cell proliferation mostly in beta cells and to a lesser extent in alpha cells. IAPP did not have any marked effect on the islet cell death frequency. These data indicate a role for IAPP as a potential regulator of beta cell proliferation in neonatal pancreatic islet.</p><p>It is concluded that IAPP may be involved in regulation of pancreatic beta cell function both in fetal and adult life.</p>
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Studies of neuropeptides in pancreatic beta cell function with special emphasis on islet amyloid polypeptide (IAPP)Karlsson, Ella January 2000 (has links)
The presence of protein amyloid in pancreas and its association to diabetes was first described 100 years ago in 1901, but was not identified as Islet Amyloid Polypeptide (IAPP) until 1986. The aim of the present work was to determine the role of the beta cell hormone, IAPP, in normal pancreatic islet physiology and during early disturbances of islet function. Intra-islet peptides, i.e. chromogranin peptides and an extra-islet peptide, i.e. leptin, were studied to identify possible endogenous regulators of IAPP and insulin secretion. Chromogranin-B, but not chromogranin-A or pancreastatin, had the ability to inhibit islet IAPP and insulin release, suggesting that chromogranin-B may serve as an autocrine regulator of IAPP and insulin secretion. Leptin had a more potent effect on IAPP secretion than on insulin secretion, which was dissociated from effects on islet glucose metabolism. Glucose oxidation rates were increased at physiological leptin concentrations, whereas higher leptin concentrations showed an inhibitory effect and chronically high leptin concentrations had no effect. Female NOD mice were studied to investigate the release of IAPP in the progression to type 1 diabetes. The release of IAPP was lower than that of insulin from immune cell infiltrated islets, indicating preferential insulin release during the early course of the disease. IAPP is expressed at an early embryonic stage. The effect of IAPP on cell proliferation in neonatal rat islets was studied in the search for a physiological role of IAPP. IAPP concentrations of (1-1000) nM stimulated neonatal islet cell proliferation mostly in beta cells and to a lesser extent in alpha cells. IAPP did not have any marked effect on the islet cell death frequency. These data indicate a role for IAPP as a potential regulator of beta cell proliferation in neonatal pancreatic islet. It is concluded that IAPP may be involved in regulation of pancreatic beta cell function both in fetal and adult life.
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Crystal Structures of Sortase A from Streptococcus Penumoniae : Insights into Domain-Swapped Dimerization. Crystal Structures of Designed Peptides : Inhibitors of Human Islet Amyloid Polypeptide (hIAPP) Fibrillization Implicated in Type 2 Diabetes And Those Forming Self-Assembled NanotubesMisra, Anurag January 2014 (has links) (PDF)
Sortases are cell-membrane associated cysteine transpeptidases that are essential for the assembly and covalent anchoring of certain surface proteins to the cell wall in Gram-positive bacteria. Thus, they play critical roles in virulence, infection and colonization by pathogens. Sortases have been classified as type A, B, C, D, E and F based on their phylogeny and the target-protein motifs that they recognize. Sortase A (SrtA) enzymes participate in cell wall anchoring of proteins involved in bacterial adhesion, immune evasion, internalization, and phage recognition and in some cases pili formation. SrtA substrates are characterised by the presence of a C-terminal cell wall sorting signal as LPXTG motif, followed by a stretch of hydrophobic residues and a positively charged tail. Experimental and bioinformatics studies show that class A sortases are housekeeping as well as virulence determining proteins. Hence, Sortase A enzymes are considered as promising antibacterial drug targets, particularly because many organisms are developing multi-drug resistance behaviour. SrtA adopts an eight-stranded β-barrel structure and the overall fold is conserved among the sortase isoforms, with some modifications.
The thesis candidate has determined the three dimensional (3D) crystal structures of wild-type and active site mutant of Sortase A from Streptococcus pneumoniae R6 strain by using X-ray diffraction method. The wild-type enzyme crystallized in P21 space group whereas active site cysteine mutant crystallized in C2 space group. In both the cases, N-terminal 81 residue deletion constructs (ΔN81) were used for crystallization. Uncommonly, both the structures showed a phenomenon of domain-swapping which resulted in the protein adopting a domain-swapped dimeric form. Two such dimers in wild-type protein and three dimers in mutant protein were observed in the asymmetric unit. To the best of our knowledge, our work reveals for the first time the occurrence of domain-swapping in sortase superfamily.
Experimental techniques like size-exclusion chromatography, native-PAGE, analytical centrifugation and thiol cross-linking (carried out in our collaborator’s laboratory at National Institute of Immunology (NII), New Delhi, India) of functionally active wild-type SrtA from S. pneumoniae showed dimerization as well as domain-swapping in solution state. These results support the possibility that the protein indeed exists in a domain-swapped dimeric form and the determined structure is not the result of crystal packing artifact but is physiologically relevant as well. The work done by the thesis candidate covering crystallization of both, the active and inactive protein constructs, their structure determination using molecular replacement method, detailed structural analyses, structural comparisons with known SrtA structures and new structural findings are described in from Chapter 2 to Chapter 4. Based on the SrtA crystal structure the author of the thesis has also proposed various point mutations which are likely to disrupt domain– swapping and result in loss of dimer formation. In addition, as a part of the ongoing project in our laboratory, molecular dynamics studies of these domain-swapped dimers containing two sets of active site residues facing each other in a very compact volume have been initiated to understand substrate binding, which in future could lead to inhibitor design.
Apart from the crystal structure analyses of SrtA structures, the author of the thesis has also carried out systematic crystal structure investigation of dipeptides and pentapeptides containing non-standard amino acids (ΔPhe, Aib and β-amino acids) along with computational studies. Conformationally restricted α,β-dehydrophenylalanine residue (ΔF) and α-aminoisobutyric acid (Aib) have been incorporated in highly amyloidogenic human Islet Amyloid Polypeptide (hIAPP) fragments. Amyloid deposits, observed in a vast majority of Type 2 diabetic patients, are primarily on account of misfolding and aggregation into fibrils of hIAPP, a 37 residue endocrine hormone secreted by pancreatic β-cells. It has been suggested that intermediates produced in the process of fibrillization are toxic to insulin producing β-cells. Hence, the inhibition of misfolding of hIAPP that involves structural transition from its native state (coil and/or helical and/or transient helical conformation) to β-sheet conformation, could be a possible strategy to mitigate Type 2 Diabetes Mellitus (T2DM). All the peptides discussed in this thesis were synthesized in our collaborator, Prof. V. S. Chauhan’s laboratory at the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.
In this work, author of the thesis has designed short peptides containing helicogenic residue, α,β-dehydrophenylalanine (ΔF) and determined their 3D crystal structures. It was found that pentapeptides, FGA∆FL and FGA∆FI act as inhibitors of hIAPP fibrillization. As revealed by crystal structure analysis, both the peptides have similar backbone conformation consisting of a ‘nest’ motif, which is an anion receptor. Molecular docking suggested that both the pentapeptides interact with the hIAPP20-27 segment, stabilizing the hIAPP in helical form by shielding the core aggregation initiation region. This reduces the possibility of oligomerization, formation of toxic intermediates and subsequently the transition to β-structure and fibrillization. Thus, the crystal structures of pentapeptide inhibitors together with computational docking studies suggest an atomic level model of the possible mode of action by which the FGAΔF(L/I) peptides manifest their fibrillization inhibition activity and this could be of value in the design of a new class of amyloid inhibitors. In another peptide design, L→U (Aib) mutation was done in core fibrillization region ANFLV i.e. hIAPP13-17. The resulting mutant peptide ANFUV as well as native fragment ANFLV was crystallized and their 3D crystal structures were determined. ANFLV crystallized in two space groups C2 and P2 adopting extended conformation. Crystal packing of ANFLV in both the crystals shows parallel beta sheet arrangement which is favoured and strengthened by hydrogen bonding between asparagine side chains of Asn-Asn pair each located in neighbouring parallel beta-strands. Hydrogen bonded Asn-Asn residue pairing in parallel beta-strands suggests its significant contribution during hIAPP fibril formation. The substitution L→U abolished its fibrillization property and the structure of ANFUV was solved by direct methods in P21 space group. The occurrence of β-bulge in ANFUV induced by Aib, as observed in crystal packing, suggests that Aib acts as a β-breaker through β-bulge inducing property in the highly amyloidogenic hIAPP segment. β-bulge forming property, an attribute of Aib as β-breaker may be responsible for the curtailment of fibrillization potential of the peptide in which the residue was incorporated. The aim of the anti-amyloid work is to design potent anti-fibrillization peptides and the work is important to design peptide based drugs to fight type II diabetes.
The utilization of ΔPhe in the molecular self-assembly offers an added benefit in terms of variety and stability. Taking advantage of the conformation constraining property of ΔPhe residue, its incorporation in dipeptide molecules has been probed. The author has studied nanotube formation through molecular self-assembly, involving two classes of non¬standard amino acids i.e. ΔF and β-amino acids. FΔF in D-form, L-form and DL-mixture crystallized in different space groups forming rectangular/hexagonal channels constituting different channel dimensions. Recently, the application of FΔF nanotubes have been demonstrated in controlled drug delivery, showing the relevance of the work in health care. Another class of dipeptides containing β-amino acids (β-FF, β-FΔF, β-AΔF, β-VΔF, β¬LΔF, β-IΔF, and β-LF) was also explored for the self-assembled nanotube formation. These β-peptides were crystallized and their 3D structures were determined solely by the author of the thesis. Except the β-AΔF & β-LΔF, these peptides self-assemble and form rectangular/ hexagonal channels. Structures of ΔF and β-amino acid containing dipeptides forming ordered nanotubes through self-assembly are detailed in Chapters 8 and 9 in the thesis. Overall, the author of the thesis has crystallized and determined structures of more than twenty peptides. Experimentally, β-peptide nanotubes were observed to encapsulate drug molecules and thus might be useful as a drug delivery system.
In the present thesis crystal structures of the following designed peptide sequences (including one natural sequence ANFLV) are reported in detail.
Table 1
Peptide sequence Representation Length Discussed in
1. Phe-Gly-Ala-ΔPhe-Leu FGAΔFL 5 Chapter 6
2. Phe-Gly-Ala-ΔPhe-Ile FGAΔFI 5 Chapter 6
3. Ala-Asn-Phe-Leu-Val (2 forms) ANFLV_P2, ANFLV_C2 5 Chapter 7
4. Ala-Asn-Phe-Aib-Val ANFUV 5 Chapter 7
5. LPhe-ΔPhe (2 forms) LFΔF1 , LFΔF2 2 Chapter 8
6. DPhe-ΔPhe DFΔF 2 Chapter 8
7. DLPhe-ΔPhe DLFΔF 2 Chapter 8
8. LTyr-ΔPhe LYΔF 2 Chapter 8
9. LSer-ΔPhe LSΔF 2 Chapter 8
10. Boc-D,LPhe-ΔPhe Boc-DLFΔF 2 Chapter 8
11. Cbz-D,LPhe-ΔPhe Z-DLFΔF 2 Chapter 8
12. D,LMet-ΔPhe DLMΔF 2 Chapter 8
13. β-Phe-ΔPhe β-FΔF 2 Chapter 9
14. β-Phe-Phe β-FF 2 Chapter 9
15. β-Val-ΔPhe β-VΔF 2 Chapter 9
16. β-Ile-ΔPhe β-IΔF 2 Chapter 9
17. β-Leu-ΔPhe β-LΔF 2 Chapter 9
18. β-Leu-Phe β-LF 2 Chapter 9
19. β-Ala-ΔPhe β-AΔF 2 Chapter 9
20. Cyclo(Phe-ΔPhe) DKP-FΔF 2 Appendix C
21. Cyclo(Ile-ΔPhe) DKP-IΔF 2 Appendix C
22. Cyclo(Cha-Cha) DKP-ChaCha 2 Appendix C
23. Cyclo(Cha-Phe) DKP-ChaF 2 Appendix C
24. Cyclo(Cha-ΔPhe) DKP-ChaΔF 2 Appendix C
25. Cyclo(S-tritylCys-ΔPhe) DKP-CΔF 2 Appendix C
Most of the dipeptides, except the N-terminal protected dipeptides, cyclic dipeptides (i.e. DKPs) and LSΔF, were found in the zwitterionic conformation and out of these, ten dipeptides resulted in tubular structures of dimensions in the nanoscale range.
The thesis is organized into nine chapters and five appendices. Chapter 1 is an introduction to the work presented in the thesis, while Chapter 2, Chapter 3 and Chapter 4 describe the crystallographic work on the protein Sortase A. Chapter 5 is an introduction to the non-standard amino acids used for peptide designs and Chapter 6, Chapter 7, Chapter 8, Chapter 9 and Appendix C describe the crystallographic work on peptides.
Chapter 1 starts with a general introduction to the Gram-positive bacteria containing sortase enzymes, and the bacterial cell-wall where sortase catalyzed proteins get attached for implicating their virulence during host-pathogen interactions. Pneumococcal diseases mostly affect children and their count has been observed to be higher than the combined total cases of malaria, AIDS and tuberculosis in child population worldwide. The chapter describes different virulence factors of S. pneumoniae out of which many are proteins. Among these, LPXTG containing proteins, which are the prime substrates of the sortase enzymes, are discussed in detail. Sortase enzymes, their classification and their structural studies with conserved ‘Sortase fold’ are discussed elaborately. A brief mention is made about the enzymatic activity of Sortase A to understand the transpeptidation mechanism. To appreciate the biomedical and biotechnological importance of the sortase enzyme, some potential applications of Sortase A are detailed in this chapter. A section is dedicated to describe the protein in the present study 'Sortase A from Streptococcus pneumoniae'. At the end, the scope of the present work, comprising of both protein and peptide crystallography, is presented.
Chapter 2 begins with a brief account of the sequence analysis of Sortase A from S. pneumoniae and phylogenetic analysis of the sortase superfamily enzymes, followed by the details of protein purification & crystallization of two different constructs, wild-type SrtA from S. pneumoniae (Spn-∆N59SrtAWT and Spn-∆N81SrtAWT) as well as that of an active site cysteine mutant (Spn-∆N81SrtAC207A). This chapter includes X-ray intensity data collection of both types of crystals and data processing.
Sortases are membrane anchored enzymes and therefore their expression as a full-length protein is a difficult task. Hence, the deletion of N-terminal transmembrane region from the enzyme is crucial for expression in its soluble form and is important for its successful crystallization. Thus, two wild-type constructs of S. pneumoniae sortase A, ∆N59SrtAWT (N-terminal 59 residue deletion) and ∆N81SrtAWT (N-terminal 81 residue deletion), and one active site mutant ∆N81SrtAC207A (N-terminal 81 residue deletion & active site Cys207 to Ala mutation) were cloned, expressed and purified. Cloning, expression and purification of the protein were done at the laboratory of our collaborator Prof. Rajendra P. Roy, Cell biology lab-II, National Institute of Immunology (NII), New Delhi, India.
Crystallization of Spn-∆N59SrtAWT (~23 kDa) construct was initiated by manual screening using sparse matrix conditions from Hampton research. Initial trials were set up by following hanging-drop vapour diffusion method. Spn-∆N59SrtAWT construct crystallized in diamond, needle, rod and wedge-shaped crystal forms in more than one crystallization condition but they failed to diffract. Further trials were set up in microbatch plates that resulted in diamond-shaped crystals again, which diffracted up to a maximum of
4.0 Å resolution. Sequence comparison of the present construct was performed to modify the construct to achieve better diffraction. Thus, we made modifications in the Spn¬∆N59SrtAWT construct by deleting additional 22 residues at the N-terminal (i.e. total 81 residues deletion in the original sequence from the N-terminal) similar to SrtA from S. pyogenes. Hence, Spn-∆N81SrtAWT construct was prepared. For further crystallization experiments, we used the new construct Spn-∆N81SrtAWT. Similar to Spn-∆N59SrtAWT construct, crystallization set up for Spn-∆N81SrtAWT were done in microbatch plates at 293 K by using the Hampton conditions. During the crystallization set up, protein concentration was varied from 6-30 mg/ml. Notably, the protein crystals grown with 25 mg/ml protein concentration diffracted very well. Thus increasing the protein concentration helped to improve diffraction quality. Crystals obtained in Index-88 condition (0.2 M tri-ammonium citrate and 20% (w/v) PEG 3350, pH 7.0) diffracted up to 2.9 Å. Additive screen was used to improve its diffraction quality. This time many diffracting crystals were obtained and the best rod-shaped crystals grown in additive screen-79 (40% v/v (±)-1,3-butanediol) diffracted well up to 2.70 Å at home source.
Thus, Spn-ΔN81SrtAWT crystallized at protein concentration of 25 mg ml-1 (in 10 mM Tris buffer, pH 7.5; 2 mM β-mercaptoethanol) with a condition containing 0.2 M tri-ammonium citrate and 20% (w/v) PEG 3350, pH 7.0, along with 40% v/v (±)¬1,3-butanediol as an additive agent by using microbatch-under-oil crystallization method.
The chapter also includes crystallization of active site mutant Cys207Ala of ∆N81SrtAWT from S. pneumoniae (Spn-∆N81SrtAC207A). Spn-∆N81SrtAC207A mutant crystallized as a beautiful rectangular block type crystal (with a diffraction up to 2.7 Å at home source and up to 2.48 Å at synchrotron) at protein concentration of 25 mg ml-1 (in 10 mM Tris buffer, pH 7.5; 2 mM β-mercaptoethanol) with a condition containing 0.2 M tri-ammonium citrate and 20% (w/v) PEG 3350, pH 7.0, along with
1.0 M guanidine hydrochloride as an additive agent by using microbatch-under-oil crystallization method. Data collection was done on home-source diffraction facility for both the crystals however; mutant data in better resolution was collected by the author of the thesis at BM-14 beamline at ESRF, Grenoble, France.
Thus, two crystals of SrtA, wild-type (Spn-∆N81SrtAWT) and its C207A mutant (Spn-∆N81SrtAC207A) were indexed satisfactorily in two space groups and their cell parameters are given in the following table 2.
Table 2
Protein Space group a (Å) b (Å) c (Å) β (°) X-ray source
Spn-∆N81SrtAWT P21 66.94 103.45 74.87 115.65 Home source
Spn-∆N81SrtAC207A C2 155.57 113.33 81.34 90.80 Synchrotron
The quality of both the data sets was assessed by SFCHECK and none of them showed twinning. Thus, the data sets collected were found appropriate and useful for structure determination as discussed in Chapter 3.
Chapter 3 details the structure determination of Sortase A from S. pneumoniae for a wild-type construct (Spn-ΔN81SrtAWT) and for an active site cysteine mutant construct (Spn-ΔN81SrtAC207A). Sortase A from S. pyogenes was used as a search model in the molecular replacement (MR) method and a single solution for each data set was obtained through PHASER program. It resulted in four-molecules in wild-type sortase structure and six-molecules in the mutant structure in the respective crystal asymmetric unit. Iterative model building and structure refinement revealed a clear case of domain-swapping as observed in the electron density map. Finally, in the asymmetric unit of wild-type structure and in mutant protein structure two and three domain-swapped dimers were located, respectively. Simulated annealing and TLS refinement resulted in the protein structure with best refinement statistics. All these are elaborately discussed in Chapter 3. The last round of refinement of Spn-ΔN81SrtAWT converged to Rwork = 18.10% and Rfree = 23.39 % for 25152 unique reflections in the resolution range 30.7-2.7 Å whereas for Spn¬ΔN81SrtAC207A structure these parameters converged to Rwork = 18.25% and Rfree = 22.39% for 50010 unique reflections in the resolution range 47.15-2.48 Å.
Chapter 4 describes the wild-type (Spn-ΔN81SrtAWT) as well as mutant (Spn¬ΔN81SrtAC207A) structures of Sortase A. The structure of Sortase A is not found in its commonly observed monomeric form but occur in a domain-swapped dimeric form. There are two dimers in Spn-ΔN81SrtAWT and three in Spn-ΔN81SrtAC207A as observed in the asymmetric unit. Each dimer contains two characteristic 8-stranded beta-barrel folds i.e. ‘sortase fold’ which is unique to the sortase superfamily. Unlike the structure of SrtA from other organisms known so far, the monomer does not form the 8-stranded beta-barrel all by itself. One monomer exchanges the β7 and β8 strands with the other monomer having β1 to β6 strands, thereby forming a complete 8-stranded β-barrel fold and such kind of two complete folds are present in each dimer. Because of the mutual swapping of strands between two monomers in a dimer, the dimer thus formed is defined as a domain-swapped dimer. This is the first time we have observed Sortase A structure in the domain-swapped dimeric form and is also the first example of domain-swapping in the sortase superfamily.
Interestingly, all the catalytic residues (His141, Cys207 and Arg215) in each sortase fold in the swapped dimer lie at the secondary interface (open interface) generated by domain-swapping. Catalytic R215 (in one fold) interacts with D209 residue (in other fold of same dimer) through salt bridge interactions. Each dimer contains two pairs of such residues at the secondary interface but only one pair shows this kind of interaction. R215 (B-chain) interacts with D209 (A-chain) in AB dimer whereas R215 (D-chain) interacts with D209 (C-chain) in CD dimer. Asymmetry in the catalytic residues for their orientations and observed interactions at the secondary interface was evidenced. These active site residues were seen buried to a great extent except Arg215 which is slightly better exposed. It was difficult to find the exact substrate-binding pocket to approach the catalytic Cys207. However, biochemical and biophysical analyses (done at NII, New Delhi) provided strong evidence for the existence of the swapped-dimeric form at physiological pH as well. The enzyme exists with an equilibrium between its monomeric and dimeric forms, and the dimeric population is the most active species of the functionally active enzyme. An important role of Glu208 (in all the chains of two dimers; e.g. Chain A) was seen in the catalytic site where its side chain wobbles between His141 and H142 (both in Chain B) residues for interaction. Due to such kind of interactions the backbone conformation between C207-E208 (Chain B) shows variability, and coordinates the distance between His141 (ND1, Chain A) and Cys207 (SG, Chain B) each belonging to opposite chains in a swapped-dimer. The nature of side chain conformations of Glu208 in all the four sets of active site residues (in wild-type as well as in cysteine mutant structure) indicates that its movement presumably regulates thiolate-imidazolium acid-base pair formation which is a crucial condition for the sortase function where cysteine thiolate acts as nucleophile. Based on the crystal structure, the thesis candidate has suggested several mutants which might disrupt domain-swapping pointing to future studies on the system.
Domain movement analyses by using HingeProt and DynDom servers indicate that the two-sortase folds joined with hinge loops in each dimer may show twist movement around the hinge axis. Possibly, such motion will affect the secondary interface covering active site residues and may allow increasing the exposure of the catalytic residues to perform catalysis. Presumably, such kind of domain movements may play a key role for the unique kind of regulatory mechanism for transpeptidase activity in sortase enzymes. However, more study has to be done to explore the role of these possibilities, if any, in the enzyme function and its regulation.
Chapter 5 provides an introduction to non-standard amino acids, their sources and their uses in de novo peptide design; this is followed by a description of outcomes of structural investigations of modified peptides and their applications in various fields of medical and material science. Specifically, α, β-dehydrophenylalanine (ΔPhe), α-aminoisobutyric acid (Aib) and β-amino acids are discussed and their structures and conformational preferences are highlighted for their use in naturally occurring peptides or peptide fragments.
Chapter 6 begins with an introduction to the human Islet Amyloid Polypeptide (hIAPP), which is an amyloidogenic protein and considered to be an important protein constituent of the amyloid plaques in pancreatic beta-cells in Type 2 diabetes patients. Therefore, fibrillization inhibition of hIAPP is considered as an important therapeutic approach to combat Type 2 Diabetes Mellitus (T2DM). In this chapter, the author of the thesis describes an approach to design peptide based inhibitors of hIAPP fibrillization using non¬standard amino acid ΔPhe (α,β-dehydrophenylalanine) residue. The first designed inhibitor has the sequence origin from hIAPP23-27 and it was developed by replacing I→ΔF (i.e. β¬favouring residue to helical conformation favouring) which resulted in FGAΔFL peptide. Fibrillization inhibition studies were done by co-incubation of hIAPP and FGAΔFL in 1:5 molar ratio and monitored by electron microscopy and thioflavin T binding assay that showed ~75% fibrillization inhibition. It suggested that the inhibitor is working effectively and thus the author determined its crystal structure by X-ray diffraction method. Peptide synthesis and experimental studies like electron microscopy and Thioflavin T binding assay were done in our collaborator’s laboratory at ICGEB, New Delhi, India. Subsequently a sequence similar peptide FGAΔFI was also designed by mutating L→I in the first inhibitor sequence. The resulting peptide FGAΔFI showed ~70% fibrillization inhibition. Following this success, crystal structures of both peptides were determined. FGAΔFL crystallized in P212121 space group whereas FGAΔFI crystallized in P21 space group. Though it was not anticipated, crystal structure analysis revealed that FGAΔFL and its analogue FGAΔFI harbour the anion receptor ‘nest’ motif. Both peptides dock with the helical form of hIAPP which may contribute to the inhibitory function of the peptides through their interaction with hIAPP in the core fibrillization region. These peptides effectively inhibit hIAPP fibrillization in vitro and it seems that these are unique examples of ‘nest-motif’ containing peptides that inhibit fibrillization. We also propose a model for fibrillization inhibition by these peptides; this has been published in Chemical Communications, a journal published by the Royal Society of Chemistry (RSC) and its reprint is enclosed within the thesis. In general, the approach described in the chapter may be applicable to target helices or helical intermediates and could be utilized in developing inhibitors useful, apart from T2DM, in other amyloid diseases including Alzheimer’s disease and Parkinson’s disease.
Table 3
Peptide Crystal system and space group Unit cell details X-ray data Structure solution and refinement Agreement factor
FGAΔFL Orthorhombic, P212121 a=8.9951 (9) Åb=13.0144 (12) Åc=27.7521 (24) ÅV=3248.82 (5) Å3 Z=4 Mo Kα(λ=0.71073Å) 4703 Unique reflections 2581 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 5.95 % for [|Fo| > 4σ (|Fo|)]
FGAΔFI Monoclinic, P21 a=8.9951 (9) Åb=13.0144 (12) Åc=27.7521 (24) Å β=92.637 (2)°V=935.59 (2) Å3 Z=2 Mo Kα(λ=0.71073Å) 4024 Unique reflections 2612 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 5.02 % for [|Fo| > 4σ (|Fo|)]
Chapter 7 describes another important but less studied core fibrillization fragment of hIAPP (hIAPP13-17) different than the hIAPP23-27 discussed in the previous chapter. It also discusses the development of fibrillization inhibitor design from this segment. The fragment hIAPP13-17 i.e. ANFLV crystallized in two space groups; C2 with one molecule in the asymmetric unit and P2 with two molecules in the asymmetric unit. In these structures, ANFLV peptide shows fully extended conformation i.e. a β-conformation. Crystal packing shows parallel β-sheet arrangement with the involvement of dry ‘steric-zippers’. The peptide prefers cross-strand Asn-Asn residue pair by side chain hydrogen bonding and is discussed in comparison with a few crystal structures of hIAPP fragments, solved by Eisenberg’s group, containing Asn residue in their sequence. It is observed that if the Asn is located in the sequence between two terminal residues the peptide will arrange itself in parallel beta sheet. This supports a structural model of hIAPP fibril in parallel beta sheet arrangement as the hIAPP sequence contains several Asn residues. To develop an inhibitor from ANFLV, a partial success was achieved where the Leu → Aib mutant i.e. ANFUV was developed. ThT (Thioflavin T) and TEM (Transmission electron microscopy) results show that the mutant peptide does not fibrilize on its own. This strongly supports the fact that the native peptide (ANFLV) lost its inherent fibrillization characteristic with the introduction of Aib in place of Leu i.e. the resultant mutant ANFUV is a non-fibrillizing peptide. The logic behind the development was to retain ANF in the same extended conformation and then break the β-strand with β-breaker residues. The structure of ANFLV showed parallel beta-sheets along with the additional side chain-side chain hydrogen bonding in the same direction as the fibril axis. Thus, we retained the ANF region to keep the sticky segment in the design and then Leu was mutated to Aib, a known β-breaker, to alter backbone conformation. The crystal structure of the peptide ANFUV resulted in the similar ANF region in beta conformation and Aib in helical conformation. Interestingly, in this situation the conformation of Aib develops a beta-bulge observed in the crystal packing and this bulge structure probably turned the peptide to have non-fibrillizing characteristics. These results will be useful in designing peptide inhibitors by using U as a beta breaker to inhibit hIAPP fibrillization.
Table 4
Peptide Crystal system and space group Unit cell details X-ray data Structure solution and refinement Agreement factor
ANFLV1 Monoclinic, C2 a=36.1350 (20) Åb=4.8050 (10) Åc=19.4190 (20) Å β=98.644 (5)°V=3333.40 (27) Å3 Z=4 Synchrotron (λ=0.77490 Å) 1982 Unique reflections 1825 [|Fo| > 4σ (|Fo|)] Direct methods: Sir92 & SHELXL97 11.71% for [|Fo| > 4σ (|Fo|)]
ANFLV2 Monoclinic, P2 a=18.7940 (80) Åb=4.7970 (10) Åc=35.4160 (50) Å β=103.929 (10)°V=3099.03 (81) Å3 Z=4 Synchrotron (λ=0.77490 Å) 2651 Unique reflections 2580 [|Fo| > 4σ (|Fo|)] Direct methods: Sir92 & SHELXL97 15.39% for [|Fo| > 4σ (|Fo|)]
ANFUV Monoclinic, P21 a=10.8140 (22) Åb=9.1330 (18) Åc=16.7540 (34) Å β=107.960 (30)°V=1574.07 (161) Å3 Z=2 Synchrotron (λ=0.97918 Å) 1426 Unique reflections 1398 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 5.45% for [|Fo| > 4σ (|Fo|)]
Chapter 8 elaborates the self-assembly of α-dipeptides containing conformationally constrained achiral amino acid, α,β-dehydrophenylalanine (ΔF). The structural polymorphism in LFΔF peptide and the resulting self-assembly are discussed. Its D-isomer (DF∆F) and its racemic mixture (DLF∆F) are also discussed as these peptides self-assemble to give channel-forming assemblies. In addition to LFΔF, crystal structures of LYΔF, DLMΔF and LSΔF peptides and their self-assemblies are presented as well. Except DLMΔF xi
and N-terminal protected DLFΔF (Boc-DLF∆F and Z-DLF∆F) peptides, the other dipeptides discussed in this chapter resulted in tubular structures of nanoscale dimensions through molecular self-aggregation.
Table 5
Peptide Crystal system and space group Unit cell details X-ray data Structure solution and refinement Agreement factor
LFΔF1 Hexagonal, P65 a=23.1873(24) Åb=23.1873(24) Åc=5.5260(8) ÅV=2573.01(5) Å3 Z=6 Mo Kα(λ=0.71073Å) 3489 Unique reflections 2915 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 6.19% for [|Fo| > 4σ (|Fo|)]
LFΔF2 Monoclinic, P21 a=5.5739(2) Åb=13.1383(4) Åc=13.5816(4) Å β=96.137(2)°V=988.90(2) Å3 Z=2 Mo Kα(λ=0.71073Å) 4865 Unique reflections 3402 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 4.35% for [|Fo| > 4σ (|Fo|)]
DFΔF Orthorhombic, P21212 a=13.1690(21) Åb=25.3673(40) Åc=5.5622(9) ÅV=1858.12(5) Å3 Z=4 Mo Kα(λ=0.71073Å) 4370 Unique reflections 3426 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 4.44% for [|Fo| > 4σ (|Fo|)]
DLFΔF Monoclinic, P21/c a=5.5392(14) Åb=26.0376(55) Åc=13.1839(27) Å β=90.278(16)°V=1901.46(8) Å3 Z=4 Mo Kα(λ=0.71073Å) 2051 Unique reflections 1264 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 7.08% for [|Fo| > 4σ (|Fo|)]
LYΔF Hexagonal, P65 a=23.5523(4) Åb=23.5523(4) Åc=5.5183(1) ÅV=2650.96(1) Å3 Z=6 Mo Kα(λ=0.71073Å) 2746 Unique reflections 1871 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 3.91% for [|Fo| > 4σ (|Fo|)]
LSΔF Monoclinic, P21 a=5.2998(20) Åb=9.6732(30) Åc=14.1827(57) Å β=95.604(27)°V=723.62(20) Å3 Z=2 Mo Kα(λ=0.71073Å) 1978 Unique reflections 1558 [|Fo| > 4σ (|Fo|)] Direct methods: SHELXS97 & SHELXL97 13.59% for [|Fo| > 4σ (|Fo|)]
DLMΔF Monoclinic, P21/c a=9.9032(5) Åb=8.6675(4) Åc=34.0283(18) Å β=90.088(3)°V=29
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16 |
Caractérisation du rôle de l’amyline (IAPP) dans le diabète de type 2 : études de dérivés peptidiques et de composés inhibiteurs de la formation d’amyloïdeFortin, Jessica 06 1900 (has links)
L’amyloïdose, une maladie progressive et incurable, implique une vaste panoplie de pathologies et de pathogénèses, qui est expliquée par la grande variabilité biologique et structurale des protéines responsables de la formation des dépôts d’amyloïde. L’amyline (polypeptide amyloïde des îlots pancréatiques, IAPP) est une protéine très susceptible de subir des changements de conformation impliquant les feuillets bêta et conférant aussi des propriétés physicochimiques distinctes. Cette protéine prend alors une forme fibrillaire et se dépose dans les îlots de Langerhans chez les humains atteints de diabète de type 2 ou d’insulinome. Ces dépôts d’amyloïde pancréatique (AIAPP) ont été décrits chez certaines espèces animales telles que les félins domestiques, les grands félins, le raton laveur et les primates non humains. La formation de dépôts d’amyloïde contribue à la pathogénèse du diabète de type 2, mais les mécanismes qui induisent la conversion de l’amyline (IAPP) en amyloïde (AIAPP) ne sont pas complètement compris.
Les hypothèses du projet sont que certaines variations présentes dans les séquences peptidiques de l’IAPP provenant de différentes espèces animales jouent un rôle critique pour la formation de fibrilles et que plusieurs composés chimiques aromatiques/phénoliques sont capables d’abroger la formation de dépôts d’amyloïde. Le projet de recherche consiste donc à caractériser la propension des différentes isoformes animales d’IAPP à former de l’amyloïde in vitro afin d’identifier les acides aminés jouant un rôle clé dans cette transformation structurale et ultimement d’inhiber la formation d’amyloïde pancréatique.
Le projet se divise en deux volets principaux. Le premier consiste à identifier les différentes séquences peptidiques de l’IAPP retrouvées chez les espèces animales. L’objectif est d’identifier les acides aminés jouant un rôle clé dans la formation d’amyloïde. Le gène de l’IAPP a été séquencé chez plus d’une quarantaine d’espèces. Le potentiel d’agrégation des séquences obtenues a été simulé à l’aide d’outils bioinformatique. Une librairie de 23 peptides a été commandée afin de procéder à des analyses physicochimiques in vitro permettant d’évaluer le potentiel amyloïdogénique (test fluorimétrique à la thioflavine T, essai de liaison au rouge Congo, dichroïsme circulaire, microscopie électronique à transmission) et cytotoxique (sur une lignée cellulaire provenant d’insulinome : INS-1). Les analyses effectuées à partir de la librairie constituée de 23 peptides ont permis d’identifier trois séquences ne formant pas d’amyloïde et qui proviennent des espèces animales suivantes : le tamarin lion doré (Leontopithecus rosalia), le grand dauphin (Tursiops truncatus) et l’alpaga (Vicugna pacos). Un site potentiellement critique est le segment 8-20 présentant le motif NFLVH qui ne forme plus d’amyloïde lorsqu’il est remplacé par le motif DFLGR ou KFLIR. Les acides aminés 29P, 14K et 18R sont également impliqués dans l’inhibition de la transformation structurale en fibrille.
La dernière partie du projet consiste à inhiber la formation de l’amyloïde en utilisant des composés chimiques commercialisés (hypoglycémiants, anti-inflammatoires non stéroïdiens) ou nouvellement synthétisés dans notre laboratoire (les aryles éthyles urées). Un criblage d’une soixantaine de composés chimiques a été conduit dans cette étude. Leur efficacité a été testée sur l’IAPP humaine, qui possède un fort potentiel amyloïdogénique. Les techniques utilisées sont les mêmes que celles exploitées précédemment. L’essai de liaison croisée photo-induite ("photo-induced cross-linking of unmodified proteins", PICUP) a été réalisé afin d’étudier les formes intermédiaires (monomères, oligomères). Un total de 11 composés chimiques a démontré un potentiel à inhiber l’agrégation des fibrilles. Pour la classe des hypoglycémiants, le glyburide, le répaglinide et la troglitazone ont montré l’activité thérapeutique la plus élevée pour retarder et réduire la formation de fibrilles. Les anti-inflammatoires antiamyloïdogènes actifs incluaient le diclofenac, le méloxicam, le phénylbutazone, le sulindac et le ténoxicam. Les aryles étyles urées les plus intéressantes étaient la EU-362 et la EU-418. Tous ces composés ont conféré une protection cellulaire contre l’activité cytotoxique des fibrilles. Les molécules actives possèdent des éléments structuraux communs tels des substituants donneurs d’électrons (alcool, amine, halogène) sur un noyau benzène.
En conclusion, ce projet de recherche a permis de caractériser l’IAPP chez diverses espèces animales, dont plusieurs chez lesquelles elle n’avait pas encore été décrite, de déterminer les sites jouant un rôle clé dans sa transformation en amyloïde et, ultimement, de tester le potentiel thérapeutique de nouveaux agents antiamyloïdogènes dans le diabète de type 2. Nous espérons que ce projet ouvrira ainsi la porte à de nouvelles stratégies de traitement. / Amyloidosis is a progressive and, as of now, incurable disease caused by the deposition of insoluble proteins. Amyloid research over the past decades focused on the characterization of the substantive biological variability of amyloid deposits. Amyloidosis encompasses a diversity of pathological manifestations, explained by the diversity of underlying causal proteins. In the pancreas of susceptible species, islet amyloid polypeptide (IAPP) is a precursor for an amyloid protein (AIAPP), which has a characteristic fibrillar structure and resistance to physical agents. This folded protein deposits in the islets of Langerhans of patients with type 2 diabetes or islet cell tumors (insulinoma). Amyloid deposits have also been well characterized, anatomically, in feline and non-human primate species. Amyloid fibril formation contributes to the pathogenesis of diabetes mellitus but the precise pathophysiologic factors involved in the fibrillization of IAPP as well as resultant islet injury remain to be elucidated. Further understanding of the causative factors in the fibrillogenesis of IAPP will be requisite in the development of therapeutic strategies to disrupt the amyloidosis process.
This project hypothesizes that the specific variations found in IAPP peptide sequences among different animal species are critical for IAPP fibrillization. Also, some aromatic/polyphenolic compounds can abrogate fibrillization. The main objective forms the basis for development of new therapeutic tactics to impede amyloid formation and associated cellular injury. Thus this project has two specific aims.
The first specific aim was to identify critical variations in IAPP amino acid sequences from different animal species and to assess their amyloidogenic potential. To accomplish this, the IAPP gene was isolated and sequenced from paraffin-embedded tissues from various animals (40 species). The aggregation potency was assessed for each sequence using in silico analysis. A library of 23 peptides was prepared from sequences that were distinctly different and their amyloidogenic potential was assessed in vitro using physicochemical analysis (thioflavin-T assay, Congo red binding assay, far-UV circular dichroism, transmission electron microscopy) and cytotoxicity assays (insulinoma cell line INS-1). Among this peptide library, three were non-amyloidogenic and corresponded to the following animal species: golden lion tamarin (Leontopithecus rosalia), commun bottlenose dolphin (Tursiops truncates) and alpaca (Vicugna pacos). Segment 8-20 of the peptide was critical for amyloid formation and the substitution of the NFLVH motif found in this region by a DFLGR or KFLIR motif impeded fibrillization. The amino acids 29P, 14K and 18R were also demonstrated to abrogate fibril formation.
The second objective consisted in abrogating IAPP fibrillogenesis using conceptualized aromatic/polyphenolic structures, specifically hypoglycemic, non-steroidal anti-inflammatory and aryl ethyl urea agents. This part of the project involved molecular screening of more than 60 compounds. Their efficacy at inhibiting amyloid formation was assessed in vitro on human IAPP, which exhibits the highest amyloidogenic potential. Techniques included the above-mentioned methods, with the addition of photo-induced cross-linking of unmodified proteins (PICUP). A total of 11 compounds showed potential in abrogating IAPP aggregation. Among the hypoglycemic agents evaluated, glyburide, repaglinide and troglitazone showed the highest potency in reducing fibril formation. The NSAIDs that displayed anti-amyloidogenic activity were diclofenac, meloxicam, phenylbutazone, sulindac and tenoxicam. EU-362 and EU-418 were the hit compounds resulting from the screening of the aryl ethyl urea (EU) class. Additionally, these anti-amyloidogenic molecules conferred a protection against fibril cytotoxicity. All of the active molecules bear a commun motif composed of benzene ring with electron donor moieties, such as alcohol, amine or halide.
To conclude, this project characterized IAPP in several animal species in which it has not been previously described and improves our understanding of the amyloidogenesis process. Moreover, the therapeutic potential of hypoglycemic, non-steroidal anti-inflammatory and aryl ethyl ureas agents as anti-amyloidogenic compounds was evaluated. It is conceivable that the additional information hereby gained on the regulation of amyloidogenesis may point towards new therapeutic strategies for diabetic patients.
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Phycocyanin protects INS-1E pancreatic beta cells against human islet amyloid polypeptide-induced apoptosis through attenuating oxidative stress and mitochondrial dysfunction. / CUHK electronic theses & dissertations collectionJanuary 2010 (has links)
Additionally, cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT) pore, failed to prevent hIAPP-induced DeltaPsim collapse, cytochrome c and AIF release and caspase-3 activation, indicating that the MPT pore was not involved in hIAPP-induced apoptosis. On the other hand, potential crosstalk between the extrinsic and intrinsic apoptotic pathways was demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP. / It is widely accepted that human islet amyloid polypeptide (hIAPP) aggregation plays an important role in the loss of insulin-producing pancreatic beta cells. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. hIAPP-induced cytotoxicity is mediated by the generation of reactive oxygen species (ROS). Phycocyanin (PC) is a natural compound from blue-green algae that is widely used as food supplement. Currently, little information is available about the effect of hIAPP on mitochondrial function of beta cells and protection of PC against hIAPP-induced cytotoxicity. In this thesis, I hypothesize that hIAPP may impair beta cell function with the involvement of mitochrondrial dysfunction, and this effects could be attenuated by PC. Therefore, the aim of this study was to investigate the role of mitochondria in hIAPP-induced apoptosis, the in vitro protective effects of PC and explore the underlying mechanisms. / It was found that hIAPP induced apoptosis in INS-1E cells with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (DeltaPsim). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS1-E cells was effectively restored by co-treatment with PC. / Our results showed that hIAPP inhibited the INS-1E cell growth in a dose-dependent manner. However, cytotoxicity of hIAPP was significantly attenuated by co-incubation of the cells with PC. hIAPP induced DNA fragmentation and chromatin condensation, which were key characteristics of cell apoptosis. These changes were inhibited by PC as examined by TUNEL assay and DAPI staining. Moreover, PC significantly prevented the hIAPP-induced overproduction of intracellular ROS and malonaldehyde (MDA), as well as changes of activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes. Furthermore, hIAPP triggered the activation of mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK) and p38 kinase, and these effects were effectively suppressed by PC. / Taken together, I have demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis, which was attenuated by PC through attenuating oxidative stress, modulating JNK and p38 pathways and reducing mitochondrial dysfunction. / Li, Xiaoling. / Adviser: Juliana Chung Ngor Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 150-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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