by Lam Suet Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 109-121). / Acknowledgements --- p.i / Table of contents --- p.ii / Abstract --- p.vii / List of Abbreviations --- p.ix / List of Tables --- p.x / List of Figures --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ribosome-inactivating proteins (RIPs) --- p.3 / Chapter 1.1.1 --- Types of RIPs --- p.4 / Chapter 1.1.1.1 --- Type I RIPs --- p.5 / Chapter 1.1.1.2 --- Type II RIPs --- p.7 / Chapter 1.1.2 --- Physicochemical properties --- p.7 / Chapter 1.1.3 --- N-glycosidase activity of RIPs --- p.8 / Chapter 1.1.3.1 --- Specificity of N-glycosidase activity --- p.10 / Chapter 1.1.3.2 --- Inhibition of protein synthesis --- p.11 / Chapter 1.1.4 --- Other enzymatic and biological activities of RIPs --- p.11 / Chapter 1.1.4.1 --- Enzymatic activities --- p.11 / Chapter 1.1.4.2 --- Multiple depurination --- p.13 / Chapter 1.1.4.3 --- RNase activity --- p.14 / Chapter 1.1.4.4 --- DNase activity --- p.15 / Chapter 1.1.4.5 --- Biological activities --- p.16 / Chapter 1.1.5 --- Storage of RIPs in plant cells --- p.17 / Chapter 1.1.5.1 --- RIPs targeted to subcellular compartments --- p.18 / Chapter 1.1.5.2 --- Cytoplasmic RIPs --- p.20 / Chapter 1.1.6 --- Physiological roles of RIPs --- p.22 / Chapter 1.1.6.1 --- Defensive role in plants --- p.22 / Chapter 1.1.6.2 --- Metabolic role of RIPs --- p.26 / Chapter 1.1.6.3 --- RIPs as storage proteins --- p.26 / Chapter 1.1.7 --- Application of RIPs --- p.27 / Chapter 1.1.7.1 --- Therapeutic applications --- p.27 / Chapter 1.1.7.2 --- Possible use of RIPs in agriculture --- p.30 / Chapter 1.2 --- Objectives of the present study --- p.31 / Chapter 1.2.1 --- Rationale of the study --- p.31 / Chapter 1.2.2 --- Outline of the thesis --- p.32 / Chapter Chapter 2 --- Screening of hitherto unexplored plant species for RIPs --- p.33 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Materials --- p.36 / Chapter 2.2.2 --- Preparation of crude powder --- p.36 / Chapter 2.2.3 --- Protein determination --- p.38 / Chapter 2.2.4 --- Preparation of rabbit reticulocyte lysate --- p.38 / Chapter 2.2.5 --- Protein synthesis inhibition assay --- p.39 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Preparation of crude powder --- p.41 / Chapter 2.3.2 --- Protein synthesis inhibition assay --- p.41 / Chapter 2.4 --- Discussion --- p.43 / Chapter Chapter 3 --- Purification of RIP from garden pea (Pisum sativum) --- p.45 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Materials --- p.50 / Chapter 3.2.2 --- Purification of RIP from garden pea --- p.52 / Chapter 3.2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.54 / Chapter 3.2.4 --- Precautions for working with RNA --- p.56 / Chapter 3.2.5 --- N-glycosidase assay --- p.57 / Chapter 3.2.6 --- Quantitation of RNA --- p.60 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Quantitation of RNA --- p.61 / Chapter 3.3.2 --- Affinity chromatography on Affi-gel Blue gel --- p.61 / Chapter 3.3.3 --- Iminodiacetic acid-agarose chromatography --- p.64 / Chapter 3.3.4 --- Cation exchange chromatography on Resource-S --- p.66 / Chapter 3.3.5 --- Gel filtration on Superose 12 HR 10/30 --- p.69 / Chapter 3.3.6 --- "Assessment of purity, yield and activity" --- p.72 / Chapter 3.4 --- Discussion --- p.74 / Chapter Chapter 4 --- Physicochemical and biological properties of garden pea RIP --- p.77 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Materials --- p.81 / Chapter 4.2.2 --- Molecular weight determination --- p.82 / Chapter 4.2.3 --- Subunit composition --- p.82 / Chapter 4.2.4 --- Isoelectric focusing (IEF) --- p.83 / Chapter 4.2.5 --- Detection of glycoproteins --- p.84 / Chapter 4.2.6 --- N-terminal amino acid sequence --- p.84 / Chapter 4.2.7 --- Inhibition of cell-free protein synthesis --- p.86 / Chapter 4.2.8 --- N-glycosidase activity --- p.86 / Chapter 4.2.9 --- Deoxyribonuclease activity --- p.87 / Chapter 4.2.10 --- Activity towards tRNA --- p.88 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Molecular weight determination --- p.89 / Chapter 4.3.2 --- Subunit composition --- p.91 / Chapter 4.3.3 --- Isoelectric focusing (IEF) --- p.92 / Chapter 4.3.4 --- Detection of glycoproteins --- p.94 / Chapter 4.3.5 --- N-terminal amino acid sequence --- p.96 / Chapter 4.3.6 --- Inhibition of cell-free protein synthesis --- p.97 / Chapter 4.3.7 --- N-glycosidase activity --- p.99 / Chapter 4.3.8 --- Deoxyribonuclease activity --- p.101 / Chapter 4.3.9 --- Activity towards tRNA --- p.102 / Chapter 4.4 --- Discussion --- p.103 / Chapter Chapter 5 --- General discussion and conclusion --- p.106 / References --- p.109

No description available.

by Tsang Kwok Yeung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 82-93). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.V / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- PURIFICATION AND CHARACTERIZATION OF A TYPE I RIP MOMORGROSVIN FROM THE SEEDS OF MOMORDICA GROSVENORI --- p.34 / GENERAL DISCUSSION --- p.80 / REFERENCES --- p.82

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: a) Characterization of traditional methodologies and testing methods used to purify and quantify trypsin and α-chymotrypsin b) Re-engineering / development of a new method for purifying trypsin and α-chymotrypsin that delivered higher product yields and improved control exercised over the process by investigating: i. Extraction methods ii. Centrifugation iii. Ultrafiltration iv. Chymotrypsinogen and trypsin crystallization v. Column chromatography vi. Investigation into different raw material sources for pancreatic enzyme production c) Development of kinetic and ELISA testing methodologies for in-process QC analysis. / AFRIKAANSE OPSOMMING: Hierdie Studie beskryf: a) Karakterisering van die ou prosessering metodes en toets metodes wat gebruik word om Tripsien en Alpha-chimotripsien te suiwer en te kwantifiseer. b) Herontwerp / ontwikkeling van 'n nuwe metode vir die suiwering Tripsien en Chimotripsien wat „n hoër opbrengs lewer en meer kontrole oor die proses uit oefen deur ondersoek in te stel na: i. Ekstraksie- metodes ii. Sentrifugering iii. Ultrafiltrasie iv. Chymotripsienogeen - en tripsien kristallisasie v. Kolom chromatografie vi. Ondersoek na verskillende rou materiaal bronne vir die produksie van pankreas ensieme. c) Die ontwikkeling van kinetiese- en ELISA toets metodes vir die in-proses kwaliteitkontrole.

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human heat shock protein 70 (Hsp70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). The two proteins are homologues of the highly conserved stress-inducible protein 1 (STI1) family of co-chaperones. The STI1 proteins interact directly and simultaneously at some stage, with Hsp70 and Hsp90 in the formation of the hetero-multi-chaperone complexes that facilitate the folding of signal transducing kinases and functional maturation of steroid hormone receptors. The interactions of mSTI1 with both Hsp70 and Hsp90 is mediated by a versatile structural protein-protein interaction motif, the tetratricopeptide repeat (TPR). The TPR motif is a degenerate 34-amino acid sequence a-helical structural motif found in a significant number of functionally unrelated proteins. This study was aimed at characterizing the structural and functional determinants in the TPR domains of mSTI1 responsible for binding to and discriminating between Hsp70 and Hsp90. Guided by data from Hop's crystal structures and amino acid sequence alignment analyses, various biochemical techniques were used to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal TPR domain (TPR1) of mSTI1 to bind to the C-terminal EEVD motif of heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. Substitutions in the first TPR motif of Lys⁸ or Asn¹² did not affect binding of mSTI1 to Hsc70, while double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn⁴³ lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys⁸ or Asn¹² reduced but did not abrogate binding. Steady state fluorescence and circular dichroism spectroscopies revealed that the double substitution of Lys⁸ and Asn¹² resulted in perturbations of inter-domain interactions in mSTl1. Together these results suggest that mSTI1-Hsc70 interaction requires a network of electrostatic interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70, but also outside the TPR1 domain. It is proposed that the electrostatic interactions in the first TPR motif collectively made by Lys⁸ and Asn¹² define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. In the first central TPR domain (TPR1A), single substitution of Lys³°¹ was sufficient to abrogate the mSTI1-Hsp90 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, residues predicted by crystallographic data to determine Hsp70 binding specificity were substituted in the TPR1 domain. The modified protein had reduced binding to Hsc70, but showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on the TPR2A domain. These data suggest that binding of Hsc70 to the TPR1 domain is more specific than binding of Hsp90 to the TPR2A domain. In addition, residues C-terminal of helix A in the second TPR motif of mSTI1 were shown to be important in determining specific binding to Hsc70. Binding assays using surface plasmon resonance spectroscopy showed that the affinities of binding of mSTI1 to Hsc70 and Hsp90 were 2 μM and 1.5 μM respectively. Preliminary in vivo studies revealed differences in the dynamics of binding of endogenous and exogenous recombinant mSTI1 with Hsc70 and Hsp90. The outcome of this study poses serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in the cell.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: During the last two and a half decades a large body of research has accumulated indicating the presence of various natural and synthetic chemical compounds within the environment capable of inducing hormone-like responses in humans and animals. Such compounds, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand binding domains of these proteins. With a view to ultimately design a portable kit for the detection of endocrine disrupting compounds in water based on the bio-specific immobilisation of nuclear receptor ligand binding domains to a stationary membrane matrix, this study specifically describes: 1. The effects on recombinant protein expression by the addition of small molecules to the cultivation media of bacteria. 2. The optimisation of conditions for the lysis of bacterial cells to increase the solubility of heterologously expressed proteins. 3. The purification of recombinant proteins from bacterial cell lysates by means of a two-step chromatographic methodology. 4. The cloning of the genes for the human androgen and estrogen receptors’ ligand binding domains into baculovirus transfer plasmids. 5. Transfer of genetic material from the created baculovirus transfer plasmids to a linearised baculovirus genome for the generation of recombinant viruses. 6. The cultivation, and baculoviral infection, of Spodoptera frugiperda and Trichoplusia ni cell lines. 7. Expression and purification of N-terminal hexahistidine-tagged human nuclear receptor LBDs from insect cell lysates by means of immobilised metal affinity chromatography. / AFRIKAANSE OPSOMMING: Die teenwoordigheid van natuurlike en sintetiese chemiese middels wat oor die vermoë beskik om die aksies van hormone in die mens en dier na te boots het toenemend aftrek gekry in navorsing gedurende die laaste twee en ’n halwe dekades. 'n Verskeidenheid van ontwikkelings-, reproduktiewe- en fisiologiese abnormaliteite ontstaan as gevolg van die aksies van hierdie molekule, genaamd endokriene-ontwrigters, op die natuurlike funksionering van die endokriene-sisteem. Gegewe dat die groep chemiese middels waaruit endokriene-ontwrigters bestaan van diverse oorsprong afkomstig is lei dit daartoe dat algemene analitiese tegnieke nie in alle gevalle geskik is vir effektiewe omgewingsmonitering is nie. Die modulasie van geentranskripsie is een van die metodes wat voorgestel word as ’n metode waarop hierdie eksogene molekule die homeostatiese regulering deur die endokriene-sisteem omverwerp. ’n Algemene metode waarop vele endokrien-ontwrigtende stowwe geentranskripsie beïnvloed, is deur interaksie met die hormoon-bindende gedeeltes van ’n belangrike groep transkripsiefaktore, die nukluêre reseptore. Hierdie studie, met die uiteindelike ontwikkeling van ’n draagbare toetsstelsel vir die opsporing van endokrien-ontwrigtende-stowwe in water, gebasseer op die bio-spesifieke immobilisering van nukluêre reseptor ligand bindingsdomeins op ’n stasionêre membraanmatriks, het ten doel om die volgende te beskryf: 1. Die effek wat die byvoeging van klein molekule tot die groeimedium van bakteriëe het op die uitdrukking van rekombinante proteïene. 2. Die optimisering van bakteriese sel-lisering in terme van verhoging in die oplosbaarheid van heteroloë proteïene. 3. Die suiwering van rekombinante proteïen vanuit bakteriese sellisate deur middel van ’n twee-stap chromatografiese sisteem. 4. Die klonering van die gene vir die menslike androgeen en estrogeen reseptore se ligand bindingsdomeine in bakulovirus oordragplasmiede. 5. Die oordrag van genetiese materiaal vanaf hierdie bakulovirus oordragplasmiede na ’n gelineariseerde bakulovirus genoom deur middel van homoloë rekombinasie vir die produksie van rekombinante virusse. 6. Die groei en infeksie van Spodoptera frugiperda en Trichoplusia ni sellyne wat lei tot die uitdrukking van menssoortgelyke nukluêre reseptor ligandbindingsdomains. 7. Suiwering van N-terminaal heksahistidien-etiket-gekoppelde menslike nukluêre reseptor ligandbindingsdomeins vanuit inseksellisate deur middel van geïmmobiliseerde metaal affiniteitschromatografie.

錯誤折疊並聚集的促澱粉樣變蛋白和多肽分子通常以β折疊含量豐富的纖維狀澱粉態存在，這種纖維狀澱粉態被認為與多種神經退行性疾病的發病有關，例如老年癡呆症，多聚穀氨醯胺症以及傳染性海綿狀腦病。澱粉態沉積物作為多種神經退行性疾病的顯著標誌，促澱粉樣變蛋白和多肽發生錯誤折疊並聚集進而導致神經毒性的機理仍未被闡明。在當前的研究中，我們選擇酵母感染性蛋白Sup35作為探索促澱粉樣變蛋白聚集機理的模型。Sup35是一種存在於釀酒酵母細胞中的感染性蛋白，作為一種翻譯終止因子，它可以通過改變自身構象，進而形成不溶的纖維狀澱粉態沉澱。根據位置和功能的不同，Sup35可被劃分為3個結構域，即N，M和C。作為控制其感染性的區域，Sup35-NM被廣泛接受為一種用於研究促澱粉樣變蛋白的模型。研究人員已經針對Sup35的聚集機理開展了很多研究，其中最為廣泛接受的是Lindquist 等人提出的β螺旋模型。在這個模型中，相鄰的氨基酸片段形成了一種頭對頭和尾對尾的構象。我們的研究目的就是要探究這種聚集機理模型是否正確。如果不正確，我們將對聚集機理提出一種新的假設。 / 作為探索促澱粉樣變蛋白聚集過程的重要前提，研究人員必須首先製備出只含有單獨的蛋白單體的樣品溶液。否則，相關的動力學過程研究將被干擾。我們通過動態激光光散射研究發現，使用現有的多種用於溶解促澱粉樣變蛋白和多肽的實驗方法並不能製備出真正的蛋白溶液，得到的樣品中總含有微量的、尺寸大約為10-10² nm的聚集體。這些聚集體會極大地影響聚集的動力學過程。這也可以在一定程度上解釋為什麼在不同的文獻報導中，同一種蛋白在相同的環境中卻表現出差異巨大的動力學過程。在當前的研究中，我們將傳統方法與我們實驗室新進開發的超濾法相結合，發展出了一套可以用於製備真正的、不含有聚集體的促澱粉樣變蛋白或多肽溶液的方法。製備出的溶液可以保持其中的蛋白或多肽處於單體狀態至少一個星期，這為研究在生理條件下蛋白的聚集過程提供了重要的基礎。 / 為了研究Sup35不同亞基之間的相互作用，我們分別在其N結構域的頭，腰和尾做了半胱氨酸點突變，並用兩種相互獨立的方法研究亞基之間的相互作用。第一種方法是在突變位點引入空間位阻，從而減弱所謂的頭對頭尾對尾的相互作用。我們的想法很直接，如果Lindquist等人提出的機理是正確的，那麼突變後的蛋白將無法形成纖維狀澱粉態沉澱。第二種方法是通過形成二硫鍵在不同蛋白的半胱氨酸突變位點之間引入連接分子，共有兩種連接分子，一種長約2 Å，另一種長約11 Å。選擇這兩種連接分子的原因是，聚集體中兩條Sup35蛋白鏈之間的距離通常約4.7 Å，連接分子長於或短于這個距離應會對聚集產生不同影響，從而反映出聚集體的結構資訊。 / 在這篇博士論文中，首先，我將介紹促澱粉樣變蛋白研究的背景和激光光散射測量的原理以及研究中用到的主要實驗方法。然後，我將闡述如何將傳統方法和我們實驗室新進發展出的超濾法相結合，從而製備出真正的、不含聚集體的蛋白溶液。接下來，我還將證明通過動態和靜態激光光散射相結合，我們可以得到更多關於促澱粉樣變蛋白的微觀參數，包括分子量，蛋白單體和聚集體的流體力學半徑等。最後，我將針對不同Sup35突變體的聚集動力學過程來研究其亞基之間的相互作用並提出Sup35的聚集模型。 / Misfolding and aggregation of amyloidogenic protein/peptide are frequently found in a β-sheet-rich fibrillar protein conformation known as amyloids, which are related to the onset of neurodegenerative diseases, ranging from Alzheimer and polyglutamine diseases to transmissible spongiform encephalopathies. While amyloid deposits are hallmarks of many neurodegenerative diseases, the mechanism by which these proteins/peptides gain their neurotoxic function upon misfolding and aggregation remains unclear. In the current study, we choose the yeast prion Sup35 as a model system to investigate the aggregation mechanism of amyloidogenic protein. The Sup35 protein is a yeast (Saccharomyces cerevisiae) prion protein, a translation termination factor that can convert into insoluble amyloid fibril. The structure of Sup35 protein can be divided into three regions; namely, N, M, and C based on their positions and different functions. Being the prion-determining region, Sup35-NM has been widely accepted as a model to study the amyloidogenic proteins. Many studies have been focused on the aggregation mechanism. The β-helix model proposed by Lindquist and her coworkers is mostly accepted. In such a model, Sup35-NM is folded to form a “head and a “tail region in the N region and different Sup35-NM chains aggregate together via a cooperative Head-to-Head and Tail-to-Tail stacking. The aim of our current study is to check whether this proposed mechanism is valid. / To gain insight into the mechanism of aggregation process, one must start with a solution that contains only individual (monomeric) protein chains. Otherwise, the kinetic study would be compromised. Our dynamic laser light scattering (LLS) study shows that the existing protocols of dissolving amyloidogenic protein/peptide do not result in a true solution; namely, there always exist a trace amount of interchain aggregates with an average size of ~10-10² nm, which greatly affect the association kinetics, partially explaining why different kinetics were reported even for a solution with identical protein and solvent. In this thesis study, by using a combination of the conventional dissolution procedure and our newly developed ultra-filtration method, we have developed a novel protocol to prepare a true solution of amyloidogenic protein/peptide without any interchain aggregates. The resultant solutions remain in their monomeric state for more than one week, which is vitally important for further study of the interchain association under the physiological conditions / To investigate the inter-subunit relationship, cysteine variants mutated at “Head, Waist, Tail" of the N region have been constructed. Two independent assessments have been proposed to study the inter-subunit interaction. One is to provide steric hindrance to the mutated sites so that the so called “Head-to-Head and Tail-to-Tail" interaction will be attenuated. Our strategy is quite straightforward, if the mechanism proposed by Lindquist and her coworkers is valid, the modified protein should lose its ability to form amyloid fibrils. The other strategy is to introduce disulfide cross-linkage between different mutation sites. Two types of disulfide cross-linkage have been chosen, one with a bond length of ~2 Å and the other, ~11 Å. The reason for such choices is that Sup35-NM has a characteristic inter-strand distance of ~4.7 Å. The disulfide bond length shorter or longer than this distance is supposed to play a different role in the protein aggregation, shedding light on the structural information. / In this Ph.D. thesis, we first introduce the background of amyloidogenic protein research and present the principle and instrumentation of laser light scattering, the main technique applied in our study. Next we show how to obtain a true solution of amyloidogenic protein with no oligomeric aggregates by combining a conventional dissolution procedure and our newly developed ultra-filtration method. We also show how to combine static and dynamic laser light-scattering measurements in the study of protein solutions, which leads to more microscopic parameters, such as the molar mass and the hydrodynamic sizes of individual protein chains and their aggregates. Our focus is on the aggregation kinetics of modified Sup35-NM variants and on the investigation of the inter-subunit interaction. Finally, we propose a model for the aggregation of Sup35-NM prion protein. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Diao, Shu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 99-101). / 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 (in Chinese) --- p.i / ABSTRACT --- p.iii / Table of content --- p.v / Acknowledgement --- p.viii / Chapter Chapter 1 --- Introduction and background --- p.1 / Chapter 1.1 --- The biological role of amyloidogenic protein --- p.1 / Chapter 1.1.1 --- The role of amyloidogenic protein in human disease --- p.1 / Chapter 1.1.2 --- The functional role of amyloidogenic protein in living system --- p.2 / Chapter 1.1.3 --- The role of amyloidogenic protein asnonchromosomal genetic elements --- p.3 / Chapter 1.2 --- The structure of amyloid fibrils --- p.4 / Chapter 1.2.1 --- Macromolecular structure of amyloid fibrils --- p.5 / Chapter 1.2.2 --- Structure models for protofilament --- p.6 / Chapter 1.2.3 --- The polymorphism of amyloid fibrils --- p.9 / Chapter 1.3 --- Aggregation mechanism of amyloidogenic protein --- p.10 / Chapter 1.3.1 --- The nucleated polymerization mechanism --- p.11 / Chapter 1.3.2 --- Multiple conformations adopted by amyloidogenic protein chains --- p.13 / Chapter 1.3.3 --- Sequence effect on amyloid formation --- p.15 / Chapter 1.4 --- The pathogenesis of amyloid diseases --- p.16 / Chapter 1.4.1 --- Prefibrillar aggregates may be the real culprits --- p.16 / Chapter 1.4.2 --- Strategies to prevent amyloid diseases --- p.17 / Chapter 1.5 --- References and Notes --- p.19 / Chapter Chapter 2 --- Principle of Laser Light Scattering and Instrumentation --- p.27 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Static Laser Light Scattering --- p.28 / Chapter 2.2.1 --- Scattering by a small particle --- p.28 / Chapter 2.2.2 --- Scattering by many small-particle system --- p.30 / Chapter 2.2.3 --- Scattering by real systems --- p.31 / Chapter 2.3 --- Dynamic Laser Light Scattering --- p.37 / Chapter 2.3.1 --- Power spectrum of scattered light --- p.37 / Chapter 2.3.2 --- Siegert relation --- p.39 / Chapter 2.3.3 --- Translational diffusions --- p.40 / Chapter 2.3.4 --- Analysis of the correlation function profile --- p.42 / Chapter 2.4 --- Combination of Static and Dynamic Light Scattering --- p.44 / Chapter 2.5 --- Practice of Laser Light Scattering --- p.45 / Chapter 2.5.1 --- Light source --- p.45 / Chapter 2.5.2 --- Optics and cell design --- p.46 / Chapter 2.5.3 --- Detector --- p.47 / Chapter 2.5.4 --- Sample Preparation --- p.47 / Chapter 2.5.5 --- Differential refractometer --- p.48 / Chapter 2.6 --- References and Notes --- p.49 / Chapter Chapter 3 --- How to obtain a true solution of amyloidogenic protein/peptide with no oligomeric aggregates --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Experimental section --- p.53 / Chapter 3.3 --- Results and discussion --- p.59 / Chapter 3.4 --- Conclusion --- p.68 / Chapter 3.5 --- References and Notes --- p.71 / Chapter Chapter 4 --- Aggregation mechanism investigation of the Yeast prion protein Sup35-NM --- p.73 / Chapter 4.1 --- Introduction --- p.73 / Chapter 4.2 --- Experimental section --- p.75 / Chapter 4.3 --- Results and discussion --- p.82 / Chapter 4.3.1 --- Aggregation kinetics of Sup35-NM protein initiated from monomeric state --- p.82 / Chapter 4.3.2 --- Does Sup35-NM protein aggregate in a head-to-head and tail-to-tail fashion? --- p.87 / Chapter 4.3.2.1 --- The effect of dimerization on Sup35-NM aggregation --- p.88 / Chapter 4.3.2.2 --- Inter-subunit investigation by Pyrene excimer fluorescence assay --- p.92 / Chapter 4.3.2.3 --- The effect of PEGylation on Sup35-NM aggregation --- p.94 / Chapter 4.4 --- Conclusion --- p.98 / Chapter 4.5 --- References --- p.100 / Publications --- p.102

Orientador: Celso Eduardo Benedetti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T14:13:17Z (GMT). No. of bitstreams: 1 Lira_NayaraPatriciaVieirade_M.pdf: 3098417 bytes, checksum: 6441392e6b6c275daba6bd89e88cbaf8 (MD5) Previous issue date: 2014 / Resumo: Xylella fastidiosa e Agrobacterium tumefaciens são bactérias fitopatogênicas que infectam, respectivamente, o interior do xilema e de tecidos vasculares de raiz, ambientes cuja tensão de oxigênio é relativamente baixa. Uma vez que Xylella e Agrobacterium são bactérias estritamente aeróbicas, elas apresentam o operon bigR, responsável pela detoxificação do sulfeto de hidrogênio ou gás sulfídrico, um potente inibidor do citocromo c oxidase e respiração aeróbica. O operon bigR codifica cinco proteínas denominadas Blh (Beta-lactamase-like hydrolase), BigR (biofilm growth-associated repressor), um repressor transcricional e regulador do operon, e MP1-3, proteínas que compõem um transportador de membrana. Em trabalho anterior, foi demonstrado que mutantes de Agrobacterium deficientes na produção de Blh acumulavam gás sulfídrico, enquanto mutantes no repressor BigR secretavam mais sulfito, indicando que a proteína Blh convertia gás sulfídrico em sulfito e que este, que também é tóxico, seria exportado pelo complexo MP1-3. Além disso, dados de modelagem molecular indicaram que Blh poderia desempenhar funções de sulfotransferase e dioxigenase de enxofre, uma vez que apresenta os domínios DUF442 (rodanase) e ETHE1 (dioxigenase). A fim de testar tais hipóteses, este trabalho teve como principais objetivos a caracterização enzimática dos domínios DUF442 e ETHE1 da Blh de Xylella e Agrobacterium, como também confirmar interações proteína-proteína entre os componentes do operon bigR. Ensaios de atividade enzimática usando-se proteínas recombinantes purificadas confirmaram a função de dioxigenase de enxofre e de rodanase dos domínios ETHE1 e DUF442, respectivamente. Além disso, verificou-se que ambos os domínios produzem sulfito como produto final da reação, embora atuando em substratos diferentes. Ainda, ensaios de duplo híbrido de levedura mostraram haver inúmeras interações entre as proteínas do operon bigR, mas não entre os dois domínios DUF442 e ETHE1 de Blh que, de acordo com os ensaios enzimáticos, atuam de forma independente. / Abstract: Xylella fastidiosa and Agrobacterium tumefaciens are phytopathogenic bacteria that infect, respectively, the xylem vessels and root vascular tissues, where the oxygen tension is relatively lower. Since Xylella and Agrobacterium are strict aerobic organisms, they use the bigR operon for the detoxification of hydrogen sulfide, a potent inhibitor of cytochrome c oxidase and aerobic respiration. The bigR operon encodes five proteins designated Blh (Beta-lactamase-like hydrolase), BigR (biofilm growth-associated repressor), a transcriptional repressor that regulates the operon, and MP1-3, proteins that act as a membrane transporter. In a previous work, it was shown that Agrobacterium mutants deficient in Blh production accumulated hydrogen sulfide, whereas BigR-deficient mutants secreted sulfite at higher levels than the wild type bacteria, indicating that Blh converted hydrogen sulfide into sulfite, which would be exported by the MP1-3 complex. In addition, molecular modeling indicated that Blh could function as a sulfur transferase and sulfur dioxigenase, since it carries a DUF442 (rhodanese) and ETHE1 (dioxygenase) domains. To test such hypothesis, this work aimed to demonstrate the enzymatic activities of the DUF442 and ETHE1 domains of Blh from Xylella and Agrobacterium, as well as to confirm protein-protein interactions between components of the bigR operon. Enzyme activity assays using the purified proteins confirmed the sulfur dioxygenase and rhodanese activities of the ETHE1 and DUF442 domains, respectively. In addition, it was found that both domains produce sulfite as a final product, although having different substrates. Furthermore, yeast two-hybrid assays showed that many of the bigR operon proteins interact with each other, suggesting the formation of a protein complex. However, no physical interactions were detected between DUF442 and ETHE1 domains, which, according to the enzyme activity assays, act independently. / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action.

Malaria poses the greatest threat of all parasites to human life. Current vaccines and efficacious drugs are available however their use is limited due to toxicity, emergence of drug resistance, and cost. The discovery of an alternative pathway of isoprenoid biosynthesis, the non-mevalonate pathway, within the malarial parasite has resulted in development of novel anti-malarial drugs. 1-Deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme in this pathway, is responsible for the synthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP) in an intramolecular rearrangement step followed by a reduction process involving NADPH as a hydrogen donor and divalent cations as co-factors. Fosmidomycin and FR900098 have been identified as inhibitors of DOXP reductoisomerase. However, they lack clinical efficacy. In this investigation recombinant DOXP reductoisomerase from Escherichia coli (EcDXR) and Plasmodium falciparum (pfDXR) were biochemically characterized as potential targets for inhibition. (His)6-EcDXR was successfully purified using nickel-chelate affinity chromatography with a specific activity of 1.77 μmoles/min/mg and Km value 282 μM. Utilizing multiple sequence alignment, previous structural data predictions and homology modeling approaches, critical active site amino acid residues were identified and their role in the catalytic activity investigated utilizing site-directed mutagenesis techniques. We have shown evidence that suggests that Trp212 and Met214 interact to maintain the active site architecture and hydrophobic interactions necessary for substrate binding, cofactor binding and enzyme activity. Replacement of Trp212 with Tyr, Phe, and Leu reduced specific activity relative to EcDXR. EcDXR(W212F) and EcDXR(W212Y) had an increased Km relative to EcDXR indicative of loss in affinity toward DOXP, whereas EcDXR(W212L) had a lower Km of ~8 μM indicative of increased affinity for DOXP. The W212L substitution possibly removed contacts necessary for full catalytic activity, but could be considered a non-disruptive substitution in that it maintained active site architecture sufficient for DOXP reductoisomerase activity. EcDXR(M214I) had 36-fold reduced enzyme activity relative to EcDXR, while its Km (~8 μM) was found to be lower than that of EcDXR. This suggested that the M214I substitution had maintained (perhaps improved) substrate and active site architecture, but may have perturbed interactions with NADPH. Rational drug design strategies and docking methods have been utilized in the development of furan derivatives as DOXP reductoisomerase inhibitors, and the synthesis of phosphorylated derivatives (5) and (6) has been achieved. Future inhibitor studies using these novel potential DOXP reductoisomerase inhibitors may lead to the development of effective anti-malarial drug candidates.