Antifungal proteins were isolated from the crude bacterial supernatant using ammonium sulfate salt precipitation followed by passage over DEAE -cellulose and Biogel P100 columns. The purified protein had an apparent molecular mass of 14 kDa. Its antifungal activity was retained even at 100°C, for 60 min. The results of protein identification using MALDI -TOF/TOF mass spectrometer suggested that the purified protein is indeed a chitin binding protein that has 206 acid amine containing chitin -bind -3 region with a relative molecular mass of 22230 Da. / Protein có hoạt tính kháng nấm được tinh sạch từ dịch ngoại bào chủng vi khuẩn Bacillus subtilis sau khi qua ba bước tinh sạch: tủa muối ammonium sulphate 30-70%, qua cột sắc ký trao đổi ion DEAE – cellulose và cột săc ký lọc gel Biogel P100. Protein tinh sạch có khối lượng phân tử đạt 22 kDa trên điện di SDS-PAGE. Hoạt tính kháng nấm của protein tinh sạch vẫn còn duy trì khi ủ ở 100°C trong 60 phút. Kết quả nhận dạng bằng khối phổ MALDI -TOF/TOF đã chỉ ra rằng protein bền nhiệt này là chitin binding protein được mã hóa bởi 206 acid amin cùng với khối lượng phân tử là 22230 Da. trị an toàn đối với Al và Atrazie trong môi trường nước tự nhiên về khía cạnh bảo vệ sức khỏe sinh thái.

No description available.

No description available.

Mucopolysaccharidosis IIIB (MPS IIIB) is a rare, metabolic disorder that results from a deficiency in the lysosomal hydrolase, α-N-acetylglucosaminidase (Naglu). Naglu is a housekeeping enzyme involved in the degradation pathway of heparan sulfate. A deficiency in active Naglu leads to an accumulation of heparan sulfate within the lysosome, initiating a pathological cascade within the cell. Patients with MPS IIIB experience progressive central nervous system degeneration and die within the first few decades of life. Presently, enzyme replacement therapy, which is a standard of care for other lysosomal storage disorders, is an ineffective treatment for MPS IIIB. This is due to impermeability of the blood-brain barrier (BBB) to exogenous recombinant enzymes. A promising approach to this therapeutic obstacle is protein transduction domains. Protein transduction domains have been shown to facilitate the delivery of active enzyme across the BBB in mice. Previously, our laboratory used Spodoptera frugiperda (Sf9) insect cell system to express human recombinant Naglu fused to a synthetic protein transduction domain (PTD4). The purpose was to use PTD4 to the facilitate the delivery of Naglu across biological membranes, including the blood-brain barrier. However, a missing stop codon following PTD4 limited its transducibility. The stop codon was re-introduced and the improved fusion enzyme, Naglu-PTD4X, was stably expressed in Sf9 cells. The overarching goal of this project is to create a large-scale production of human recombinant Naglu that has the potential to be used to treat the neuropathology of patients with MPS IIIB. This project used a three-step purification system to purify Naglu-PTD4X. Uptake of Naglu-PTD4X was assessed in MPS IIIB fibroblasts using a fluorogenic activity assay, immunoblotting, and immunocytochemistry. Our purification system was successful at purifying Naglu-PTD4X to homogeneity with a 26% yield and specific activity of 84,000 units/mg. An increase in Naglu activity was detected in MPS IIIB fibroblasts following incubation with Naglu-PTD4X. Future directions will focus on optimizing immunodetection and conducting BBB penetration studies in murine models. / Graduate / 2020-06-21

Pseudomonas aeruginosa est un pathogène opportuniste responsable de nombreuses maladies nosocomiales chez les patients immunodéprimés ainsi que d'infections graves chez les patients atteints de la mucoviscidose (CF). La colonisation des voies respiratoires des patients CF est souvent mortelle car une fois installée, cette bactérie est difficile à éradiquer et provoque le déclin des fonctions respiratoires des patients. L'antibiothérapie devient inefficace face au développement de souches multi-résistantes et sa capacité à former un biofilm. L'étape cruciale initiant l'infection ou la formation du biofilm est l'adhésion durant laquelle des interactions spécifiques lectines/oligosaccharides permettent la fixation de la bactérie à la surface de la cellule hôte. Bloquer l'adhésion serait un moyen de lutter contre l'infection. Dans l'approche d'une thérapie anti-adhésive, plusieurs lectines impliquées dans l'adhésion et l'élaboration du biofilm sont prises comme cibles. Dans un premier temps, des lectines fimbriales putatives identifiées récemment, CupB6 et CupE6, ont fait l'objet d'une étude. Des essais d'expression, de purification et de cristallisation ont été réalisés dans l'objectif de résoudre leur structure cristallographique. Une étude visant à identifier le ligand naturel de CupB6 a également été entreprise. Puis une étude a été menée pour caractériser le potentiel d'inhibition de plusieurs molécules dérivées du galactose sur la lectine soluble, PA-IL. Certaines de ces molécules pourraient être utilisées comme glycomimétiques offrant une alternative aux antibiotiques. Une étude par microcalorimétrie et cristallographie aux rayons X a permis d'étudier la spécificité d'une lectine de légumineuse, PELa. / P. aeruginosa is an opportunistic pathogenic bacterium responsible for numerous nosocomial infections in immunosuppressed patients. It is the first mortal pathogen in cystic fibrosis (CF) patients. The invasion of the respiratory tract of CF patients by the bacterium is often lethal because it is hard to eradicate and it rapidly impairs the respiratory functions of the patients. None of the current antibiotherapy procedures are efficient against multiresistant, biofilm forming P. aeruginosa. The first step leading to infection or biofilm formation involves the initial adhesion of bacterial cells to the host pulmonary cells via specific lectin/oligosaccharid interactions. Blocking the adhesion would be a way to fight against the infection. The anti-adhesion therapy targets several bacterial lectins involved in adhesiveness and biofilm formation. In this work, the recently identified putative fimbrial lectins CupB6 and CupE6 have been studied. Expression and purification tests followed by crystallization trials have been performed. In parallel, attempts to identify the natural ligand of CupB6 were also carried out. This work also presents a systematic characterization of the inhibitory effects of various galactose-derived molecules on the PA-IL lectin. Some of these molecules could be used as glycomimetic drugs thus offering an interesting alternative to standard antibiotics. Finally, the combination of microcalorimetry together with X-ray crystallography enabled us to gain insights into the ligand specificity of PELa, a legume lectin.

Orientadores: Jorg Kobarg, Jose Andres Yunes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T22:20:50Z (GMT). No. of bitstreams: 1 Trindade_DanielMaragno_D.pdf: 7352494 bytes, checksum: 5b03d143205e747b05e3d4b6a8ed8e34 (MD5) Previous issue date: 2009 / Resumo: Staniocalcinas (STCs) representam uma pequena família de hormônios glicoprotéicos, encontrados em todos os vertebrados e composta por STC1 e STC2, que foram inicialmente implicados na homeostase de cálcio e recentemente também foram implicados em vários outros processos. Stanniocalcina-1 (STC1), o primeiro membro encontrado, foi originalmente descoberto em peixes ósseos e posteriormente identificado em humanos onde parece ter um papel, embora ainda obscuro, na carcinogênese e angiogênese. Nos seres humanos STC1 pode ser encontrado em duas formas: um dímero ou um grupo de variantes de alto peso molecular coletivamente chamados bigSTC. Uma vez que ambas as células leucêmicas e os tumores sólidos dependem vascularização, a angiogênese é um passo fundamental na interação tumorhospedeiro e essencial para a progressão do câncer. Análises prévias de microarray resultaram na identificação de vários genes ativados em células endoteliais da medula óssea (BMEC) modulados pela presença de células leucêmicas. Apresentamos aqui STC1 como um marcador microambiente de medula óssea (BMM) durante leucemia linfoblástica aguda (LLA) pela validação dos dados prévios de microarray através de PCR em tempo real quantitativos. Em vista da falta de informações funcionais e estruturais sobre STC1 realizamos: (1) um screen no sistema de duplo híbrido em levedura, a fim de encontrar algumas das proteínas que interagem com a STC1 humana, e (2) uma caracterização estrutural inicial à baixa resolução desta proteína. Fomos capazes de construir um mapa interação proteína-proteína, obtido a partir dos 22 interactantes encontrados em screen de duplo híbrido em levedura. As proteínas encontradas apresentam-se em vários compartimentos celulares nos quais STC1 já foi demonstrada para estar presente, e essas novas informações podem ajudar a esclarecer como e qual o papel STC1 desempenha nesses locais. A fim de fornecer informação estrutural sobre STC1, realizamos análises bioquímicas e estruturais da proteína recombinante STC1 com 6xHis tag produzida em células de inseto utilizando o sistema baculovírus. A análise de dicroísmo circular confirmou a predição in silico do elevado conteúdo de alfa-helices. A análise por espectrometria de massas forneceu os dados experimentais que confirmaram o padrão conservado de pontes dissulfeto anteriormente descrito para STC1 de peixes. Finalmente, os dados de espalhamento de raios-X a baixo ângulo demonstraram que, STC1 adota uma estrutura dimérica, ligeiramente alongada em solução fornecendo deste modo os primeiros dados estruturais à baixa resolução desta família de proteínas. Além disso, foi possível obter proteína suficiente e de elevado grau de pureza para realizar ensaios cristalização que resultaram em cristais os quais difrataram a boa resolução. / Abstract: Staniocalcins (STCs) represent a small family of glycoprotein hormones found in all vertebrates composed by STC1 and STC2, which have been initially implicated in calcium homeostasis and also recently implicated in several other processes. Stanniocalcin-1 (STC1), the first member found, was originally discovered in bony fishes and later identified in humans were it seems to have a role, although still unclear, in carcinogenesis and angiogenesis. In humans STC1 can be found in two forms: a dimer or a group of higher molecular weigh variants collectively called bigSTC. Since both leukemic cells and solid tumors depend on vascularization, angiogenesis is a fundamental step in tumor-host interaction and essential to the cancer progression. Previous microarray analysis resulted in the identification of several activated genes in bone marrow (BM) endothelial cells (BMEC) modulated by presence of leukemic cells. Here we present STC1 as a BM microenvironment marker during acute lymphoblastic leukemia (ALL) by validating previous microarray data by quantitative RealTime-PCR. In view of the lack of functional and structural information on STC1 we performed (1) a yeast two hybrid screen in order to find some of the human STC1 interacting proteins and (2) a initial structural lowresolution characterization of this protein. We were able to construct a protein-protein interaction map, derived from the 22 interactants found in our yeast two-hybrid screen. The proteins found are located in several cellular compartments in which STC1 has already been shown to be present, and the new information might help to clarify how and which role it performs at these sites. As a means to provide structural information about STC1, we performed biochemical and structural analyses of recombinant STC1 6xHis tagged protein produced in insect cells by using the baculovirus system. Circular dichroism analysis confirmed the in silico predicted high alpha-helical content. By mass spectroscopy analysis we provided experimental data that confirmed the conserved disulfide pattern previously described for fish STC1. Finally Small Angle X-ray Scattering data demonstrated that STC1 adopts a dimeric, slightly elongated structure in solution, providing there by the first low resolution structural data of this family of proteins. Additionally, we could obtain enough protein of high purity to perform crystallization trials that resulted in crystals diffracting at good resolution. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

La CO déshydrogénase (CODH) de Desulfovibrio vulgaris est une métalloenzyme qui catalyse la réduction réversible du CO2 en CO. C’est un homodimère composé de deux sites actifs Ni-4Fe-4S et de trois centres fer-soufre. Durant ma thèse, nous avons étudié la maturation de la CODH à nickel et les propriétés catalytiques de la CODH à nickel de D. vulgaris.Pour comprendre le mécanisme de maturation de la CODH à nickel, nous avons caractérisé deux formes de la CODH à nickel produites en présence ou en absence de CooC par des approches biochimiques, spectroscopiques, électrochimiques et cristallographiques. Notre caractérisation montre que la présence de CooC est nécessaire à l’obtention d’une CODH mature et activable. Nous avons également mis en évidence un processus d’activation en présence de nickel dans des conditions réductrices qui n’implique apparemment pas de changement structural du site actif.Notre étude de la CODH à nickel par électrochimie nous a permis de mettre en évidence plusieurs phénomènes d’activations/inactivations de l’enzyme dans des conditions aérobies et anaérobies, et l’existence d’une hétérogénéité fonctionnelle : plusieurs formes de l’enzyme qui montrent des propriétés catalytiques différentes peuvent être présentes simultanément. Cette observation pourrait éclairer d’une façon nouvelle l’hétérogénéité structurale observée par cristallographie et remettre en question les mécanismes proposés sur la base de ces structures. / The monoxide carbon dehydrogenase (CODH) from Desulfovibrio vulgaris is a metalloenzyme which catalyses the reversible reduction of CO2 into CO. It is a homodimer containing two active sites and three iron-sulfur clusters. During my thesis, we studied the maturation of CODH nickel and catalytic properties of Ni-CODH from D. vulgaris.In order to understand, the maturation mechanism of Ni-CODH, we have characterized two forms of Ni-CODH produced in the presence or absence of CooC by biochemical, spectroscopic, electrochemical and crystallographic approaches. Our characterisation shows that the presence of CooC is necessary to obtain a mature Ni-CODH which can be activated. We have also identified an activation process in the presence of nickel in reducing conditions that apparently involves no structural change in the active site.Our study of the Ni-CODH by electrochemistry has shown several phenomena of activation/inactivation of the enzyme under aerobic and anaerobic conditions, and the existence of a functional heterogeneity : several forms of the enzyme which show different catalytic properties may be present simultaneously. This observation could illuminate the structural heterogeneity observed by crystallography and question the proposed mechanisms on the basis of these structures.

Les acides ribonucléiques (ARN) subissent plusieurs modifications posttranscriptionnelles avant de remplir leur rôle dans la cellule. Un des acteurs responsables de ces modifications sont les ligases à ARN. Il existe deux grandes familles de ligases à ARN soit les « ATP-grasp » et les « RtcB-like ». Malgré le fait que ces enzymes ont des rôles biologiques similaires dans la cellule, leurs mécanismes moléculaires sont très différents. Notre équipe a découvert chez un eucaryote marin la présence de trois gènes codant pour des homologues de ligases à ARN de type RtcB. Ceci est pour le moins inhabituel considérant que la plupart des espèces eucaryotes n’ont qu’un seul gène codant pour des ligases de ce type. Diplonema papillatum, l’organisme en question, est un eucaryote dont l’étude a gagné en popularité au courant des dernières années dû au mode d’expression particulier de son matériel génétique mitochondrial. Celui-ci est fragmenté en plusieurs morceaux appelés modules qui, durant le processus de maturation de l’ARN, sont joints ensemble via épissage en trans. Nous supposons que l’une des ligases de type RtcB présente chez D. papillatum, plus spécifiquement DpRTCB1, est un des acteurs principaux de ce phénomène d’épissage en trans. Nous pensons aussi que les deux autres RtcB présentes chez cet organisme, soit DpRTCB2 et DpRTCB3, ont chacune leur propre rôle dans la cellule. Nous avons donc modélisé la structure tertaire de ces protéines in silico donnant ainsi des indices quant à ce qui pourraient être requis comme cofacteurs par ces trois enzymes. Nous proposons aussi un système de classification des ligases de type RtcB en fonction de leurs rôles biologiques et de leurs variations au niveau des résidus composant le site actif de l’enzyme. Nous avons tenté de purifier des protéines de fusion DpRTCB pour de futurs essais enzymatiques afin de déterminer les rôles biologiques potentiels de ces enzymes. Toutefois, ces protéines formaient des corps d’inclusion rendant leur purification difficile. Ce faisant, nous démontrons les différentes techniques qui existent actuellement pour purifier des protéines à partir d’agrégats insolubles. Ce mémoire prédit les potentiels cofacteurs et substrats nécessaires pour de futurs essais biochimiques des ligases DpRTCB. Nous établissons aussi une base robuste pour un système de classification des ligases de type RtcB. Ce document prodigue entre autres des solutions de base aux chercheurs désireux de purifier des protéines qui forment des corps d’inclusion avant de considérer passer à des méthodes de purification plus laborieuses et coûteuses. / Ribonucleic acids (RNA) undergo several post-transcriptional modifications before fulfilling their role in the cell. One of the actors responsible for these modifications are RNA ligases. There are two main families of RNA ligases, namely “ATP-grasp” and “RtcB-like”. Despite the fact that these enzymes have similar biological roles in the cell, their molecular mechanisms are very different. Our team has discovered in a marine eukaryote the presence of three genes encoding RtcB RNA ligase homologs. This is unusual considering that eukaryotes generally have only one gene encoding for an homolog of this ligase. Diplonema papillatum, the organism in question, is a eukaryote that has grown in popularity in recent years due to the particular mode of expression of its mitochondrial genetic material. Its genome is fragmented into several pieces called modules which, during the RNA maturation process, these modules undergo ligation via trans-splicing. We posit that one of the RtcB-type ligases present in D. papillatum, more specifically DpRTCB1, is a major player in this trans-splicing phenomenon. We also believe that the other two RtcB ligases present in this organism, DpRTCB2 and DpRTCB3, each have its own role in the cell. We therefore established in silico models for these proteins which could hint at the cofactors required by these three enzymes. We also propose a classification system for RtcB-type ligases according to their biological roles and variations in known active site residues. We attempted to purify DpRTCB fusion proteins for future enzymatic assays in order to get a better understanding in the biological role of these enzymes. These proteins, however, formed inclusion bodies making their purification difficult. Thus, we demonstrate various techniques that currently exist to attempt to purify proteins from insoluble aggregates. This Master’s thesis attempts to predict the potential cofactors and substrates necessary for future biochemical assays of DpRTCB enzymes. We also establish a robust foundation for a classification system of RtcB-type ligases. Among other things, this document provides basic solutions to researchers wishing to purify proteins that form inclusion bodies before considering switching to more laborious and expensive purification methods.

Les protéines Binder of Sperm (BSP) appartiennent à une superfamille de protéines exprimées dans le système reproducteur masculin, plus particulièrement dans les vésicules séminales chez les ongulés, et dans l’épididyme chez l’humain et la souris. Jusqu'à présent, des rôles variés chez différentes espèces ont été démontrés pour les protéines BSP, tels que dans la motilité et la capacitation chez le bovin. Cependant, leur rôle demeure élusif chez d’autres mammifères comme la souris et l’humain. Des études in vivo récentes ont démontré que la délétion des gènes Bsph1 et Bsph2 chez la souris n’a aucune conséquence sur la fertilité, et n’induit aucune anomalie au niveau de l'appareil reproducteur masculin. Afin d'élucider le rôle spécifique de la protéine BSP chez l'humain (BSPH1), nous avons d’abord développé une méthode de purification efficace permettant d’obtenir la protéine BSPH1 fonctionnelle car ces protéines ne sont présentes qu'en quantité infime dans l’épididyme humain. Suite, a la purification de BSPH1, j’ai réalisé des expériences in vitro et cherché à identifier son réseau d'interaction protéique. Il a été démontré que les protéines BSP interagissent avec des groupes pseudo-choline tels que le diéthylaminométhyle par affinité plutôt que par des interactions ioniques. Le diéthylaminoéthyle est chargé positivement et par conséquence, est un échangeur d'anions faible, mais les BSP interagissent avec affinité à la résine. Cette étude présente également une nouvelle méthode de purification rapide et peu coûteuse, qui fournit des protéines BSP recombinantes de grande pureté qui peuvent être utilisées pour étudier leurs rôles dans la fécondation chez les mammifères. Nous avons montré que la pré-incubation des ovocytes avec la protéine BSPH1 recombinante peut diminuer le taux de fécondation de manière dose-dépendante. Les spermatozoïdes ont également été pré-incubés avec un anticorps anti-BSPH1 et ont montré une diminution du taux de fécondation. Pour identifier le réseau d’interaction protéique de BSPH1, j'ai utilisé la méthode « Proximity-dependent biotin identification » (BioID) couplée à la spectrométrie de masse. Les résultats de la spectrométrie de masse ont démontré une interaction entre BSPH1 et toutes les sous-unités du complexe CCT / TRIC (Chaperonin containing tailless complex polypeptide 1 (CCT) ou tailless complex polypeptide 1 ring complex (TRiC)). Ce complexe interagit avec un autre complexe appelé BBSome (Bardet–Bied syndrome complex), qui joue un rôle important dans le transport de protéines à travers les cils primaires. BSPH1 a également interagi avec un grand nombre de protéines de la famille CEP (centrosome-associated proteins), importantes dans la formation des cils primaires par les microtubules et de la maturation du centrosome, qui soutiennent le rôle de BSPH1 dans les cils primaires. Dans l’ensemble, cette étude démontre que BSPH1 pourrait avoir un nouveau rôle en tant que chaperonne, à travers les cils primaires dans les cellules qui l’expriment dans l’appareil reproducteur masculin. / Binder of SPerm (BSP) proteins belong to a superfamily of proteins expressed in the male reproductive tract, particularly in seminal vesicles of ungulates (e.g., bovine, ram) and in the epididymis of humans and mice. So far, BSP proteins have been shown to play different roles in different species such as in motility and capacitation in bovine; however, their role remains unclear in other mammals. For instance, depletion of Bsph1/Bsph2 in mice had no effect on fertility. In order to elucidate the specific role of BSP protein in humans (BSPH1), I sought to investigate a purification method to produce functional human BSP protein, as these proteins are only present in minute amounts in the human epididymis. Following purification of BSPH1, I carried out in vitro experiments and sought to identify its protein interaction network. BSP proteins have been shown to interact with pseudo-choline groups such as diethylaminomethyl through affinity rather than ionic interactions. Diethylaminoethyl is positively charged and therefore is a weak anion exchanger, but BSPs interact through affinity to this resin. This study presents a new, rapid and cost-effective purification method that provides recombinant BSP proteins of a high purity level, which can be used to study their roles in mammalian fertilization. We showed that pre-incubation of oocytes with recombinant BSPH1 can decrease fertilization rate in a dose-dependant manner. Sperm were also preincubated with anti-BSPH1 antibody and showed a decrease in fertilization rate. Secondly, I used BioID (proximity-dependent biotin identification), coupled with mass spectrometry to identify the protein-protein interaction network of BSPH1 by proximity labeling. Mass spectrometry results showed an interaction between BSPH1 and all subunits of the CCT/TRIC complex (Chaperonin containing tailless complex polypeptide 1 (CCT) or tailless complex polypeptide 1 ring complex (TRiC). This complex interacts with another complex called BBSome (Bardet–Biedl syndrome complex), which plays a role in protein trafficking through primary cilium. I also identified BBS proteins, as well as other proteins, that interact with the BBSome complex and regulate protein trafficking in the cilia. BSPH1 also interacted with a large number of CEP (centrosome-associated proteins) family proteins, important in the formation of primary cilium through microtubules and centrosome maturation, which further support the potential implication of BSPH1 with the primary cilia. Overall, this study demonstrates that BSPH1 may have a new role as a chaperone involved in protein trafficking through the primary cilia in cells that express it in the male reproductive system

6 Abstract Transcription factors play a key role in the management of cell growth and differ- entiation and their deregulation is associated with many cancers. TEAD proteins utilise highly conserved DNA binding domain to recognise specific DNA sequences. This domain could facilitate new drug design and development. The goal of this master thesis includes recombinant preparation of DNA binding domain of transcriptional factor TEAD4 extended by a part of an unstruc- tured variable sequence, which connects this domain with transactivation domain. Purification steps include affinity chromatography followed by size exclusion chro- matography. The characterization of produced protein was performed by mass spectrometry and finally, native gel electrophoresis was used to prove the ability of the produced protein to bind DNA. During purification steps, a fragmentation from C-terminus was observed. Based on analysis of the mass spectra, three most represented forms of produced protein were described all of which were fragmented. The most abundant form (55%) consisted of amino acids 30-131 from TEAD4 protein. Second most abun- dant form (18%) consisted of amino acids 30-144 and the third form consisted of amino acids 30-81. Native gel electrophoresis verified the ability to bind DNA, the efficiency was however lower...