Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 189-242). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 189-242). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 143-164). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 143-164). Also available on the Internet.

La cardosine A est une protéase aspartique identifiée il y a plus de 20 ans dans les cellules du chardon Cynara cardunculus. Sa distribution dans tous les tissus de la plante et ses caractéristiques enzymatiques ont été caractérisées par approches biochimiques. La cardosine A a des fonctions essentielles dans la reproduction, la mobilisation de réserves protéiques, et le remaniement de membranes. Pour assumer ces différentes fonctions, la cardosine A doit pouvoir transiter et s’accumuler dans différents compartiments intracellulaires : vacuole de stockage, vacuoles lytiques, ou autres compartiments membranaires. Il n’y a cependant que très peu de données disponibles sur les mécanismes de biosynthèse, de tri, de transport et d’adressage aux différents compartiments cellulaires. De récents travaux suggèrent que l’expression en modèle hétérologue pourrait être utilisée pour une meilleure compréhension de la biologie intracellulaire de la cardosine A. Les résultats de cette étude montrent que l’expression transitoire de la cardosine A dans les feuilles de Nicotiana tabacum est un bon modèle expérimental pour explorer le transport de la cardosine A dans la cellule. En effet dans ce système les mécanismes de maturation et de transport de la protéine à la vacuole sont conservés. De plus, une lignée stable d’Arabidopsis thaliana exprimant la cardosine A sous promoteur inductible s’est également avérée un bon modèle d’étude du transport intracellulaire de la cardosine A. L’utilisation de ces systèmes hétérologues a permis de combiner l’expression de formes mutées de la cardosine A (dans lesquelles des séquences spécifiques ou des acides aminés avaient été tronqués ou modifiés) avec des approches de biochimie et d’imagerie cellulaire pour identifier des signatures moléculaires responsables de l’adressage vacuolaire de la protéine. Nos résultats montrent que la cardosine A a deux déterminants vacuolaires dans sa séquence protéique : le domaine “PSI”, qui définit un déterminant d’adressage vacuolaire original et propre à certaines protéases aspartiques, et un peptide C-terminal appartenant à la classe bien définie des ctVSD. De plus, les résultats montrent que la présence de ces deux déterminants illustre la capacité d’emprunter deux routes distinctes pour atteindre la vacuole : le domaine PSI peut permettre d’attendre la vacuole sans passer par le Golgi, tandis que le domaine C-ter négocie un transport classique Reticulum, Golgi, Prévacuole, Vacuole. Cette capacité à choisir deux routes différentes n’est pas observée pour la cardosine B, autre protéase aspartique du chardon présentant une haute homologie de séquence avec la cardosine A. Pour expliquer cette capacité de la cardosine A à emprunter deux routes vacuolaires différentes, l’hypothèse d’un rôle possible de la glycosylation dans le tri des protéines entre les deux routes vacuolaires est alors étudiée. L’expression de la cardosine A dans les graines en germination d’Arabidopsis thaliana révèle que la protéine peut s’accumuler d’une manière différentielle dans les graines en absence de germination ou pendant la germination, tout au long du système endomembranaire jusqu’à la vacuole de réserve ou dans les vacuoles lytiques en formation. Les expériences de blocage de transport du Reticulum au Golgi n’ont pas permis de conclure d’une manière certaine si les accumulations vacuolaires dérivaient d’un transport pouvant court-circuiter le Golgi comme dans les feuilles de Nicotiana. Au total, la cardosine A constitue une protéine modèle pour étudier les transports vacuolaires chez Nicotiana tabacum and Arabidopsis thaliana, deux systèmes hétérologues qui permettent de développer des méthodes complémentaires pour une exploration fonctionnelle des mécanismes impliqués. Cette étude permet de contribuer à une meilleure connaissance de la biologie des cardosines en particulier, et des protéases aspartiques en général. / The aspartic proteinase cardosin A is a vacuolar enzyme found to accumulate in protein storage vacuoles and lytic vacuoles in the flowers and in protein bodies in seeds of the native plant cardoon. Cardosin A has been first isolated almost two decades ago and has been extensively characterized since, both in terms of distribution within the tissues and of enzyme biochemistry. In the native system, several roles have been addressed to cardosin A in reproduction, mobilization of reserves and membrane remodeling. To participate in such diverse events, cardosin A must accumulate and travel to different compartments inside the cell: protein storage vacuoles, lytic vacuoles, cytoplasmic membrane (and eventually outside the cell). However, not much information is available regarding cardosin A biogenesis, sorting or trafficking to the different compartments. Recent studies have approached the expression of cardosin A in Arabidopsis thaliana and Nicotiana tabacum. These preliminary observations were the starting point of a detailed study of cardosin A expression, localisation, sorting and trafficking routes, resourcing to several and very different methods. It has been showed that transient expression of cardosin A in Nicotiana tabacum leaf is a good system to explore cardosin A trafficking inside the cell, as the protein is processed in a similar manner as the control and accumulates in the vacuole. Furthermore, an Arabidopsis thaliana line expressing cardosin A under an inducible promoter was explored to understand cardosin A dynamics in terms of vacuolar accumulation during seed germination events. Similarly to the Nicotiana tabacum one, this system was also validated for cardosin A expression and it allowed to conclude that the protein’s expression did not retrieved any phenotype to the cells or individuals. However, experiments conducted in BY-2 cells revealed to be inconclusive since cardosin A expression in this system is not predictable. The data obtained along this work using several cardosin A mutated forms, lacking specific domains or point-mutated, allowed to determine that cardosin A has two Vacuolar Sorting Determinants in its protein sequence: the PSI, an unconventional sorting determinant, and the C-terminal peptide, a C-terminus sorting determinant by definition. Furthermore, it was also demonstrated that each domain represents a different route to the vacuole: the PSI bypasses the Golgi Apparatus and the C-terminal peptide follows a classic Endoplasmic Reticulum-Golgi Apparatus-Prevacuole route to the vacuole. This difference in the trafficking routes is not observed for cardosin B sorting determinants as both the PSI and C-terminal peptide from cardosin B needs to pass the Golgi Apparatus to reach the vacuole. A putative role for glycosylation in the trafficking routes is further discussed as cardosin A PSI, contrary to cardosin B, is not glycosylated. The production of mutants affecting cardosin A glycosylation sites supported this idea. Moreover, cardosin A expression in germinating Arabidopsis thaliana seeds revealed a differential accumulation in non-germinated and germinated seedlings. Cardosin A was detected along the secretory pathway to the Protein Storage Vacuole in association with the Endoplasmic Reticulum, Golgi Apparatus, Prevacuole and newly formed Lytic Vacuoles. The drug Brefeldin A caused the protein to be retained in the Golgi Apparatus, despite some amount being still detected in the vacuole, not being clear if the Golgi Apparatus bypass observed in Nicotiana tabacum leaves occurs in this system. As a whole, cardosin A confirmed to be a good model to study vacuolar sorting in these two systems that complement each other in terms of approaches available. This study provided good results in order to understand in more detail cardosin A biology in particular and vacuolar trafficking of plant Aspartic Proteinases as a group.

published_or_final_version / Public Health / Master / Master of Public Health

Structural investigations on two mycobacterial enzymes were carried out. Tuberculosis still remains a major threat to mankind even though drugs against it have been in use for many decades. The emergence of drug resistant strains of the bacteria calls for the identification of new targets based on which new drugs can be developed to combat the disease. A thorough understanding of the functioning of the target molecules is essential for this approach. We have taken up the structural studies on two such molecules, aspartic proteinases and adenylyl cyclases, of Mycobacterium tuberculosis with a view to obtain insights into their mechanisms of action at the atomic level. The work presented in the thesis includes (i) the identification, cloning, expression, purification and structure determination of a putative aspartic proteinase domain of M. tuberculosis and (ii) the crystal structure of an adenylyl cyclase of M. tuberculosis and its mutant; and also of an adenylyl cyclase from M. avium. Chapter 1 presents an overview of aspartic proteinases and nucleotide cyclases with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2. Work on the putative aspartic proteinase domain identified in M. tuberculosis is presented in Chapter 3. The structure of the aspartic proteinase domain is the first structural report of such domain from any bacteria. A search in the genome of M. tuberculosis showed a weak similarity to the HIV aspartic proteinase sequence. This region corresponds to the C-terminal domain of a PE family protein in M. tuberculosis. The presence of two signature motifs, DTG and DSG, of aspartic proteinases in the full sequence of this domain encouraged us to take up further studies on this domain. Previous reports identifying the protein as a surface antigen and our findings on the occurrence of similar domains in two other PE proteins of M. tuberculosis and also in other pathological strains of Mycobacteria indicated that these domains probably play an important role in infecting the host. The crystal structure of one of the domains showed that it has a pepsin-like fold and the catalytic site architecture of known aspartic proteinases. However, no proteolytic activity was detected. The size of the molecule is intermediate to eukaryotic pepsins and the homodimeric retroviral pepsins. A close examination of the binding site revealed subtle differences when compared to the active enzyme structures. Appropriate mutations of some of the residues in this region to convert it to an active enzyme did not make it active. Once the in vivo function of these putative aspartic proteinase domains is established, their potential to act as drug targets can be probed as the PE proteins are present exclusively in pathogenic Mycobacteria. As part of an ongoing project on adenylyl cyclases of Mycobacteria, we have taken up the structure analysis of the catalytic domains of two adenylyl cyclases; Rv1625c from M. tuberculosis and Ma1120 from M. avium. This work is described in Chapter 4. The wild-type of Rv1625c crystallized as a domain swapped head to head inactive dimer even though it is an active dimer in solution and expected to have a head to tail arrangement as in the previously reported structures of the active forms of the enzyme. Mutation of a phenylalanine residue presumed to occur at the subunit interface of the active dimeric structure of the enzyme to an arginine residue, a conserved residue of guanylyl cyclases, resulted in reduced adenylyl cyclase activity. This mutant crystallized as a monomer though it was expected to be an active dimer. Similarly, Ma1120 also has a monomeric structure in the crystal in spite of showing activity in solution. Though our aim was to capture the active dimers in the crystalline state we did not succeed in this effort in any of the three cases. The catalytic reaction probably takes place very rapidly with the formation of a transient active form of the dimer which cannot be easily crystallized. However, the analysis revealed new structures which are likely to represent the stable states of the enzyme when it is required to stay inactive in certain conditions. We have also established that the N-terminal segments of the enzyme, a loop at the dimeric interface and external factors like pH are involved in determining the oligomeric status of the enzyme thereby regulating its function. Publications 1 Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16; Deivanayaga V. Barathy and K. Suguna; FEBS Open Bio (In Press) 2 New structural forms of mycobacterial adenylyl cyclases (in preparation)

Sabe-se que a hemoglobina é uma rica fonte de peptídeos antimicrobianos (hemocidinas). A primeira hemocidina derivada da hemoglobina bovina caracterizada em carrapatos foi o peptídeo Hb33-61, que é ativo contra bactérias gram-positivas e fungos. Acredita-se que tais hemocidinas sejam geradas proteoliticamente no intestino do carrapato. Neste trabalho nós caracterizamos bioquimicamente uma catepsina D, designada BmAP. A análise da expressão gênica por qPCR mostrou que ela é expressa predominantemente no intestino. Através de LC-MS/MS, determinamos a especificidade de clivagem da BmAP utilizando Hb bovina, e verificamos que resíduos hidrofóbicos foram preferencialmente clivados nos subsítios P1 e P1. Também investigamos a especificidade de clivagem da catepsina L intestinal BmCL1, utilizando uma biblioteca combinatória de tetrapeptídeos e através de hemoglobinólise in vitro. A BmCL1 preferiu resíduos alifáticos no P2 e polares no P1 e P1. Além disso, hidrolisou a cadeia da Hb bovina entre A63/A64, gerando peptídeos com estrutura primária similar ao Hb 33-61. A hemoglobinólise com a BmAP e/ou BmCL1 resultou na formação de algumas hemocidinas, corroborando a hipótese do seu envolvimento na geração endógena de peptídeos antimicrobianos. / It is known that hemoglobin is a rich source of antimicrobial peptides (hemocidins). The first hemoglobin-derived hemocidin characterized in ticks was the peptide Hb33-61, which is active against Gram-positive bacteria and fungi. It is believed that hemocidins are endogenously generated in the tick gut. In this work we biochemically characterized a cathepsin D, designated BmAP. Expression analysis by qRT-PCR showed that it is expressed predominantly in the gut. Through LC-MS/MS, we determined the cleavage specificity of BmAP using bovine hemoglobin, and we verified that hydrophobic residues were preferentially cleaved at the subsites P1 and P1. We also investigated the cleavage specificity of the intestinal cathepsin L BmCL1, using a positional scanning synthetic combinatorial library and through in vitro hemoglobinolysis. BmCL1 preferred aliphatic residues at P2 and polar residues at P1 and P1. Also, it hydrolysed the subunit of bovine hemoglobin at A63/A64, generating peptides with a primary structure similar to Hb 33-61. Hemoglobinolysis with BmAP and/or BmCL1 resulted in the formation of some hemocidins, corroborating the hypothesis that these proteinases are involved in the endogenous generation of antimicrobial peptides

Sabe-se que a hemoglobina é uma rica fonte de peptídeos antimicrobianos (hemocidinas). A primeira hemocidina derivada da hemoglobina bovina caracterizada em carrapatos foi o peptídeo Hb33-61, que é ativo contra bactérias gram-positivas e fungos. Acredita-se que tais hemocidinas sejam geradas proteoliticamente no intestino do carrapato. Neste trabalho nós caracterizamos bioquimicamente uma catepsina D, designada BmAP. A análise da expressão gênica por qPCR mostrou que ela é expressa predominantemente no intestino. Através de LC-MS/MS, determinamos a especificidade de clivagem da BmAP utilizando Hb bovina, e verificamos que resíduos hidrofóbicos foram preferencialmente clivados nos subsítios P1 e P1. Também investigamos a especificidade de clivagem da catepsina L intestinal BmCL1, utilizando uma biblioteca combinatória de tetrapeptídeos e através de hemoglobinólise in vitro. A BmCL1 preferiu resíduos alifáticos no P2 e polares no P1 e P1. Além disso, hidrolisou a cadeia da Hb bovina entre A63/A64, gerando peptídeos com estrutura primária similar ao Hb 33-61. A hemoglobinólise com a BmAP e/ou BmCL1 resultou na formação de algumas hemocidinas, corroborando a hipótese do seu envolvimento na geração endógena de peptídeos antimicrobianos. / It is known that hemoglobin is a rich source of antimicrobial peptides (hemocidins). The first hemoglobin-derived hemocidin characterized in ticks was the peptide Hb33-61, which is active against Gram-positive bacteria and fungi. It is believed that hemocidins are endogenously generated in the tick gut. In this work we biochemically characterized a cathepsin D, designated BmAP. Expression analysis by qRT-PCR showed that it is expressed predominantly in the gut. Through LC-MS/MS, we determined the cleavage specificity of BmAP using bovine hemoglobin, and we verified that hydrophobic residues were preferentially cleaved at the subsites P1 and P1. We also investigated the cleavage specificity of the intestinal cathepsin L BmCL1, using a positional scanning synthetic combinatorial library and through in vitro hemoglobinolysis. BmCL1 preferred aliphatic residues at P2 and polar residues at P1 and P1. Also, it hydrolysed the subunit of bovine hemoglobin at A63/A64, generating peptides with a primary structure similar to Hb 33-61. Hemoglobinolysis with BmAP and/or BmCL1 resulted in the formation of some hemocidins, corroborating the hypothesis that these proteinases are involved in the endogenous generation of antimicrobial peptides

Candida parapsilosis is an emerging human opportunistic pathogen causing a wide spectrum of potentially life-threatening infections in immunocompromised hosts. One of the most important virulence factors of Candida spp. is a production of secreted aspartic proteinases (Saps). Presented thesis is mainly focused on the study of secreted aspartic proteinase 1 (Sapp1p) of C. parapsilosis, its processing and secretion under variable conditions and by use of various experimental models. Sapp1p is secreted by C. parapsilosis cells into the extracellular space as a completely processed and fully active enzyme. Experiments studying the C. parapsilosis cell wall (CW) confirmed the prolonged presence of completely processed Sapp1p on the cell surface (CW- Sapp1p). Proteolytic activity assay performed with the intact cells showed that CW-Sapp1p is proteolytically active prior to its release into the extracellular space and is capable of substrate cleavage. Biotinylation experiments with consecutive MS analysis revealed that CW-Sapp1p biotinylation is incomplete but saturable process, leaving partially unlabelled molecules. The accessibility of individual lysine residues in the Sapp1p molecule varied, with exception of four residues that were labelled in all of our experiments performed. The final step of...