Els dominis d'activació són presents en la porció N-terminal de la forma inactiva de les procarboxipeptidases de la subfamília de proteases M14A, i les seves funcions descrites comprenen tant el manteniment d'aquest estat inactiu com l'assistència en el plegament de l'enzim complet. La limitada mida i la notable estabilitat derivada de la gran quantitat d'estructura secundària han fet que un d'aquests dominis, el de la procarboxipeptidasa A2 humana (ADA2h) hagi estat un bon model per a l'estudi del plegament proteic, en treballs anteriors, i de l'agregació en forma de fibres amiloides en la present tesi. Els estudis cinètics de dicroisme circular de la variant salvatge i una extensa bateria de mutants puntuals revelen la seqüència corresponent a la cadena beta-4 com la responsable de la direcció del procés d'agregació ordenada en conformació beta. Mutacions en aquesta zona tenen la capacitat tant d'accelerar dramàticament la velocitat d'agregació, com d'abolir-la totalment. Les correlacions amb algorismes predictius de l'agregació basats en les propietats físico-químiques de la seqüència polipeptídica posen de manifest la rellevància principal de l'estructura primària en el govern del procés. La cinètica d'agregació per dicroisme circular mostra una naturalesa dual, amb una segona etapa pràcticament independent de concentració proteica, indicant una etapa tardana de reorganització conformacional. Estudis cinètics complementaris per espectroscòpia d'infraroig mostren igualment una reorganització molecular i assenyalen que la conformació agregada en beta present a la fibra amiloide sembla originar-se primerament a partir de les hèlixs-alfa parcialment desplegades. L'estudi de mutants amb diferent propensió a l'agregació i estabilitat ha mostrat que les mutacions comporten canvis en la via d'agregació, però que finalment arriben al mateix punt de fibril·lació.Una possible funció alternativa per a un d'aquests dominis, els quals tenen una extraordinària complexitat en comparació amb altres seqüències inhibidores en cis, ha estat cercada mitjançant eines bioinformàtiques. Certa similitud seqüencial i estructural sembla ser trobada entre el domini d'activació de la procarboxipeptidasa A4 humana (ADA4h) i un domini d'unió a RNA (RRM), suggerint una possible funció ancestral per al primer, bé que no tots els residus claus per a la interacció són presents a ADA4h. La hipòtesi ha estat comprovada experimentalment mitjançant un gel de retardament, observant-se una unió a RNA dèbil i inespecífica per al RNA assajat, d'acord amb el que s'havia predit bioinformàticament. Aquest fet no exclou, emperò, una unió fisiològica i específica amb un altra molècula de RNA diferent a l'assajada. / Los dominios de activación están presentes en la porción N-terminal de la forma inactiva de las procarboxipeptidasas de la subfamilia de proteasas M14A, y sus funciones descritas comprenden tanto el mantenimiento de este estado inactivo como la asistencia en el plegamiento de la enzima completa. Su limitada longitud y notable estabilidad, derivada de la gran cantidad de estructura secundaria, han hecho que uno de estos dominios, el de la procarboxipeptidasa A2 humana (ADA2h) haya sido un buen modelo para el estudio del plegamiento proteico, en trabajos anteriores, y de la agregación en forma de fibras amiloides en la presente tesis. Los estudios cinéticos de dicroísmo circular de la variante salvaje y una extensa batería de mutantes puntuales revelan la secuencia correspondiente a la cadena beta-4 como la responsable de la dirección del proceso de agregación ordenada en conformación beta. Mutaciones en esta zona tienen la capacidad tanto de acelerar dramáticamente la velocidad de agregación, como de abolirla totalmente. Las correlaciones con algoritmos predictivos de agregación basados en las propiedades fisicoquímicas de la secuencia polipeptídica ponen de manifiesto la relevancia principal de la estructura primaria en el gobierno del proceso. La cinética de agregación por dicroísmo circular muestra una naturaleza dual, con una segunda etapa prácticamente independiente de concentración proteica, indicando una etapa tardía de reorganización conformacional. Estudios cinéticos complementarios por espectroscopia de infrarrojo muestran igualmente una reorganización molecular y señalan que la conformación agregada en beta presente en la fibra amiloide parece originarse primeramente a partir de las hélices alfa parcialmente desplegadas. El estudio de mutantes con diferente propensión de agregación y estabilidad ha demostrado que las mutaciones producen cambios en la vía de agregación, pero que finalmente convergen en el mismo punto de fibrilación.Una posible función alternativa para uno de estos dominios, las cuales tienen una extraordinaria complejidad en comparación con otras secuencias inhibidoras en cis, ha sido buscada mediante herramientas bioinformáticas. Cierta similitud secuencial y estructural parece haber sido encontrada entre el dominio de activación de la procarboxipeptidasa A4 humana (ADA4h) y un dominio de unión a RNA (RRM), sugiriendo una posible función ancestral para el primero, aunque no todos los residuos clave para la interacción son presentes en ADA4h. La hipótesis ha sido comprobada experimentalmente mediante un gel de retardo, observándose una unión a RNA débil e inespecífica para el RNA ensayado, de acuerdo con lo que había sido predicho bioinformáticamente. Este hecho no excluiría una unión fisiológica y específica con otra molécula de RNA diferente a la ensayada. / The activation domains are found in the N-terminal portion of the inactive form of the M14A subfamily procarboxypeptidases, and their described functions comprise the maintenance of the inactive state and the folding assistance of the proenzyme. Their limited size and notable stability, derived from the high amount of secondary structure, promoted the use of one of these domains, the activation domain of procarboxypeptidase A2 (ADA2h), as a folding model, in previous works, and as an amyloid formation model in the present thesis. The kinetic experiments of aggregation on the wild-type protein and a big battery of point mutants indicate that the sequence corresponding to beta-strand 4 is the main responsible of the direction of the ordered aggregation process. Mutations in this zone can either accelerate or decelerate the velocity of the aggregation process, and even abolish aggregation completely. The correlations with prediction algorithms based on the physicochemical properties of the polypeptide sequence show the relevance of the primary structure in the lead of the process. The kinetics of aggregation followed by circular dichroism exhibit a dual nature, with a second phase almost independent of protein concentration, thus indicating a late phase of conformational reorganisation. Complementary kinetic studies on ADA2h followed by infrared spectroscopy also reveal a molecular reorganisation phase, and identify the partially exposed alpha-helices as the origin of the aggregated conformation. These experiments were carried out with 3 other point mutants with different stability and aggregation velocities (according to the previous experiments), and proved that the mutations introduce changes in the aggregation pathway although the same final point is reached for all of them.A possible alternative function for one of these domains, which have an extraordinary complexity compared to other inhibitory sequences found in cis, has been investigated using bioinformatic tools. Some sequential and structural similarity has been found between the activation domain of procarboxypeptidase A4 (ADA4h) and a RNA binding domain (RRM), thus suggesting a possible ancestral function for the activation domain. However, not all the key residues required for an interaction with RNA can be found in ADA4h, hence a weaker interaction was predicted. This hypothesis has been experimentally tested using electrophoretic mobility shift assays (EMSA), observing a weak and unspecific RNA binding. It remains to be tested, however, if a physiological (and therefore specific) binding could be carried out for ADA4h using a different RNA molecule from the one assayed.

Schistosomiasis is a serious parasitic disease caused by blood flukes of the genus Schistosoma. It is a global health problem with more than 200 million people infected and 750 million people at risk. Current therapy relies on a single drug, praziquantel, for which there are concerns of emerging drug resistance. Proteases of schistosoma are promising target molecules for the development of new therapeutic strategies against schistosomiasis. This work focuses on the comprehensive characterization of proteolytic systems of Schistosoma mansoni and determination of their role in the interaction with the human host. First, the major proteolytic activities secreted by individual developmental stages of schistosoma that parasitize the human body were classified using functional proteomics. This analysis demonstrated their complex and specific distribution with predominant serine and cysteine proteases and metalloproteases. Second, tegumental and digestive proteases, namely prolyl oligopeptidase and cathepsins B, C and D, were identified by chemical genomics as suitable target molecules for therapeutic intervention. Prolyl oligopeptidase was biochemically characterized using a recombinant protein, its effective inhibitors were developed as templates for antischistosomal drugs, and a biological role of the...

Introduction: Since its introduction, Highly Active Antiretroviral Treatment (HAART) has been shown to prolong the life expectancy of HIV-infected patients. HIV and HAART, especially protease inhibitors (PIs), have been associated with the occurrence of Metabolic Syndrome (MS). The objective of this systematic review and meta-analysis was to determine whether there is an association between the use of PIs and the incidence of MS in HIV-infected patients. Methods: A comprehensive search (including databases such as MEDLINE/PubMed, CENTRAL, LILACS and EMBASE) was performed. Observational studies published until November 2015 were included. Inclusion criteria for primary studies were: study population comprised HIV-infected patients aged 18 years or older and who were receiving HAART; patients assessed according to their use of PIs; DM as defined by the primary study. Heterogeneity was assessed and a pooled analysis was performed using a random-effects model. Results: 3 articles met the inclusion criteria, describing 586 HIV patients. Use of PIs was associated with the development of MS (RR: 2.11; 95% CI 1.28 to 3.48; 〖Chi〗^2:0.04, I^2: 0%; p-value 0.003). Conclusion: Use of PIs in HIV-infected patients is associated with an increased risk of MS. These findings are of relevance for future public policy because it will increase the interest in screening and prevention of MS in an expanding population. / Tesis

Blood flukes of the genus Schistosoma are causative agents of the disease schistosomiasis, which affects more than 250 million people worldwide and together with malaria represents the most important parasitic infection. There is a high risk of resistance development against the only drug in use, therefore novel therapeutic approaches for schistosomiasis are intensively researched. Proteolytic enzymes of schistosomes are crucial for their survival in the host and thus are promising drug and vaccine targets. This thesis is focused on two proteases of the human blood fluke Schistosoma mansoni, which were produced as recombinant proteins and functionally characterized. The first one is serine protease SmSP2, which is localized at the surface of the adult worms and secreted into the blood of the host. It was identified as a vasodilatory and fibrinolytic agent, and its modulatory role in host-parasite interactions was proposed. The second one is cysteine cathepsin SmCL3, which is involved in the digestion of host blood proteins serving schistosomes as nutrients. Potent peptidomimetic inhibitors of SmCL3 were identified, and their antischistosomal activity was demonstrated in an assay with live parasites. The thesis provides new important information about S. mansoni proteases, their pathobiochemistry...

Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.

Serine beta-lactamase-like protein (LACTB) is a tumour suppressor that modulates mitochondrial lipid metabolism and induces differentiation of breast cancer cells. This is achieved by the LACTB-dependent downregulation of phosphatidylserine- decarboxylase (PISD) which subsequently leads to decreases in the amounts of phosphatidylethanolamines and lysophosphatidylethanolamines in mitochondrial membranes. However, PISD was shown to not be a direct substrate of the LACTB enzyme what leaves the identity of the LACTB substrate an open question. To fill this important gap in the mechanism of the LACTB tumour suppressive pathway, this diploma thesis was focused on finding a physiological substrate of LACTB via Proteomic Identification of protease Cleavage Sites (PICS) assay. For this purpose, the other sub-aims of this project were to isolate recombinant wild-type LACTB and its catalytic mutant, to reveal ideal in vitro conditions for LACTB activity and to find out the requirements needed for LACTB multimerization. My results show that in vitro activity of LACTB is increased in the presence of higher pH and calcium ions. I also show that higher LACTB multimeric forms are bound together via disulfide bonds as they disintegrate after treatment with dithiothreitol. Furthermore, and most importantly, I show...

Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...

Blood fluke Schistosoma mansoni is one of the most important human parasites. Proteolytic system of schistosoma is crucial for parasite - host interactions. Therefore some of the proteases became potential therapeutic targets. This work is focused on not yet characterized serine protease SmSP2. SmSP2 is newly discovered protease of S. mansoni, whose biological role is unknown. This protease is highly expressed in developmental stages parasitizing humans. SmSP2 was recombinantly expressed in prokaryotic and eukaryotic expression system (E. coli a P. pastoris) and purified using chromatographic methods. Recombinant SmSP2 was used for polyclonal antibody production. Conditions for refolding were optimized. Basic biochemical properties of the protease were detected and substrate amino acid preferences for P1 - P4 sites for single aminoacids were identified using synthetic fluorogenic peptides for positional scanning substrate combinatorial library (PS-SCL). (In Czech)

Tot i que s'han assolit molts reptes en la lluita contra el SIDA, actualment encara se'n desconeix la cura definitiva. Tots els fàrmacs assequibles comercialment i llur combinació en l'anomenada teràpia antiretroviral altament activa (HAART) milloren la qualitat de vida de les persones infectades però l'actual teràpia té uns costos molt elevats, sobretot si tenim en compte que els 95% de la gent infectada viu en països en vies de desenvolupament, i pateix altres limitacions com ara la necessitat de dosis elevades, els efectes secundaris i sobretot, el problema de la resistència als fàrmacs. A més a més aquests inhibidors, tot i tenir una forta activitat, no són ideals, ja que en general tenen una baixa solubilitat aquosa, s'eliminen fàcilment, no són assequibles per via oral i tenen una durada d'acció breu.La present Tesi doctoral té per objectiu el disseny, síntesi i avaluació de nous inhibidors de la proteasa del VIH-1 que actuïn per un mecanisme d'inhibició diferent als comercialitzats fins al moment i que ajudi a combatre els inconvenients associats als inhibidors comercials.En aquest treball es descriu en primer lloc la síntesi de diferents estructures pseudodipeptídiques: l'oxazolopiperidona 80, el lactam de Freidinger 82 i les pirrolizidinones (6R)- i (6S)-79.A continuació es descriu la incorporació de l'oxazolopiperidona 80 i el lactam de Freidinger 82 en nous inhibidors de dimerització de la HIV-1 PR, mitjançant síntesi peptídica en fase sòlida.Els inhibidors sintetitzats es poden subdividir en 3 categories: a) inhibidors patró formats per aminoàcids naturalsb) inhibidors de tipus I, formats per una sola cadena peptídica i que incorporen una de les estructures pseudodipeptíquesc) inhibidors de tipus II, formats per dues cadenes peptídiques i que incorporen una de les estructures pseudodipeptídiques d) inhibidors de tipus II, formats pels subproductes de reacció simètrics fruit de l'entrecreuament entre cadenes peptídiques.Aquests nous compostos han estat avaluats in vitro mitjançant un assaig de fluorescència sobre la HIV-1 PR expressada en E.coli. Aquest estudi ha mostrat 8 dels inhibidors presenten baixa activitat i 7 activitat moderada. D'aquests compostos amb activitat moderada, els compostos CH-50, 176 i 181 han mostrat ser inhibidors dissociatius.Addicionalment, s'ha assajat l'activitat in vivo dels inhibidors sintetitzats. Els assaigs in vivo han mostrat que 3 dels compostos tenen activitat baixa anti-VIH sobre el virus wild type, mentre que 4 presenten activitat moderada (176, 186, 187cc i 189). Els assaigs in vivo també han evidenciat la baixa toxicitat dels inhibidors sintetitzats.Entre els compostos sintetitzats destaca l'inhibidor 176, que ha resultat tenir activitat anti-VIH sobre un virus resistent als inhibidors de proteasa comercials. Es tracta, doncs, d'un bon cap de sèrie per a la investigació de nous inhibidors de la proteasa del VIH-1 actius en línies cel·lulars resistents als inhibidors comercials.Finalment, s'han dut a terme experiments de microcalorimetria per tal d'estudiar la interacció del compost 176 amb la HIV-1 PR, on s'ha observat que aquest compost presenta un perfil d'interacció amb la proteasa similar a l'inhibidor comercial ritonavir, tot i presentar menor afinitat (Ka). Aquests experiments també han posat de manifest que l'inhibidor 176 actua sobre la forma dimèrica de la proteasa, sense arribar a desplaçar un dels monòmers. / Dimerization of HIV-1 protease is essential to attainment of the mature structure, an enzymatically active C2-symmetric homodimer. The dimerization interface is largely composed of interdigitated N- and C-terminal portions of the protease, which thus form a four-stranded antiparallel ß-sheet. By targeting this ß-sheet portion of the protease, a region which is highly conserved among HIV-1 isolates, agents may be generated which block the assembly of the homodimer or disrupt the dimeric interface, and so should lead to a loss of biological activity.Dimerization inhibitors have been developed in the context of this research based on a structure composed by the native sequences of HIV-1 protease N- and C-termini and by substitution of some of the dipeptides in these structures for pseudopeptides with restricted conformation (3-aminolactams derivatives), to increase the conformational constraint and convert them, this way, in more active agents and less prone to degradation.First of all, the synthesis of oxazolopiperidone 80, Freidinger lactam 82 and pyrrolizidinones (6R)- i (6S)-79 has been achieved and these structures have been incorporated by SPPS into the N- and C-terminal peptide chains of HIV-1 PR.The resulting compounds have been tested in vitro by means of a fluorescence assay, using HIV-1 PR expressed in E. Coli. As a result, 7 compounds with moderate activity have been identified.An in vivo test has been also carried out, showing that 4 of the synthesized compounds (176, 186, 187cc i 189) present moderate activity against the HIV wild type and that none of the compounds show significant toxicity at the assayed concentrations. The compound 176 has additionally shown to present activity against a mutated strain of HIV-1, proving its potential as a lead compound for future research of resitant HIV-1 PR inhibitors.

Accurate, timely replication of a DNA molecule is a pivotal moment in the life cycle of every living organism. Any temporal or spatial defect putting the fine-tuned replication machinery off balance causes the so-called replication stress. As the replication machinery consists mainly of enzymes and other proteins, it is not surprising that many of the obstacles most severely blocking the replication machinery progress are of protein origin. Therefore, specialized proteases responsible for relieving replication stress matured during evolution. However, neither the full repertoire of proteolytic enzymes and their particular substrates taking place in countering the DNA replication stress nor detailed molecular mechanisms involved remain unknown. This thesis describes how conserved putative aspartic proteases of the Ddi1-like family engage in countering DNA replication stress via a proteolysis dependent mechanism. We structurally and biophysically characterized yeast and human members of the Ddi1-like family, explored their interactions with ubiquitin and polyubiquitin chains, and identified hypersensitivity to DNA replication inhibitor hydroxyurea in a yeast strain double deleted for DDI1 gene together with a DNA dependent metalloprotease WSS1. Detailed analysis of the DDI1 role in hydroxyurea...