High levels of accuracy in transcription, aminoacylation of tRNA, and mRNA translation are essential for all life forms. However, high accuracy also necessarily means large energy dissipation and slow kinetics. Therefore, in vivo there is a fine tuned balance between rate and accuracy of key chemical reactions. We have shown that in our optimized in vitro bacterial protein synthesis system we have in vivo compatible rate and accuracy of ribosomal protein elongation. Our measurements of the temperature and the pH dependence of peptide bond formation with native substrates also suggest that the chemical step of peptidyl transfer, rather than tRNA accommodation, limits the rate of peptide bond formation. This work has made it possible to study ribosomal peptidyl transfer with native substrates. Furthermore, we have developed a general theoretical model for the rate-accuracy trade-off in enzymatic reactions. When considering this trade-off for protein synthesis in the context of the living bacterial cell, where cognate aa-tRNAs compete for ribosome binding with an excess of non-cognate aa-tRNAs, the model predicts an accuracy optimum where the inhibitory effect of non-cognate substrate binding and the efficiency loss due to high discard rate of cognate aa-tRNAs are minimized. However, these results also show that commonly used biochemical systems for protein synthesis studies operate at exceptionally suboptimal conditions. This makes it difficult, if not impossible, to relate the biochemical data to protein synthesis in the living cell. To validate our theoretical model we developed a method, based on variation of the concentration of Mg2+ ions in the buffer, to study the rate-accuracy trade-off of bacterial protein synthesis in vitro. We found a linear trade-off between rate and accuracy of tRNA selection on the ribosome, from which we could estimate the maximal accuracy. Exploiting this method for a complete set of single-mismatch readings by one tRNA species, we found simple patterns of genetic code reading, where the accuracy was highest for the second and lowest for the third codon position. The results bridge the gap between in vivo and in vitro protein synthesis and allow calibration of our test tube conditions to those of the living cell.

Peptide bond formation and translational termination on the ribosome have been simulated by molecular mechanics, free energy perturbation, empirical valence bond (MD/FEP/EVB) and automated docking methods. Recent X-ray crystallographic data is used here to calculate the entire free energy surface for the system complete with substrates, ribosomal groups, solvent molecules and ions. A reaction mechanism for peptide bond formation emerges that is found to be catalyzed by the ribosome, in agreement with kinetic data and activation entropy measurements. The results show a water mediated network of hydrogen bonds, capable of reducing the reorganization energy during peptidyl transfer. The predicted hydrogen bonds and the structure of the active site were later confirmed by new X-ray structures with proper transition states analogs. Elongation termination on the ribosome is triggered by binding of a release factor (RF) protein followed by rapid release of the nascent peptide. The structure of the RF, bound to the ribosomal peptidyl transfer center (PTC), has not been resolved in atomic detail. Nor is the mechanism known, by which the hydrolysis proceeds. Using automated docking of a hepta-peptide RF fragment, containing the highly conserved GGQ motif, we identified a conformation capable of catalyzing peptide hydrolysis. The MD/FEP/EVB calculations also reproduce the slow spontaneous release when RF is absent, and rationalize available mutational data. The network of hydrogen bonds, the active site structure, and the reaction mechanism are found to be very similar for both peptidyl transfer and termination. New structural data, placing a ribosomal protein (L27) in the PTC, motivated additional MD/FEP/EVB simulations to determine the effect of this protein on peptidyl transfer. The simulations predict that the protein N terminus interacts with the A-site substrate in a way that promotes binding. The catalytic effect of L27 in the ribosome, however, is shown to be marginal and it therefore seems valid to view the PTC as a ribozyme. Simulations with the model substrate puromycin (Pmn) predicts that protonation of the N terminus can reduce the rate of peptidyl transfer. This could explain the different pH-rate profiles measured for Pmn, compared to other substrates.

Licentiatavhandling (sammanfattning) Linköping : Linköpings universitet, 2009. / Härtill 2 uppsatser.

Artificial ribonucleases (ARs) are recognised as a potential strategy to selectively target and cleave biologically significant RNA in cells. However, in order to work as true enzymes they must exhibit catalytic turnover. Many of the reported ARs incorporate metal-containing centres (e.g. dysprosium, copper) in order to induce substantial phosphodiester cleavage, which is not amenable to use in vivo. Therefore, new strategic directions employing metal-independent ARs, such as peptidyl-oligonucleotide conjugates (POCs), need to be investigated. Previous work has shown that poor or non-complementary POCs demonstrate catalytic turnover of a HIV-1 substrate; however, sequence specificity is an issue. For POCs to be useful from a therapeutic standpoint they must only cleave specific RNA molecules and do so in a catalytic fashion, therefore removing the requirement for stoichiometric drug delivery and binding. Consequently, novel POC design strategies are required that allow selective RNA targeting but promote dissociation of the POC following phosphodiester cleavage. In this research, three types of different peptidyl-oligonucleotide conjugate designs have been implemented with the attempt to find an appropriate balance between selectivity and catalytic turnover.(i) Selective targeting and quantitative cleavage (97-100%) of a tRNAPhe target was achieved through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to catalytic amphiphilic peptide sequences containing leucine, arginine and glycine. Although the half-life of tRNAPhe was less than 1 h on exposure to some of these POCs, hybridisation studies reveal that the POCs bind too tightly to their target RNA sequences and thus an excess of POC is required for efficient cleavage activity. The effect of peptide and oligonucleotide sequence variations as well as the role of enhanced conformational freedom via incorporating an abasic deoxyribose linker between the oligonucleotide targeting motif and catalytic peptide is also investigated. (ii) Most of ‘Dual’ peptidyl-oligonucleotide conjugates containing an amphiphilic RNA-cleaving peptide placed between two RNA recognition motifs directed towards the TΨC loop and 3’ acceptor stem of tRNAPhe demonstrate marked RNA binding and cleavage activities. Interestingly, those dual conjugates which showed poor or negligible binding ability in electrophoresis assays, demonstrated sufficient RNA cleavage (70%) within the vicinity of the 65GACAC61 target region. Therefore, weak POC:RNA complexes may exist which could facilitate substrate turnover. (iii) Finally, POCs were designed which induce bulge-loops in their target RNA region upon hybridisation. By introducing regions of non-complementarity into the oligonucleotide sequences, 2- to 5- membered bulges sizes were formed. Via attachment of a catalytic peptide to an internally modified oligonucleotide residue, catalytic peptides were placed directly adjacent to single-stranded RNA regions to promote cleavage by nuclease mimics. Through probing the hybridised complexes with RNase H, the presence of bulges were confirmed for all bulge-loop sizes, which will be followed by cleavage experiments to assess the possibility for reaction catalytic turnover. In conclusion, a variety of POCs have been synthesised, characterised and partially tested for their RNA cleaving and turnover activity. Based on the encouraging results presented POCs could be further developed to target disease specific RNA sequences such as micro- or messenger RNAs.

Dans certaines situations physiologiques et pathologiques, la concentration tissulaire en oxygène diminue grandement, créant ainsi une situation d'hypoxie. Heureusement, les cellules de mammifères possèdent un mécanisme de réponse efficace envers un stress hypoxique assuré par le facteur de transcription inductible par l'hypoxie, HIF-1. Le facteur HIF-1 est constitué de deux sous-unités : HIF-1β et HIF-1α. La sous-unité HIF-1β demeure invariable en fonction des niveaux d'oxygénation. En revanche, la sous-unité HIF-1 a est hautement régulée par les niveaux d'oxygénation. En normoxie, HIF-1α est rapidement hydroxylée par les prolyl-hydroxylases (PHD). L'hydroxylation de HIF-1α mène à sa reconnaissance par la protéine E3-ubiquitine ligase pVHL (von Hippel-Lindau protein), ciblant ainsi la protéine HIF-1 a ubiquitinylée vers la dégradation par le protéasome. À l'inverse, en hypoxie, l'hydroxylation de HIF-la est fortement diminuée, ce qui provoque la stabilisation de HIF-1α et la formation du dimère HIF-1 responsable de l'expression des gènes cibles impliqués dans la réponse adaptatrice des cellules à l'hypoxie. Outre l'hydroxylation et l'ubiquitinylation, la sous-unité a subie également plusieurs autres modifications telles que la phosphorylation, la sumoylation et la S-nitrosylation. Les travaux présentés dans cette thèse visent à démontrer l'impact de nouvelles modifications post-traductionnelles de HIF-1α sur l'activité du facteur HIF-1. Dans un premier temps, des travaux portant sur l'acétylation de la sous-unité HIF-1α par l'acetyltransferase ARD1 (Arrest-defective-1) seront présentés. En 2002, une étude démontrait que la protéine ARD1 était responsable de l'acétylation de HIF-1α, contribuant ainsi à son association avec pVHL et à sa dégradation. Cependant, nos travaux démontrent plutôt qu'ARDl n'est pas impliquée dans la stabilité de HIF-1α. Dans un deuxième temps, nous démontrons un rôle clé de la peptidyl-prolyl isomérase PIN1 sur l'activité transcriptionnelle du facteur HIF-1 et l'expression d'un gène cible important, le facteur de croissance des cellules endothéliales vasculaires (VEGF). Finalement, nous présentons des travaux impliquant la methylation de HIF-1α et le rôle important de cette modification sur l'activité du facteur HIF-1. Bref, l'étude de nouvelles modifications de la protéine HIF-1α s'avère donc fondamentale pour la compréhension de la régulation de HIF-1, tant au niveau des fonctions physiologiques et pathologiques importantes de HIF-1 qu'au niveau du développement de stratégie thérapeutique.

No description available.

No description available.

Rhomboids are intramembrane serine proteases that belong to the evolutionarily widespread rhomboid superfamily. Rhomboids developed a slightly different catalytic mechanism compared to classical serine proteases; they utilise a catalytic dyad (Ser/His) instead of the common triad (Ser/His/Asp), and the rhomboid active site is buried in the membrane. This, coupled with their hydrophobicity, makes them quite difficult to study. Therefore, even though they are known to be involved in several important biological processes it is still not clear how exactly most of them are involved in the regulation of or in the pathologies of diseases related to these processes (such as malaria, Parkinson's disease or cancer). Our understanding is hindered by the lack of tools for their characterisation both in vitro and in vivo. In my thesis I present new fluorogenic substrates based on the LacYTM2 sequence, which is hydrolysed by several different rhomboid proteases. Using Förster resonance energy transfer (FRET)-based methods, these substrates are suitable for continuous monitoring of rhomboid activity in vitro. Modifications in the P5-P1 residues can improve selectivity for a specific rhomboid, the choice of FRET pair of fluorophores that absorbes light of longer wavelengths makes them suitable for high throughput...

La peptidyl-ARNt hydrolase est une enzyme qui hydrolyse les peptidyl-ARNt issus d'une terminaison prématurée de la traduction. Cette protéine est essentielle à la viabilité des bactéries, mais pas à celle des eucaryotes, ce qui fait d'elle une cible potentielle pour l'action d'anti-bactériens. Cela justifie également qu'on cherche à cartographier l'interaction de cette protéine avec son substrat, pour faciliter la conception d'inhibiteur. Les tentatives d'obtention de cristaux de complexes enzyme:analogue de substrat étant restées vaines, nous avons choisi d'étudier de tels complexes en solution, par RMN. Grâce à un double marquage 15N/13C, nous avons tout d'abord attribué les fréquences de résonance des atomes du squelette de la protéine et d'une grande partie des chaînes latérale. Nous avons ensuite étudié l'interaction entre la PTH d'E. coli et un analogue de son substrat synthétisé chimiquement, la diacétyl-Lys-(3'NH)-adénosine. Cette étude nous a permis de caractériser le rôle de nombreux résidus du site actif, notamment celui d'une phénylalanine (F66) interagissant via son cycle aromatique avec l'adénine 3'-terminale du substrat, celui d'une asparagine (N114) stabilisant une molécule d'eau responsable de l'hydrolyse du substrat et celui d'une autre asparagine (N10) permettant à la PTH de discriminer positivement les peptidyl-ARNt par rapport aux aminoacyl-ARNt. Nous avons aussi étudié l'interaction entre la protéine et des mini-ARNt mimant la tige acceptrice et le bras TΨC d'un ARNt. Ce travail a permis de cartographier la surface de la PTH où l'ARN s'ancre à la protéine. Il a confirmé l'importance de deux résidus basiques, la lysine K105 et l'arginine R133, pour la reconnaissance du phosphate en 5' de l'ARNt. Il a également révélé une interaction entre l'hélice C-terminale de la protéine et le bras TΨC de l'ARNt, à 30 Å du site actif. La pertinence fonctionnelle de ce dernier contact a pu être établie par mutagenèse dirigée. L'ensemble de ces résultats permet de proposer un modèle complet de l'interaction entre la PTH et un peptidyl-ARNt.

O polimorfismo de inserção/deleção (I/D) do gene da enzima de conversão da angiotensina I foi estudado em coorte de sobreviventes de 333 portadores de insuficiência cardíaca, de idades entre 13 e 68 (43,3 ± 10,5) anos, 262 (78,7%) dos quais eram homens e 71 (21,3%) mulheres. O grupo controle foi constituído de 807 doadores voluntários de sangue com idades entre 18 e 60 (31,5 ± 9,3) anos, 557 (69%) dos quais eram homens e 250 (31%) mulheres. A insuficiência cardíaca foi atribuída à cardiomiopatia dilatada idiopática em 125 (37,6%) pacientes. Nos demais pacientes as etiologias foram: a cardiomiopatia insquêmica em 63 (18,9%), a cadriomiopatia da doença de Chagas em 58 (17,4%), a cardiomiopatia hipertensiva em 41 (12,3%), a cardiomiopatia alcoólica 24 (7,2%), a cardiomiopatia valvar em 11 (3,3%) e a cardiomiopatia periparto em 11 (3,3%). A determinação dos genótipos associados ao polimorfismo I/D foi realizada pela reação em cadeia da polimerase. Foram estudadas a distribuição dos genótipos entre os indivíduos do grupo controle e os pacientes e as prováveis associações do polimorfismo I/D com diferentes variáveis clínicas e com a evolução. Essas análises foram realizadas de acordo com o padrão de herança genética atribuído ao alelo D (co-dominante, recessivo ou dominante). Na análise estatística foram utilizados o teste do qui-quadrado, o teste t-Student, a análise de variância (ANOVA), o método de Kaplan-Meier, o teste log-rank e a regressão de Cox. A freqüência do genótipo DD foi menor no grupo de pacientes, considerando-se o caráter recessivo atribuído ao alelo D (p=0,034). Os portadores do genótipo DD apresentaram maior média de valores do diâmetro sistólico do ventrículo esquerdo determinado pelo ecocardiograma, quando foi atribuído caráter recessivo ao alelo D (p=0,031). O intervalo decorrido do nascimento até o aparecimento dos sintomas foi menor nos portadores do genótipo DD com cardiomiopatia alcoólica, ao ser atribuído caráter recessivo ao alelo D (p=0,033). O intervalo entre o nascimento e o aparecimento dos sintomas foi menor nos portadores do genótipo DD com cardiomiopatia hipertensiva, quando foi atribuído caráter co-dominante (p=0,048) ou recessivo (p=0,024) ao alelo D. Os portadores do genótipo DD apresentaram maior mortalidade após os 50 anos, ao ser atribuído caráter co-dominante (p=0,007) ou recessivo (p=0,002) ao alelo D. As variáveis independentes relacionadas com a maior mortalidade após os 50 anos de idade, ao ser atribuído caráter recessivo ao alelo D, foram: a idade (p<0,001), o genótipo DD (p=0,003), o diabetes melito (p=0,003) e a doença de Chagas (p=0,005). Atribuindo-se caráter co-dominante ao alelo D, as variáveis independentes relacionadas com maior mortalidade foram: a idade (p<0,001), a doença de Chagas (p=0,004), o diabetes melito (p=0,005) e o genótipo DD (p=0,015). Os resultados obtidos permitem sugerir que o genótipo DD está associado com maior morbidade e mortalidade em determinados grupos de pacientes com insuficiência cardíaca. / Insertion/deletion (I/D) polymorphism of the angiotensin I- converting enzyme gene was studied in a cohort of survivors of 333 patients with heart failure of different etiologies and in a control group of 807 volunteer blood donors. The age of the patients ranged from 13 to 68 (43,3 ± 10,5) years, 262 (78.7%) were men and 71 (21.3%) women. The age of the control group ranged from 18 to 60 (31,5 ± 9,3) years, 557 (69%) were men and 250 (31%) women. Idiopathic dilated cardiomyopathy was diagnosed in 125 (37.6%), ischemic cardiomyopathy in 63 (18.9%), Chagas\' disease cardiomyopathy in 58 (17.4%), hypertensive cardiomyopathy in 41 (12.3%), alcoholic cardiomyopathy in 24 (7.2%), valvular cardiomyopathy in 11 (3.3%) and peripartum cadiomyopathy in 11 (3.3%). The genotypes associated with the I/D polymorphism were determined by polymerase chain reaction. The distribution of the genotypes was determined in the control and patient groups as well the possible associations of the I/D polymorphism with clinical variables and the evolution. The chi-square test, the t-Student test, the analysis of variance (ANOVA), the Kaplan-Meire method, the log-rank test and Cox regression were used in the statistical analysis. The DD genotype was less frequent in patients, assuming a recessive effect of the D allele (p=0,0034). The left ventricular en-systolic diameter by echocardiography was higher in patients with the DD genotype, .... And the DD genotype, assuming a recessive effect of the D allele (p=0,033). The time elapsed until the onset of symptoms was shorter in patients with hypertensive cardiomyopathy and the DD genotype, assuming a codominant (p=0,048) or recessive (p=0.024) effect of the D allele. Patients older than 50 years with the DD genotype showed increased mortality, assuming a codominant (p=0,007) or recessive (p=0,002) effect of the D allele. The independent variables associated with increased mortality in patients older than 50 years were: age (p<0,001), the DD genotype (p=0,003), diabetes mellitus (p=0,003) and Chagas\' disease (p=0,005), assuming a recessive effect of the D allele. Assuming a codominant effect of the D allele, the independent variables associated with increased mortality in patients older than 50 years were: age (p<0,001), Chagas\' disease (p=0,004), diabetes mellitus (p=0,005) and the Dd genotype (p=0,015). These results suggest that the DD genotype may be associated with higher morbidity and mortality in some groups of patients with heart failure.