The structure of the formiminotransferase (FT) domain of formiminotransferase-cyclodeaminase (EC 2.1.2.5) - (EC 4.3.1.4) has been solved in complex with the product analogue, folinic acid, and in complex with the substrate analogue, C5,C10-dideazatetrahydrofolate. The protein is arranged as a homodimer, with each subunit comprising two a/(3 subdomains which adopt a novel protein fold. Within each subunit, an electrostatic tunnel which traverses the width of the molecule is observed and comprises the ligand binding sites. The distribution of charged residues in the tunnel enables us to propose the mode of binding for the natural substrate, tetrahydropteroylpolyglutamate. Modeling studies indicate that a gamma-linked triglutamate form of the tetrahydrofolate substrate can be accommodated through the length of the tunnel. The electron density also indicates that a single molecule of glycerol is bound to each protomer at the base of a second tunnel contacting the main electrostatic tunnel. This second tunnel is the expected entrance for formiminoglutamate to the active site of the enzyme. The structure has enabled us to propose that the residue His82 may be important in the catalytic mechanism of the transferase reaction.

The evolutionary conserved and abundant p24 proteins cycle through the early secretory pathway, but their cellular functions are still poorly understood. They are not essential in yeast, but in mice, p23 knockout is embryonic lethal. Moreover, p23 is involved in Alzheimer’s disease pathogenesis. The p24 proteins have the ability to form hetero-complexes. Here, the pairwise interactions among p24s, in yeast and human, were determined by yeast two-hybrid. The p24 interactions are specific and occur mostly through their GOLD domain with some contribution from their DOG sequence. Mutagenesis experiments suggested that two p24s interact differently with a common p24 partner. Finally, co-overexpression of p24s in yeast has a harmful effect on a subset of combinations. Some have a growth defect whereas others have a cell size increase phenotype or both. I propose that the p24 proteins might participate in transport of GPI-anchored proteins involved cell wall maintenance or cell cycle pathways. / Les abondantes protéines de la famille p24 sont conservées dans l’évolution et circulent dans la voie sécrétoire précoce, mais leurs fonctions cellulaires sont encore mal définies. Ils sont dispensables chez la levure, mais un knock-out de p23 est létal embryonnaire chez la souris. De plus, p23 est impliqué dans la pathogénèse de la maladie d’Alzheimer. Les protéines p24 peuvent former des hétéro-complexes. Dans cette étude, les interactions des p24s sont déterminées par double hybride. Elles sont spécifiques et se font surtout par leur domaine GOLD avec une contribution de leur séquence DOG. Les expériences de mutagénèse révèlent que deux p24s interagissent différemment avec un p24 commun. La co-surexpression de certains p24s dans la levure cause la mort ou l’élargissement des cellules. Je propose que les protéines p24s participent dans le transport des protéines à ancrage GPI impliquées dans l’entretien de la paroi cellulaire et dans les voies du cycle cellulaire.

An alignment/phylogeny of the papain superfamily of cysteine proteases was created from which the existence of at least 11 families of mammalian cysteine proteases was inferred, including two as yet uncharacterized ones. Character weighting increased the average speed of maximum parsimony phylogenetic searches two- to three-fold. To study a sequence motif in pro-cysteine proteases and mammalian cystatins, cystatin C, a cysteine protease inhibitor, was expressed and purified using a new affinity method giving $>$99% purity. Methionine oxidation to the sulfoxide occurring during protein production was eliminated. Reduced stability caused by Met14 oxidation was demonstrated. No function for the sequence motif was found, however cystatin C underwent a conformational change at acidic pH that rendered the His86-Asp87 peptide bond labile to proteolysis. This could constitute a mechanism for clearing inappropriately localized cystatins from the acid environment of lysosomes and explain differences between the X-ray and NMR structures of the homologous chicken cystatin.

The prominent role played by the cap structure in ribosome binding is mediated by the cap binding protein complex (eIF-4F). The importance of eIF-4F in the regulation of gene expression has been demonstrated in both mammalian and yeast cells. Nevertheless, the function of the high molecular weight subunit of eIF-4F is unknown. Here we describe the isolation and characterization of yeast eIF-4F (24- and 150-kD) as well as a novel CBP of 96-kD. The yeast gene TIF4631 encoding p150 and a closely related gene, TIF4632 were isolated. TIF4631 and TIF4632 are 53% identical, carry out an essential function, display sequences closely resembling the RNA recognition motif (RRM) and are homologous to the high molecular weight subunit of human eIF-4F (p220). The presence of an RRM-like sequence in TIF4631 is consistent with its RNA binding properties and promises to challenge the current views on how cap-dependent and cap-independent ribosome binding operate in eukaryotes.

No description available.

The important connection between dynamics, structure and function of enzymes still remains unclear and is, hence, an interesting relationship to investigate. In this thesis, the enzymes NADH oxidase (NOX) from Thermus thermophilus and mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (PtpB) were chosen as systems to study the influence of solution conditions on protein structure and flexibility in relation to their catalytic activity. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) techniques were used in the study of NOX. NMR and differential scanning fluorimetry (DSF) were utilized to assess the sample stability of PtpB under various buffer conditions.NOX is a 54 kDa homodimeric enzyme with many potential biotechnology applications. It accepts cofactors flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD). It has high thermal stability and its activity increases with the addition of low concentrations of urea. Using enzymes in vitro provides us a lot of freedom to control its environment, which is useful to look at changes occurring under different conditions. Thus, NMR allowed us to investigate these interesting characteristics on NOX dynamics and structure. When Mtb infects host cells it secrets among others, PtpB, to disrupt signalling pathways and blunts immune responses. PtpB contains a two-helix lid that completely buries the active site and it is proposed that it has evolved as a novel mean of protection against host chemical defences and affords substrate access. An investigation of conformational dynamics of the lid domain of PtpB using NMR would elucidate a relation between conformation and function of Ptpb, which to date, is still unknown and would contribute to the development of inhibitors as potential drugs. In order to do this, it is essential to obtain a stable sample that will give optimal quality NMR spectra. NMR and DSF were used to optimize PtpB NMR sample conditions and spectral quality. We employed a suite of novel 15N NMR relaxation experiments to estimate the extent of broadening due to microsecond-timescale motions and to measure exchange-free transverse relaxation rates on a per-residue basis of NOX. These measurements allowed us to identify several residues involved in dynamical processes. Moreover, order parameters, S2, determined from standard 15N T1, T2 and {1H}-15N steady NOE experiments, allowed the identification of additional residues characterized by motions on nanosecond-picosecond time scales. We then investigated the effects of temperature, urea addition, cofactor and NAD+ product binding to NOX using NMR and ITC. For each condition studied, chemical shift displacement of NMR peaks is significant relative to all other peaks only for a reoccurring small subset of residues. This cluster of residues is located around the active site of the enzyme and thus, suggests evidence of a relationship between concerted conformational rearrangement of NOX structure and NOX catalytic activity. An increase in NMR spectral quality and a NMR sample of PtpB with increased thermal stability, higher yields and reduced aggregation was obtained for further NMR dynamic experiments. This was achieved by optimizing the sample conditions by performing several NMR titrations and DSF experiments. Assessing the thermal stability of enzymes with different additives using DSF could be easily introduced during the elaboration of protein purification protocols as a routine method to optimize NMR sample conditions for NMR studies. / Le lien important entre la dynamique, la structure et la fonction des enzymes n'est pas encore bien défini et est donc une relation intéressante à étudier. Dans cette thèse, les enzymes NADH oxydase (NOX) de Thermus thermophilus et mycobacterium tuberculosis protéine tyrosine phosphatase B (PtpB) sont les systèmes choisis pour étudier l'influence des conditions de solution sur la structure et la flexibilité des protéines par rapport à leur activité catalytique. Les techniques de résonance magnétique nucléaire (RMN) et de calorimétrie de titration isothermique (ITC) ont été utilisées dans l'étude de NOX. La RMN et la fluorimétrie différentielle à balayage (DSF) ont été utilisées pour évaluer la stabilité de l'échantillon de PtpB dans diverses conditions de tampons. NOX est une enzyme homodimérique avec de nombreuses applications biotechnologiques potentielles. NOX accepte les cofacteurs flavine mononucléotide (FMN) ou flavine adénine dinucléotide (FAD). L'enzyme a une stabilité thermique élevée et son activité augmente avec l'addition de faibles concentrations d'urée. Utiliser des enzymes in vitro donne beaucoup de liberté pour contrôler leur environnement, utile pour examiner les changements qui se produisent dans différentes conditions. Ainsi, RMN nous a permis d'étudier ces caractéristiques intéressantes sur la dynamique et la structure de NOX. Lorsque Mtb infecte les cellules, il secrète PtpB qui perturbe les voies de signalisation et affaibli les réponses immunitaires. PtpB contient un couvercle de deux hélices qui enterre complètement le site actif et il est proposé qu'il ait évolué ainsi en protection contre les défenses chimiques et permet un accès au substrat. Une étude de la dynamique du domaine du couvercle de PtpB utilisant la RMN éluciderait la relation entre la conformation et la fonction de PTPB et contribuerait au développement d'inhibiteurs en tant que médicaments potentiels. Pour ceci, il est essentiel d'obtenir un échantillon stable qui donnera des spectres RMN de qualité optimale. RMN et DSF ont été utilisés pour optimiser les conditions d'échantillonnage de PtpB pour la RMN et la qualité spectrale. Nous avons utilisé une suite de nouvelles expériences de relaxation de 15N par RMN pour estimer l'ampleur de l'élargissement des signaux en raison de mouvements à l'échelle de temps des microsecondes et de mesurer les taux de relaxation transversale sans échange pour chaque résidu de NOX. Ces mesures nous ont permis d'identifier plusieurs résidus impliqués dans les processus dynamiques. De plus, les paramètres d'ordre, S2, déterminées à partir des expériences standards de T1, T2 et NOE, ont permis d'identifier des résidus additionnels caractérisés par des mouvements sur des échelles de temps de nanosecondes à picosecondes. Nous avons ensuite étudié les effets de la température, urée, la liaison de cofacteur et du produit à NOX en utilisant la RMN et le ITC. Pour chaque condition étudiée, la variation de déplacement chimique des signaux RMN n'est significative par rapport à tous les autres signaux que pour un petit sous-ensemble récurrent de résidus. Cet amas de résidus est situé autour du site actif de l'enzyme et ainsi, suggère preuve d'une relation entre un réarrangement conformationnel concertée de la structure de NOX et de son activité catalytique. Une augmentation de la qualité spectrale de RMN et un échantillon de PtpB pour la RMN avec une meilleure stabilité thermique, des rendements plus élevés et une agrégation réduite ont été obtenus pour de futures expériences dynamiques par RMN. Ceci a été réalisé en optimisant les conditions de l'échantillon en effectuant plusieurs titrages par RMN et des expériences de DSF. L'évaluation de la stabilité thermique des enzymes avec différents additifs à l'aide de DSF pourrait facilement être introduit lors de l'élaboration de protocoles de purification de protéines comme une méthode routinière pour optimiser les conditions d'échantillons pour les études par RMN.

Aminoglycosides are a class of broad-spectrum antibiotics that first came to prominence in the 1950s as the first effective treatment for tuberculosis. Aminoglycoside resistance in bacteria, however, is increasingly becoming a difficult problem to treat. One of the most common mechanisms of aminoglycoside resistance in bacteria is the expression of the clinically widespread enzyme, aminoglycoside N-6ʹ-acetyltransferase (AAC(6ʹ)). To address this problem, aminoglycoside-coenzyme A bisubstrates were previously reported by the Auclair lab and found to be potent inhibitors of AAC(6ʹ). Unfortunately, the negatively charged phosphate groups of these molecules likely prevent penetration through cellular membranes. Further research then replaced the phosphate groups of the aforementioned molecules with mimics that would not impede cell membrane penetration. One of these compounds was shown to be the first biologically active compound in blocking AAC(6ʹ) in a resistant strain of bacteria. However, the inhibitory activity of this derivative against AAC(6ʹ) was poor. To combat aminoglycoside resistance, this work focuses on the use of prodrugs to produce potent aminoglycoside-coenzyme A bisubstrates within bacterial cells, thereby resensitizing resistant strains of bacteria. This approach is based on the hypothesis that cell-permeable aminoglycoside-pantetheine molecules can be transformed by the bacterial coenzyme A (CoA) biosynthetic pathway to produce potent AAC(6ʹ) inhibitors within cells. Chapter 2 describes the design and synthesis of two generations of AAC(6ʹ) prodrug inhibitors. In Chapter 3, various in vitro enzymatic studies were performed to verify the mechanism of prodrug activation. Results show that prodrug transformation by the CoA biosynthetic enzymes, PanK, PPAT, and DPCK, was possible with a significant trend observed in relation to the carbon linker length of these molecules. Finally, to demonstrate the biological activity of the prodrugs, Chapter 4 focuses on experiments aimed at determining the potentiation activity of the prodrugs in blocking resistance within cells, thereby resensitizing bacteria to aminoglycosides. Results show considerable potentiation of aminoglycoside activity by the prodrugs against an aminoglycoside-resistant strain of E. faecium, with a reduction in the MIC50 by as much as 30 fold. Overall, this work successfully validates the research hypothesis and presents a proof-of-concept for the potential of AAC(6ʹ) prodrug inhibitors to resensitize select resistant bacteria to aminoglycoside antibiotics. / Les aminoglycosides sont une classe d'antibiotiques à large-spectre qui ont vu leur utilisation augmenter dans les années 1950, surtout en raison de leur efficacité contre la tuberculose. La résistance aux aminoglycosides dans les bactéries devient cependant un problème de plus en plus important. Un des méchanismes les plus communs de résistance aux aminoglycosides résulte de l'expression de l'enzyme aminoglycoside N-6'-acetyltransférase (AAC(6ʹ)) par les bactéries. Pour addresser ce problème, des bisubstrats d'aminoglycoside-coenzyme A ont précédemment été reportés par le groupe Auclair et ce sont avérés être des inhibiteurs puissants de l'enzyme AAC(6ʹ). Malheureusement, la charge négative des groupements phosphate de ces molécules semble empêcher leur pénétration cellulaire. Ces groupements phosphate ont ensuite été remplacés par différents bioisostères. Un des ces composés a été démontré être le premier composé capable de bloquer AAC(6') dans des souches bactériennes résistantes. Malheureusement, l'activité inhibitrice de cette molécule envers AAC(6') était faible. Pour combattre la résistance aux aminoglycosides, les travaux de cette thèse se concentrent sur la génération de prodrogues activèes en bisubstrats aminoglycoside-coenzyme A dans les cellules bactériennes. Étant de puissants inhibiteurs d'AAC(6ʹ), ces bisubstrats vont resensibiliser les bactéries à l'effect des aminoglycosides. Cette approche est basée sur l'hypothèse que les molécules aminoglycoside-pantothéine perméables peuvent être transformées par les enzymes responsable de la synthèse du coenzyme A (CoA) dans les bactéries, pour produire des puissants inhibiteurs d'AAC(6ʹ) dans les cellules. Le chapitre 2 décrit le design et la synthèse de la première série d'inhibiteurs prodrogues, ainsi que les dérivés utilisés pour obtenir une relation structure-activité (SAR). Dans le chapitre 3, plusieurs études enzymatiques permettant de vérifier le méchanisme d'activation des dites prodrogues sont décrites. Les résultats montrent que la transformation de la prodrogue par les enzymes biosynthétiques de CoA (PanK, PPAT, DPCK) est possible avec une corrélation significative entre l'activité et la longueur de la chaine de carbone de ces molécules. Finalement, pour démontrer l'activité des prodrogues, le chapitre 4 traite d'expériences permettant de déterminer l'activité potentiatrice des prodrogues sur l'effet antibacterien des aminoglycosides contre les souches resistante. Les résultats montrent un niveau considérable d'augmentation de l'activité des aminoglycoside contre E. faecium, une souche bactérienne résistante aux aminoglycosides, en presence des prodrogues. Cet effet produit une déduction du MIC50 allant jusqu'à 30 fois. En resumé, les travaux presentes dan cette thèse, valident l'hypothèse de recherche et présente une preuve de concept pour le potentiel d'inhibiteurs prodrogues d'AAC(6ʹ) dans le but de resensibiliser certaines souches bactériennes résistantes aux antibiotiques aminoglycosides.

The activation mechanisms for zymogens belonging to the papain family of cysteine proteases were investigated. This was accomplished using site-directed mutagenesis, kinetic measurements, the identification of processing intermediates, and the analysis of the various X-ray crystal structures reported to date. Procathepsin B is a unique precursor of papain-like enzymes in that it is composed of a shorter prodomain; i.e., 62 residues versus >90 residues for those belonging to the cathepsin L-subfamily, and the mature enzyme is composed of a twenty residue insertion termed the occluding loop. In this study, the pH dependence of cathepsin B inhibition by its propeptide was shown to be eliminated upon the removal of this enzyme's occluding loop. Furthermore, variants of cathepsin B carrying the mutation Asp22Ala or His110Ala also displayed a loss of pH dependence for their affinity to the propeptide inhibitor. Similarly, the overall rate of autoprocessing for full-length procathepsin B was shown to be affected by the occluding loop mutations. These results suggest a possible influence of the pH-dependent stability of the occluding loop on the overall rate of processing for this precursor. / The addition of the protein-proteinase inhibitor, cystatin C, impeded the overall rate of autoactivation for procathepsin B and procathepsin S and caused the accumulation of processing intermediates for these precursors which were subsequently identified by automated Edman degradation. The N-terminal sequences of these processing intermediates correspond to an area of the prodomains which binds through the substrate-binding clefts of these enzymes, thus suggesting a plausible intramolecular step of processing for this family of zymogens. This unimolecular mechanism was found to rely on the conformational mobility of prosegment residues (i.e., at the C-terminal end). Furthermore, in contrast to what has been observed for zymogens belonging to the aspartic protease family, it was determined that charged residues located at the N-terminus of the mature segment found in propapain do not contribute to the overall pH-triggering mechanism of activation for this precursor. / Procathepsin H was determined to be an unusual mammalian member of the cathepsin L-subfamily due to its inability to autoprocess, and the aminopeptidase activity of mature cathepsin H was found to be incapable of converting its own precursor. Furthermore, prosegment residues located near the pro/mature junction of procathepsin H are highly resistant to the proteolytic action of secondary proteases. These findings are consistent with the pre-formation of a disulfide bond within the cathepsin H precursor which links the prodomain to the enzyme using Cys82p and Cys214. Consistent with the findings for procathepsins B and S, the unrestricted conformational mobility at the C-terminal end of the prosegment (i.e., near the pro/mature junction) is an important prerequisite for efficient autoactivation to occur among zymogens of papain-like enzymes.

The involvement of protein phosphorylation and gene regulation in signal transduction pathways are examined. In particular the role of the novel STY kinase in signal transduction networks is suggested, followed by a study of the liver-specific and inducible expression of the carbamyl phosphate synthetase I (CPSI) gene. In a screen of an embryonal carcinoma (EC) cell cDNA expression library with an antibody to phosphotyrosine, the STY kinase was identified. STY is represented by a single 1.8 kb transcript in undifferentiated P19 cells. During differentiation of these cells, mRNAs of 3.2 and 5.6 are induced and persist to adulthood. Sequence analysis of the STY kinase revealed a catalytic domain homologous to kinases with serine/threonine phosphorylating specificity especially those involved in cell-cycle control, such as the FUS3 gene. Biochemical analysis of recombinant STY synthesized in bacteria or in vitro indicated a serine-, threonine-, and tyrosine-phosphorylating capability, suggesting that it belongs to a previously unappreciated family of kinases. / The promoter region of the liver specific, glucocorticoid and cAMP inducible gene, CPSI was analyzed for transcriptional activity. Sequences extending from the in vivo start site of CPSI gene to $-$1200 bp, were shown to support in vitro transcription with liver nuclear extracts. A region proximal to transcription initiation, from $-$104 to $-$124 was shown to specifically interact with the abundant liver nuclear factor C/EBP. Double stranded oligonucleotides corresponding this cis element abolishes in vitro transcription reactions in a competitive manner. The C/EBP and related factors, LAP and LIP, interaction with CPSI promoter elements has implications in the developmental, liver-specific, as well as the inducible aspects of CPSI gene expression.

Four microbial lipases were examined to test a proposed substrate model based on the relative size of the substituents on the stereogenic carbon of $ alpha$-substituted carboxylic acids (Fig. 5). Literature and our results for the kinetic resolution of carboxylic acids with the lipases from Candida rugosa, Aspergillus niger, Mucor meihei and Pseudomonas cepacia showed that sterics alone do not account for enzymatic enantioselectivity. / In the course of this work, it was found that the lipase from Aspergillus niger showed appreciable enantioselectivity (E = 19 $ pm$ 3 at pH 7/10 mM phosphate buffer) in the hydrolysis of methyl pipecolinate. Given that there are limited ways to obtain optically pure pipecolinic acid, our results offered a viable alternative. Consequently, three strategies were used to improve this enantioselectivity. Altering reaction conditions (pH, concentration and type of buffer) and substrate modifications did not improve enantioselectivity significantly. Partial purification of the crude lipase from Aspergillus niger improved the enantiomeric ratio from ca. 20 to greater than 100 for the hydrolysis of octyl pipecolinate.