Several classes of antimetabolites have been developed for the treatment of cancer, including numerous inhibitors of nucleotide biosynthesis. N-(phosphonacetyl)-L-aspartate (PALA) and hydroxyurea (HU) are two antimetabolites that inhibit nucleotide biosynthesis; PALA inhibits de novo pyrimidine synthesis and HU inhibits the conversion of ribonucleotide diphosphates to deoxyribonucleotide diphosphates. Due to the similar mechanisms, it was thought that cancer cells would respond similarly to HU and PALA treatment. However, studies in this dissertation revealed strikingly different responses to either HU or PALA treatment in HCT116 cells. A cytoprotective S-phase arrest was activated upon HU treatment while PALA treatment failed to activate the S-phase checkpoint, resulting in p53-dependent apoptosis. The checkpoint effector kinase, Chk1, was not significantly phosphorylated during PALA treatment due to a failure to recruit ATR, the upstream kinase, to chromatin sites. The post-translational modifications of p53, phosphorylation of serines 46 and 392, suggested that PALA treatment promotes the accumulation of a transcriptionally active p53 while HU does not. ChIP analysis showed that p53 bound to pro-apoptotic promoters, therefore activating p53-dependent apoptosis during PALA treatment. To gain more insight into these differential cellular responses, we developed a tandem-affinity purification (TAP) tagged p53 cell line in which a TAP tag was inserted into the C-terminus of the endogenous p53 genetic locus through homologous recombination. This technology allows purification of p53 with its protein binding partners at endogenous expression levels. The tagged p53 accumulated and bound to promoters in response to DNA damage similar to the untagged p53, suggesting that the TAP tag did not interfere with the normal cellular functions of p53. Using mass spectrometry, we can identify the different p53 protein binding partners in response to PALA or HU treatment. We can also determine the variable pattern of post-translational modifications on different drug-stabilized p53 and determine which modifications are responsible for promoting apoptosis versus cytoprotective arrest. We can then exploit the identified proteins and post-translational modifications in the development of new chemotherapeutic agents.

The complexity of all biomolecules in existence today can be attributed to the variation of the amino acid repertoire. In nature, 20 canonical amino acids are translated to form these biomolecules, however, many of these amino acids have revealed posttranslational modifications (i.e. acetylation, methylation) after incorporation. Amino acids that exhibit PTM are known for their involvement in cellular processes such as DNA repair and DNA replication; these PTMs are commonly found on histones within the chromatin complex. Utilization of in vivo site-specific incorporation has recently reported functionality of post-translationally modified amino acids.1 xii Here we report the synthesis and in vivo site-specific incorporation of the histone PTM, 2-hydroxyisobutyrl lysine (Khib), with the pyrrolysyl tRNA/ RNA synthetase system. This translational machine can better serve to probe Khib for functional benefits. Additionally, this thesis focuses much of its attention on the development of unnatural amino acids (UAA) with optogenetic characteristics. These UAAs, if site-specifically incorporated, can be used to control enzymes and proteins through rapid light perturbation (365nm UV light). Furthermore, discussed is the synthesis of photo-caged threonine and photo-caged serine as potential substrates for the pyrrolysyl translational machinery.

Differences have been shown in the steady-state accumulation and half-lives between Brassica FAD3 (BF3) and tung FAD3 (TF3) proteins expressed in yeast cells cultured at 30°C. TF3 has a greater steady-state accumulation and longer half-life than BF3. These differences are attributed to post-translational modification and have been shown to be controlled by an Nterminal element. I attempted to determine specific amino acids important for regulation, and further characterize the mechanism contributing to the differences. Through site-directed mutagenesis, it was shown that replacing lysine residues with asparagines in the BF3 and TF3 Ntermini increased protein stability, while replacing an asparagine with lysine in the TF3 Nterminus decreased its stability. Furthermore, I showed that the TF3 polyglutamic region (six consecutive glutamic acid residues) is primarily responsible for the higher steady-state amount of TF3 in comparison to BF3. This negatively charged region likely acts as an electrostatic shield protecting the protein from degradation.

Ces travaux de recherche visent à mettre au point par une nouvelle « Approche Chimique Inverse » des antigènes synthétiques pour le diagnostic de maladies auto-immunes; utilisant les autoanticorps spécifiques de ces pathologies en tant que biomarqueurs.L'efficacité de ces peptides modifiés en tant qu'outils pour le diagnostic est évaluée par un screening au moyen de tests immunoenzymatiques (SP-ELISA), de sérums de patients atteints de pathologies auto-immunes sélectionnées.Trois maladies ont été étudiées en particulier.La cirrhose primitive biliaire est une maladie auto-immune cholestatique affectant le foie, caractérisée par une destruction progressive des canaux intra-hépatiques, pouvant mener à une cirrhose. Il existe des autoanticorps antimitochondriaux spécifiques à cette maladie qui reconnaissent un epitope lipoylé de la PDC-E2, protéine impliquée dans le cycle énergétique mitochondrial. La synthèse de sondes moléculaires lipoylées basées sur la PDC-E2(167-186) a été mise au point et optimisée. Les antigènes synthétiques ont ainsi été testés sur des sérums de patients de PBC. Les résultats montrent que: 1) la séquence a une importance pour la reconnaissance des anticorps; 2) la chiralité du dithiolane de la lipoyl-lysine ne semble pas avoir d'influence majeure sur la reconnaissance des autoanticorps; 3) l'absence de lipoylation sur le mime de la protéine native semble donner de meilleurs résultats que l'antigène synthétique lipoylé.Le diabète est une maladie caractérisée par une hyperglycémie provoquée par l'action réduite ou inexistante de l'insuline. L'excès de sucre dans le sang peut provoquer des modifications de diverses natures sur les protéines circulantes (glycation, O-glycosylation, N-glycosylation).La synthèse de sondes moléculaires portant ces structures en tant que modifications post-traductionnelles a été mise au point. Les antigènes synthétiques ont ainsi été testés sur des sérums de patients atteints de diabète. Les résultats montrent que trois des peptides testés permettent de différencier les patients atteints de diabète de type 1 (forme autoimmune) de ceux de type 2 (forme non autoimmune) ainsi que des controles sains.La sclérose en plaques est une maladie impliquant une démyélinisation des fibres nerveuses du système nerveux central. Le système immunitaire détruit les protéines composante cette gaine de myéline. La kynurénine est un des métabolites principaux du Tryptophane, et il a été montré que dans la sclérose en plaques, la voie métabolique du Tryptophane pourrait être déréglée.Ainsi, la synthèse de peptides modifiés portant une kynurénine comme modification post-traductionnelle aberrante a été menée à bien. Les résultats ne montrent pas de détection particulière d'anticorps dirigés contre cette modification dans le cas de la sclérose en plaques. / This research work aims to apply the novel concept of “Chemical Reverse Approach” to the design, the production, and the immunological screening of synthetic antigens able to specifically detect autoantibodies in sera of patients affected by immune-mediated diseases. Such specific autoantibodies are considered disease biomarkers and can be used to develop novel diagnostic/prognostic tools for the aforementioned pathologies.In particular, three diseases have been investigated.Primary Biliary Cirrhosis is an autoimmune cholestatic disease of the liver, characterized by progressive destruction of intrahepatic bile ducts. Specific antimitochondrial autoantibodies directed against a lipoylated epitope of the PDC-E2 protein, are considered relevant for the disease. The PDC-E2 protein is involved in the energetic cycle of mitochondria. Synthesis of lipoylated molecular probes based on PDC-E2(167-186) was carried out and optimized. These new synthetic antigens were tested on PBC patients' sera. The results showed that: 1) the sequence is fundamental for antibody recognition; 2) dithiolane lipoyl-lysine chirality does not seem to have any significant influence on antibody recognition; 3) the unlipoylated analogue of the native protein appears to detect a more relevant antibody titre than the lipoylated one.Diabetes is a disease characterized by hyperglycaemia. This condition is caused by the reduced or inexistent action of insulin. Hyperglycaemia can cause various modifications on circulating proteins (such as glycation, O-glycosylation, N-glycosylation).The synthesis of post-translationally modified peptides containing such structures was carried out. These new synthetic antigenic probes were tested on sera from patients suffering from diabetes. The results showed that three peptides among those tested can differentiate patients with type-1 diabetes (autoimmune form) from those with type-2 (non-autoimmune form) and healthy patients.Multiple sclerosis is a demyelinating disease of the central nervous system. The immune system destroys proteins components of myelin sheath. Kynurenine is a major metabolite of Tryptophan, and it has been shown that in multiple sclerosis, the metabolic pathway of Tryptophan may be deregulated.Thus, the synthesis of modified peptides incorporating a kynurenine as an aberrant post-translational modification was carried out. The results show no specific antibody detection in multiple sclerosis sera.

Du fondamental à la clinique, cette thèse a été construite autour d’un seul mot clefs : KCC2, grâce à plusieurs projets collaboratifs. Ce co-transporteur d’ion est une molécule à plusieurs facettes dont la multiplicité des rôles et ses implications dans diverses pathologies font d’elle un élément clef de l’organisme vivant. En plus des études phylogénétiques et thérapeutiques effectuées au cours de cette période, mon principal travail a été de déterminer le rôle physio-pathologiques in vitro et in vivo d’un mécanisme de régulation post-traductionnelle de KCC2. Nous avons dans un premier temps montré que la déphosphorylation des Thréonines (Thr) 906 et 1007 in vitro était un puissant activateur de la protéine. En effet, nous avons montré que l’état de phosphorylation des sites dicté par la voie Wnk-Spak/OSR1 était impliqué dans le niveau d’expression en surface de la protéine. Par la suite, nous avons pu révéler que les souris porteuses d’une mutation phospho-mimétique Glu906 et Glu1007 “(KCC2E/+)” sur un allèle de la protéine, présentaient un décalage de l’émergence de la force inhibitrice GABAergique, une altération de la balance excitation/inhibition, ainsi qu’une augmentation de la susceptibilité à générer des crises. De plus, ces même souris développent des troubles de la communication chez le jeune ainsi qu’un défaut de sociabilité chez l’adulte, deux symptômes clefs des TSA. Ces résultats suggèrent que la régulation post-traductionnelle est un mécanisme physio-pathologique clef de la protéine. / From basic to clinical aspects, this thesis comprises different collaborative projects focusing on KCC2. KCC2 is a complex protein with multiple roles and implications in various pathologies that makes this molecule a key element of living organisms. In addition to the phylogenetic and therapeutic studies performed during this period, my main work has been to determine the in vitro and in vivo physio-pathological role of a KCC2 post-translational regulatory mechanism. We first showed in vitro that dephosphorylation of Threonines (Thr) 906 and 1007 was a potent activator of the protein. We have shown that phosphorylation state by the Wnk-Spak/OSR1 pathway of these two residues is implicated in the level surface expression of KCC2. Subsequently we have revealed that mice carrying in one allele a phospho-mimetic mutations Glu906 and Glu1007 “(KCC2E/+)”, preventing the developmental dephosphorylation at these sites, exhibited a delayed onset of fast synaptic GABA inhibition, a decreased ratio of spontaneous GABA- to glutamate-driven post-synaptic responses, and a significantly reduced flurothyl-induced seizure threshold. Furthermore, KCC2E/+ pups and adult mice, respectively, exhibited impaired communication and sociability, classic ASD phenotypes. These results suggest that post-translational regulation is a key physio-pathological mechanism of KCC2.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 131 pages. Includes vita. Includes bibliographical references.

Throughout this thesis, we studied the biochemical and structural impact of the essential residues on the activity of class D beta-lactamases. The production of these enzymes plays a major role in the bacterial resistance. Our work is subdivided in two parts : the study of the post-translational modification of lysine 70 and the screening of new potential inhibitors for the class D β-lactamases. The first part concerns the impact of the residues tryptophan 154 and valine 117 located in the hydrophobic core. Our data indicate that the mutation of tryptophan 154 in alanine or glycine lead to a large decrease of the catalytic efficiencies of the beta-lactamase. The apo-enzyme structures of these mutants show that the lysine 70 is not carboxylated. This absence of carboxylate group induces a modification of the hydrogen network of the active site. The analysis of the complex structure of W154A-benzylpenicillin demonstrates that the deacylation step is clearly the most affected by the mutation. The mutation of tryptophan 154 in histidine leads to a slight decrease of catalytic efficiencies because the imidazol group of histidine mimics the indole group of tryptophan 154. The apo-enzyme structure reveals that lysine 70 is partially carboxylated and stabilized by an hydrogen bond between the carboxylate group and the imidazol group. In the case of the V117T mutant, a strong increase of the catalytic constant values is observed at 50 mM in NaHCO3. The structure of this mutant at pH 8.0 shows that the lysine 70 is partially carboxylated in the monomer A. The determination of individual rate constants of acylation and deacylation steps indicates that the deacylation is the limiting step for the class D beta-lactamase. The k2/k3 ratio is similar between the V117T mutant and the wild-type enzyme. The mutation of lysine 70 in alanine or cysteine leads to a large decrease of the deacylation constants inducing a poorly efficient enzyme. The obtaining of the K70C-Ampicillin complex by X-ray cristallography and the trapping of acyl-enzyme by reaction with fluorescent ampicillin are supplemental proofs that the deacylation step is the limiting rate. By crystallographic and kinetic studies, we demonstrate that the chloride inhibition of the class D beta-lactamases is due to a competition between the carboxylate group of lysine 70 and the chloride ions. At high concentration in bicarbonate, this inhibition is abolished for the wild-type enzyme. The second part of this work concerns the screening of the citrate and aminophosphonate derivated molecules for the class D beta-lactamases. In the case of OXA-10, a citrate molecule is strongly stabilized by hydrogen bonds in the active site. The benzyl esters derivatives of citrate inhibits OXA-10(KI = 20 µM) but the hydrophobic substituents are necessary to obtain a good inhibition.

The SUMO protein was named Small Ubiquitin-like MOdifier because its 3-D structure was similar to Ubiquitin. In human, three SUMO proteins were discovered, namely, SUMO-1/2/3. The recombinant ¡µ1-8, 94-95 SUMO-2 protein with 10 histidine residues at its N-terminus was expressed using E. coli. BL-21(DE3), purified at 4 oC and crystallized at room temperature. The surface properties of human SUMO-1/2/3 proteins and 3-D structure of ¡µ1-8, 94-95 SUMO-2 protein were analyzed using computer modeling and X-ray diffraction technology respectively. The two-step purification by immobilized metal ion affinity chromatography(IMAC) was developed to yield ¡µ1-8, 94-95 SUMO-2 protein that reached 60 mg/ml for crystallization. On protein expression, 120 mg protein was obtained from 6 L bacterial growth broth. Crystals of ¡µ1-8, 94-95 SUMO-2 were obtained by the hanging-drop vapor diffusion method and many different crystal forms were observed. One of single crystal with triangular plate polyhedron form diffracted to 1.6 Å resolution, the other one with rectangular polyhedron form diffracted to 1.2 Å. Analysis of the diffraction pattern suggests the crystals belong to R3 space group, the former one owned unit cell parameters a= b=75.3 Å, c=29.2 Å, £\=90¢X, £]=90¢X,£^=120¢X, and the later one owned unit cell parameters a= b=74.9 Å, c=33.2 Å and the same angles respectively. The R factor and Rfree of refinement are 0.133 and 0.190 with highly precise phase on 3-D structure of SUMO-2 protein. Comparison of crystal structure between human SUMO-2 and yeast SMT3 showed that the r.m.s. deviation of C£\ coordinate is 1.054 Å. In addition, comparison of SUMO-1 NMR structure and SMT3 crystal structure showed that the r.m.s. deviation of C£\ coordinates is 2.736 Å. Hence, the structures of SUMO-2 and SMT3 are more similar each other than those of SUMO-1and SMT3.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Epigenetic control of gene expression by histone post-translational modifications (PTMs) is a complex process regulated by proteins that can 'read', 'write' or 'erase' these PTMs. The histone lysine demethylase (KDM) family of epigenetic enzymes remove methyl modifications from lysines on histone tails. The Jumonji C domain (JmjC) family is the largest family of KDMs. Investigating the scope and mechanisms of the JmjC KDMs is of interest for understanding the diverse functions of the JmjC KDMs in vivo, as well as for the application of the basic science to medicinal chemistry design. The work described in this thesis aimed to biochemically investigate the inhibitor and substrate diversity of the JmjC KDMs, it led to the identification of new inhibitors and substrates and revealed a potential combinatorial dependence between adjacent histone PTMs. Structure-activity relationship studies gave rise to an n-octyl ester form of IOX1 with improved cellular potency and selectivity towards the KDM4 subfamily. This compound should find utility as a basis for the development of JmjC inhibitors and as a tool compound for biological studies. The rest of this thesis focused on the biochemical investigations of potential substrates and inhibitors for KDM3A, a JmjC demethylase with varied physiological functions. Kinetic characterisation of reported KDM3A substrates was used as the basis for evaluations of novel substrates and inhibitors. Further studies found TCA cycle intermediates to be moderate co-substrate competitive inhibitors of KDM3A. Biochemical investigations were carried out to study potential protein-protein interactions of KDM3A with intraflagellar transport proteins (IFTs), non-histone proteins involved in the formation of sperm flagellum. Work then addressed the exploration of novel in vitro substrates for KDM3 (KDM3A and JMJD1C) mediated catalysis, including: methylated arginines, lysine analogues, acetylated and formylated lysines. KDM3A, and other JmjC KDMs, were found to catalyse novel arginine demethylation reaction in vitro. Knowledge gained from studies with unnatural lysine analogues was utilised to search for additional novel PTM substrates for KDM3A. These results constitute the first evidence of JmjC KDM catalysed hydroxylation of an Nε-acetyllysine residue. The H3 K4me3 position seems to be required for acetyllysine substrate recognition, implying a combinatorial effect between PTMs. Preliminary results provide evidence that JMJD1C, a KDM3 protein previously reported to be inactive, may catalyse deacetylation in vitro. An additional novel reaction, observed with both KDM3A and JMJD1C, is deformylation of N^ε-formyllysine residues on histone H3 fragment peptides. Interestingly, H3 K4 methylation was also observed to enhance the apparent deformylation of both KDM3A and JMJD1C catalysed reactions. Overall, findings in this thesis suggest that the catalytic activity of JmjC KDMs extends beyond lysine demethylation. In a cellular context, members of the KDM3 subfamily might provide a regulatory link between methylation and acylation marks. Such a link will further highlight the complex relationships between histone PTMs and the epigenetic enzymes that regulate them. The observed dependency of H3 K9 catalysis on H3 K4 methylation adds another layer of complexity to the epigenetic regulation by histone PTMs.