As the most common motor neuron disease, Amyotrophic lateral sclerosis (ALS) affects around 4 in every 100,000 people worldwide with reports of increasing prevalence over the years. Characterized by progressive degeneration of motor neurons, ALS patients suffer impairments of movement and typically die from respiratory failure 2-5 years after diagnosis. Curiously, ALS exists on a disease continuum with Frontotemporal Dementia (FTD) where 30-50% of patients will be diagnosed with both diseases. In FTD, degeneration of cortical neurons results in diverse behavioural changes including deficits in executive and social skills as well as language and memory. A central connection between ALS and FTD is TDP-43 (encoded by TARDBP), an essential DNA/RNA binding protein known to serve critical functions in numerous cellular processes. Despite mutations in TARDBP constituting a small percentage of familial cases, TDP 43 nuclear-to-cytoplasmic mislocalization is a pathological hallmark of most ALS-FTD cases. Therefore, therapeutic targets to rectify pathology and disease may be uncovered by identifying factors that regulate TDP-43. While it is currently established TDP-43 is ubiquitinated and phosphorylated in diseased states, our lab recently found TDP-43 is SUMOylated in response to stress. Of note, perturbations in the stress response are becoming increasingly implicated in neurodegenerations. Furthermore, TDP-43 plays critical roles in the stress response which become perturbed in ALS/FTD. We developed a TDP-43 "SUMO dead" mouse allele to gain an understanding of how disrupting this may contribute to the pathogenesis of ALS-FTD. Longitudinal characterization of the model explored behavioural and histological in vivo consequences following loss of TDP-43 SUMOylation. However, the phenotypes observed in the mutant mice were less robust in comparison to established ALS/FTD mouse models. Mutant mice did not have consistent differences in tests for similar outcomes, trials of the same test, or across age. Female mutant mice presented with early hyperactivity and disinhibition along with altered social grooming behaviour. At later age, these female mice developed impairments in spatial working memory. Male mice developed apathetic behaviour and motor deficits at the middle age timepoint. Histologically, various forms of pathological TDP-43 were observed in the absence of neurodegeneration. These data reveal that TDP-43 SUMOylation may play an important role in ALS/FTD pathogenesis.

Peptides are small proteins that are crucial in many biological pathways such as antimicrobial defense, hormone signaling, and virulence. They often exhibit tight specificity for their targets and therefore have great therapeutic potential. Many peptide-based therapeutics are currently available, and the demand for this type of drug is expected to continue to increase. In order to satisfy the growing demand for peptide-based therapeutics, new engineering approaches to generate novel peptides should be developed. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of peptides that have the potential to be effective scaffolds for in vivo peptide engineering projects. These natural RiPP peptides are enzymatically endowed with post-translational modifications (PTMs) that result in increased stability and greater target specificity. Many RiPPs, such as microcin J25 and micrococcin, can tolerate considerable amino acid sequence randomization while still being capable of receiving unique post-translational modifications. This thesis describes how we successfully engineered E. coli to produce the lasso peptide microcin J25 using a two-plasmid inducible expression system. In addition, we characterized the protein-protein interactions between PTM enzymes in the synthesis of micrococcin. The first step in micrococcin synthesis is the alteration of cysteines to thiazoles on the precursor peptide TclE. This step is accomplished by three proteins: TclI, TclJ, and TclN. We found that a 4-membered protein complex is formed consisting of TclI, TclJ, TclN, and TclE. Furthermore, the TclI protein functions as a central adaptor joining two other enzymes in the Tcl pathway with the substrate peptide.

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<p dir="ltr">Post-translational modifications (PTMs) play a crucial role in regulating protein function and location. Protein AMPylation, the addition of adenosine monophosphate (AMP), significantly influences protein trafficking, stability, and pathogenic virulence. The Fic Domain family of proteins targets hydroxyl-containing amino acid residues (Ser, Thr, or Tyr), catalyzing the addition of various phosphate-containing moieties such as nucleoside monophosphates (NMPs), phosphocholine, and phosphate. Using gene mining techniques, Dr. Seema Mattoo’s group has identified a clade of Fic domain containing proteins typified by the enzyme originating from <i>Bordetella bronchiseptica</i> (BbFic) which prefers utilizing guanosine triphosphate (GTP) as a substrate over other nucleotides. To understand the physiological role of GMPylation, identifying the proteins modified by BbFic is a first critical step and can be accomplished via mass spectrometry-based proteomics. For a low stoichiometry PTM like GMPylation, however, there is a need to develop chemical tools that enable the targeted enrichment of modified protein. Identifying key interactions between substrate proteins and the BbFic nucleotide binding site will enable development of highly specific molecular tags for Fic substrates.</p><p dir="ltr">The goal of this research project, therefore, is to design chemical probes to tag Fic enzyme substrates, thereby facilitating the identification of GMPylated proteins in chemical proteomics workflows. A set of ATP and GTP analogues carrying either alkyne or azide handles were proposed as possible probes. While 8-azido guanosine showed a high docking score in our in-silico study, literature reports highlight its chemical instability upon exposure to air and light. An alternative probe, the 8-ethynyl guanosine, also showed a high docking score and docks in the same position and orientation as guanosine (the natural ligand) but necessitates synthetically challenging via cross-coupling reactions.</p><p dir="ltr">We considered multiple GMP analogues as potential molecular tags with the assistance of molecular docking with the BbFic enzyme. With predicted binding affinities in hand, we prioritized candidate GTP analogs for synthesis to probe the BbFic-mediated protein GMPylation process. While N6 propargyl guanosine serves as a lead probe for AMPylation, computational analysis reveals challenges with O6 due to its altered hydrogen bond donor/acceptor presentation. The distinctive chemical properties of guanosine, compared to adenosine, require a thorough evaluation of protective group strategies, as not all synthetic methodologies used for ATP analogue synthesis are applicable to GTP analogues. Isolating the triphosphate analogue proved challenging, although purification of the monophosphorylated counterpart is feasible. The Protide analogue benefits from phosphate charge masking, which facilitates purification. While much work remains until the physiological role of GMPylation can be determined, important progress has been made in the design and synthesis of chemical tools for studying this newly discovered PTM.</p>

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.

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.

Proteins and RNAs are two of the most versatile macromolecules that carry out almost all functions within living organisms. In this thesis I have explored evolutionary and regulatory aspects of proteins and RNAs by studying their structures, modifications and interactions. In the first chapter of my thesis I investigate domain atrophy, a term I coined to describe large-scale deletions of core structural elements within protein domains. By looking into truncated domain boundaries across several domain families using Pfam, I was able to identify rare cases of domains that showed atrophy. Given that even point mutations can be deleterious, it is surprising that proteins can tolerate such large-scale deletions. Some of the structures of atrophied domains show novel protein-protein interaction interfaces that appear to compensate and stabilise their folds. Protein-protein interactions are largely influenced by the surface and charge complementarity, while RNA-RNA interactions are governed by base-pair complementarity; both interaction types are inherently different and these differences might be observed in their interaction networks. Based on this hypothesis I have explored the protein-protein, RNA-protein and the RNA-RNA interaction networks of yeast in the second chapter. By analysing the three networks I found no major differences in their network properties, which indicates an underlying uniformity in their interactomes despite their individual differences. In the third chapter I focus on RNA-protein interactions by investigating post-translational modifications (PTMs) in RNA-binding proteins (RBPs). By comparing occurrences of PTMs, I observe that RBPs significantly undergo more PTMs than non-RBPs. I also found that within RBPs, PTMs are more frequently targeted at regions that directly interact with RNA compared to regions that do not. Moreover disorderedness and amino acid composition were not observed to significantly influence the differential PTMs observed between RBPs and nonRBPs. The results point to a direct regulatory role of PTMs in RNA-protein interactions of RBPs. In the last chapter, I explore regulatory RNA-RNA interactions. Using differential expression data of mRNAs and lncRNAs from mouse models of hereditary hemochromatosis, I investigated competing regulatory interactions between mRNA, lncRNA and miRNA. A mutual interaction network was created from the predicted miRNA interaction sites on mRNAs and lncRNAs to identify regulatory RNAs in the disease. I also observed interesting relations between the sense-antisense mRNA-lncRNA pairs that indicate mutual regulation of expression levels through a yet unknown mechanism.