Over the past three decades, nitric oxide (NO) has been recognised as one of the most versatile and important players in many aspects of physiology, including immune responses. More recently, S-nitrosylation, the incorporation of a NO moiety into a protein thiol group, has emerged as a major post-translational modification (PTM) during pathophysiological responses in plants and animals. The main goal of this work was to investigate the role of S-nitrosylation in physiology and innate immunity of animals using the genetic reference system, Drosophila melanogaster. The S-nitrosylated derivative of glutathione (GSH), S-nitrosoglutathione (GSNO), is the main non-protein S-nitrosothiol (SNO) in the cell and extracellular fluids. GSNO can trans-S-nitrosylate other thiols and is considered a reservoir of NO bioactivity. The levels of GSNO and total S-nitrosylation have been shown to be controlled by S-nitrosoglutathione reductase (GSNOR) in yeast, plants and mammals. By employing an overlapping deletion technique to knock-out gsnor, a role for S-nitrosylation in the immune response of D. melanogaster is proposed. Compared to wild type flies, gsnor overlapping deletion flies presented lower expression of antimicrobial peptides in response to infections, and succumbed more rapidly to both Gram-positive bacterial and fungal pathogens. As the Toll pathway mediates responses against these pathogens, key components of this network were tested for their propensity to being S-nitrosylated. Two CLIP-domain serine proteases of the Toll signalling pathway, Persephone (PSH) and Spätzle-Processing Enzyme (SPE), were shown to be S-nitrosylated both in vitro and in vivo and this process seemed to control the quaternary structure of these proteins and interfere with the immune response of D. melanogaster. At least for PSH, S-nitrosylation at C254 has an immune significance as the expression of non-Snitrosylable PSHC254S in gsnor knock-out flies partially recovered the resistance of these animals to infections with the entomopathogenic fungus Beauveria bassiana. These findings might represent a novel mechanism by which NO and S-nitrosylation regulate immunity. Further results presented in this thesis reveal an interplay between reactive oxygen species (ROS) and reactive nitrogen species (RNS) in D. melanogaster physiology and immunity. Similarly to what has been reported in Arabidopsis thaliana, gsnor knock-out flies presented higher tolerance to the herbicide paraquat, an inducer of superoxide (O2 -) production. Moreover, additional mutations in Catalase (Cat), a hydrogen peroxide (H2O2) scavenger enzyme, partially restored the immunodeficiency phenotypes of gsnor knock-out flies. These findings suggest an inter-relation between the levels of ROS and RNS during stress responses of plants and animals. In addition, CRISPR/Cas9 technology was employed to generate gsnor knock-outs in the genome of D. melanogaster. These flies were shown to have no GSNOR activity, presented lower tolerance to pharmacological-induced nitrosative stress and succumbed faster to infections with B. bassiana compared to wild type flies. These results support the role played by GSNOR in regulating NO homeostasis and immunity in D. melanogaster.

A biofilm is a complex community of cells enveloped in a self-produced polymeric matrix. Entry into a biofilm is exquisitely controlled at the level of transcription and in Bacillus subtilis it requires the concerted efforts of several major transcription factors including the repressor SinR and activator DegU. I initially identified that these transcriptional regulators control biofilm formation via parallel pathways. Through investigating the regulation of biofilm formation by SinR and DegU, I discovered that biofilm formation is also regulated at the post-translational level. This was achieved by identifying three key proteins which are needed for biofilm formation. These proteins are PtkA, a bacterial tyrosine kinase; TkmA, the cognate modulator of PtkA; and PtpZ, a bacterial tyrosine phosphatase. By introducing amino acid point mutations within the catalytic domains of PtkA and PtpZ it was identified that the kinase phosphatase activities, respectively, are essential function.In addition, PtkA contains a conserved C-tyrosine cluster that is the site autophosphorylation. Investigation of the role of the C-terminal tyrosine cluster tentatively suggests that this domain acts to block access to the active site of PtkA, thus affecting the ability of PtkA to phosphorylate its targets. Deletion of the gene coding for TkmA demonstrated that this modulator was also required for biofilm formation. It was also demonstrated that TkmA may interact with other protein partners, at least in the absence of PtkA, raising the question of how signal specificity is maintained. Finally, a systematic mutagenesis approach was used with the aim of identifying the target(s) of PtkA and PtpZ during biofilm formation but,despite extensive efforts, it remained elusive. The findings presented in this thesis highlight the complexity of biofilm formation by B. subtilis by revealing an additional level of regulation in the form of protein tyrosine phosphorylation.

The O-linked N-acetylglucosamine post-translational modification (O-GlcNAcylation) is the dynamic and reversible attachment of N-acetylglucosamine to serine and threonine residues of target proteins. It is abundant in metazoa, involving hundreds of proteins linked to a plethora of biological functions with implications in human diseases. The process is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), that add and remove the sugar moiety, respectively. Ogt gene knock-out is embryonic lethal in a range of animal models, hampering the study of the biological role of O-GlcNAc. O-GlcNAcylation of nuclear and cytoplasmic proteins has been extensively studied, however little is known about the role of O-GlcNAc in mitochondria. A previous report suggested the presence of a mitochondrial OGT isoform (mOGT) in human cell lines in addition to the well-characterised nucleocytoplasmic one (ncOGT). Since this report more than one decade ago, this putative mOGT has not been studied further. Similarly, hundreds of O-GlcNAcylated nucleocytoplasmic proteins have been identified by high-throughput proteomic screens, whereas only a few mitochondrial proteins have been detected. Nevertheless, several studies suggest that altered O-GlcNAc signalling affects mitochondrial function and morphology, with potential clinical implications. The aim of this thesis work was to study and characterise the biological role of mOGT and determine the mitochondrial O-GlcNAc proteome. Firstly, the presence of mOGT in human cell lines and mouse tissues was investigated. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed at protein level in most of the species analysed, except human and some primates. In fact, the putative mOGT cDNA in most of the genomes analysed contains a stop codon that excludes the presence of such isoform. In addition, mOGT was not detected at protein level in a wide range of human cell lines. Knock-down experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, overexpression of ncOGT in HEK 293 suspension cells led to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the mitochondrial O-GlcNAc proteome. These data point to a model where O-GlcNAc cycling of mitochondrial proteins occurs in the cytosol, followed by their import into mitochondria. Alternatively, ncOGT itself might be transported into mitochondria where it can take part to O-GlcNAc cycling inside the organelle. In parallel, some advance in determining the O-GlcNAc mitochondrial proteome has been undertaken. Different mitochondrial fractionation protocols, combined with O-GlcNAc enrichment methods have been explored in order to map novel glycosylation sites on mitochondrial proteins. A novel technique established in our research group, employing a bacterial OGA orthologue as a bait to trap O-GlcNAcylated proteins, has been applied to crude mitochondrial fractions allowing the identification of several hits, although site mapping has not been yet achieved. The second chapter describes the work that has been done to improve and optimise novel O-GlcNAc inhibitors previously designed in the laboratory, called goblins. The original objective was to make these molecules cell-permeable and possibly target them to mitochondria in order to inhibit mOGT. Several strategies were explored to deliver the compounds into living cells, including the use of transfection reagents and covalent linkage to linear cell-penetrant peptides. The above methods did not achieve cellular uptake, although recently designed cyclic cell-penetrant peptides, linked to fluorescein, were internalised by HeLa cells with immediate diffuse nucleocytoplasmic staining. These molecules will be linked to goblins aiming to use the inhibitors for cell biology studies. A different approach, based on the permeabilisation of Drosophila embryos, enabled the penetration of goblins into the organisms with consequent reduction of global O-GlcNAc levels. This method allowed the use of these novel bisubstrate inhibitors in vivo for the first time, with potential applications in studying the role of O-GlcNAc in Drosophila development and possibly for future therapeutic purposes after further development of the scaffold.

Abstract Human ubiquitin-like SUMO-1/2/3 proteins have been identified. The 3-D structure of the SUMO-1 has been shown to be very similar to that of ubiquitin, although their sequences share only 18 % identity. Unlike ubiquitination targets proteins for degradation, sumoylation appears to regulate a number of cellular processes such as protein-protein interaction, subcellular localization, protein stability, apoptosis, cell cycle and so on . Our laboratory has cloned cDNAs encoding human SUMO-2, mouse SUMO-2 and SUMO-3, as well as a single SUMO gene from nematode and Drosophila. Recently (Su & Li, Gene 296:65-73,2002), Su & Li have performed data-mining on current human genomic sequence and found the presence of only three SUMO-1/2/3 functional genes located at chromosome no. 2q33, 17q25.1 and 21q22.3, respectively, as well as eight SUMO-1 pseudogenes and 23 SUMO-2 pseudogenes. The protein-coding sequence of SUMO-1 gene is interrupted by four introns , while those of SUMO-2/3 genes are interrupted by only three introns. In this study , most of SUMO-1/2/3 proteins were show to be localized on nuclear membrane, nuclear bodies and cytoplasm, respectively. The N-terminus-deleted SUMO-1 proteins was further shown to be localized on nuclear membrane and in cytosol, while the mutant SUMO-2/3 proteins were localized only in the cytosol. The inactive precursor form of SUMO-3 was exclusively localized in the cytosol. The activation of SUMO-3 in HeLa cells was triggered by actinomycin D and its location was shifted from cytosol to nucleus. Further, the inactive precursor of SUMO-3 was reduced in HeLa cells treated with nocodazole and arsenic trioxide.

Thesis (Ph.D.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 182-202). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR39002

The 2-micron circle of Saccharomyces cerevisiae is one of a small number of similar DNA plasmids found only in budding yeast. To understand how this cryptic parasite persists, despite conferring no advantage to the host, I investigated the plasmid-encoded Rep1 and Rep2 proteins. Interaction of Rep1 and Rep2 with each other and with the plasmid STB locus is required for equal partitioning of plasmid copies at mitosis. The Rep proteins also repress expression of Flp, the recombinase that mediates plasmid copy-number amplification. In this study, absence of Rep1 and Rep2, or over-expression of the plasmid-encoded Raf antirepressor, increased expression of a longer, novel FLP transcript. Translation of this mRNA may explain elevated Flp activity at low plasmid copy number. Raf competed for Rep2 selfassociation and interaction with Rep1, suggesting the mechanism of Raf anti-repression. Deletion analysis identified a target site for Rep protein repression of FLP that is also repeated in the STB locus, suggesting this as the sequence required for Rep protein association with both regions of the plasmid. Distinct roles for Rep1 and Rep2 were identified; Rep1 was found to depend on Rep2 for post-translational stability, with Rep2 dependent on Rep1 for stable association with STB. Lysine-to-arginine substitutions in Rep1 and Rep2 impaired their association with the host covalent-modifier protein SUMO, suggesting these were sites of sumoylation. The substitutions did not affect interaction of the Rep proteins with each other or their stability but did perturb plasmid inheritance, suggesting that Rep protein sumoylation contributes to their plasmid partitioning function. When Rep1 was mutant, both Rep proteins lost their normal localization to the nuclear foci where 2-micron plasmids cluster, and were impaired for association with STB, supporting this as the cause of defective plasmid inheritance. The potential sumoylation-dependent association of the Rep proteins with the 2-micron plasmid partitioning locus suggests the plasmid has acquired a strategy common to eukaryotic viral and host genomes that depend on sumoylation of their segregation proteins for faithful inheritance. Collectively, my results shed light on how the 2-micron plasmid maintains the delicate balance of persisting without harming its host.

Vaccinia virus (VV) is a large DNA virus belonging to the Orthopoxvirus family. The viral replicative life cycle takes place solely within the cytoplasm of a mammalian host cell. The VV genome contains 196 open reading frames which are expressed in a highly regulated and temporal fashion in order to bring about the production of a mature virion. In the process of viral replication many VV proteins are synthesized that require posttranslational modifications to become functional. A few of these modifications include, glycosylation, ADP-ribosylation, phosphorylation, fatty acid acylation, and proteolytic processing. This last modification is especially important with regard to the structural proteins of the virus in that they undergo prysis for an infectious virus particle to be formed, a common theme in viral systems. In order to understand these events in more detail, three abundant virion protein constituents 4a, 4b, and 25K were chosen as models for study. The three main questions we wanted to answer were: Is there a cleavage consensus site within the precursors, what protease(s) and/or factors are necessary for the process, and how are the events regulated in vivo? Our approach included development of specific immunological reagents to identify cleavage products as well as to show where these core proteins are located during virion assembly. We have subsequently identified cleavage products by N-terminal microsequence from each of the three structural proteins and this information has elucidated a putative cleavage consensus site of Ala-Gly- X, where cleavage is proposed to take place between the Gly and X and X is usually an aliphatic residue. The immunological reagents were used in conjunction with immunofluorescent and immunogold labeling analyses to identify the location of these core proteins during virion assembly. Core proteins were localized to the virosomes in VV infected cells, to the viroplasm of immature virus particles, and to the center of mature virions. Precursor specific antiserum indicated that the larger molecular weight precursors of core proteins are within immature virions as well. From these results the following conclusions can be made. Identification of a putative cleavage consensus site suggests that proteolytic processing is an endoproteolytic event. The observation that precursor structural proteins were found within immature particles indicates that the proteinase responsible for cleavage is also present. The fact that assembly has to occur before proteolytic processing of VV structural proteins suggests that the cleavage events are dependent upon a specific core protein conformation. However the nature of this conformational requirement is not known. Further research is underway to develop a full understanding of the proteolytic events during virion morphogensis. / Graduation date: 1993

Abstract Lysyl hydroxylase (EC1.14.11.4, LH) catalyzes post-translationally the hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. Hydroxylysyl residues may also be glycosylated by hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) or galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT) to form galactosylhydroxylysyl or glucosylgalactosylhydroxylysyl residues, structures unique to collagen. Three LH isoenzymes (LH1, LH2a/2b, LH3) have been characterized so far. We analyzed mRNA levels of these isoforms, as well as the mRNAs of the main collagen types (I, III, IV, V) and the α subunit of PH-4 in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. The mRNA levels of LH1, LH2, and the α subunit of PH-4 showed significant correlation with each other whereas LH3 correlated with none. No correlation was observed between the LH isoforms and individual collagen types. Three human LH isoforms were expressed in different expression systems. The purified recombinant protein produced by LH3 cDNA was found to be the only one possessing LH, GT and GGT activities. The molecular weight of the partially purified LH3 expressed in Sf9 or Cos-7 cells corresponded to about 85 kDa whereas that in E.coli cells was about 81 kDa probably due to a deficiency of glycosylation in bacterial cells. The recombinant protein of C. elegans LH cDNA was expressed in a cell-free translation system and in E.coli cells. The data indicated that the glycosyltransferase activities, GT and GGT, were also associated with this gene product. The sequence alignment of LH isoforms from different species revealed that there are 29 amino acids conserved between human LH3, mouse LH3 and C. elegans LH sequences and scattered evenly in the molecule, but differing from those of LH1 and LH2. In vitro mutagenesis data showed that the amino acids important for the glycosyltransferase activities were located at the amino-terminal part of the molecule, being separate from the LH active site. Mutation of a conserved LH3 specific, non-disulfide linked cysteine to isoleucine caused a dramatic reduction in GT and GGT activity but had no effect on LH activity. Mutations of the amino-terminal DxD motif (D187-191) characteristic of many glycosyltransferases eliminated both GT and GGT activities, showing the importance of this motif for collagen glycosyltransferases and suggesting that it might serve as the Mn2+ binding site in the molecule.

Lysine acetylation is a conserved post-translational modification (PTM) which was traditionally believed to be limited to histones and the regulation of gene expression. However, recent proteomic studies have identified lysine acetylation on proteins implicated in virtually all cellular processes indicating that this PTM plays a global regulatory role. Indeed, in humans, aberrance of lysine acetyltransferase (KAT) activity is associated with various pathogenesis. To date, over 2500 human proteins are known to be acetylated in vivo, but very few acetylations have been linked to specific KATs. Hence, to understand the biological relevance of KATs and acetylation in human pathology, it is important to learn about the mechanism regulating KAT activity and the identity of their in vivo targets. This is a complex task and will require the use of model organisms and system biology approaches. The work presented here explores the significance of self-acetylation in regulating KAT function by focusing on the highly NuA4 lysine acetyltransferase in the model organism Saccharomyces cerevisiae or budding yeast. Using genetics and biochemical assays I have identified NuA4 subunit Epl1 as a novel in vivo NuA4 substrate. I have also shown that Epl1 acetylation regulates NuA4 function at elevated temperatures. In an attempt to identify new biological processes regulated by yeast KATs and putative novel substrates, I have also performed a genome-wide synthetic dosage lethality screen with six non-essential yeast KATs; Hat1, Rtt109, Hpa2, Sas3, Sas2, and Elp3. My screen identified largely distinct sets of genetic interactions for each KAT suggesting that each KAT has specific cellular functions. Together, this study demonstrates the importance of auto-acetylation in regulating KAT function and the diversity of cellular processes impacted by KAT activity in vivo.

Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative disorder characterized by the selective degeneration of upper and lower motor neurons. Patients typically die from respiratory failures within 2-5 years after diagnosis. One of the milestones in ALS research is the discovery Fused in Sarcoma (FUS), an ALS causative gene. FUS is an RNA/DNA-binding protein and predominantly resides in the nucleus. Majority of the FUS mutations are located in the C-terminus and causing aberrant misdistribution to the cytoplasm. Currently, only a few binding partners of FUS are known, which makes it difficult to speculate on the function and interaction of the protein. In this study, we conducted a kinome-wide RNAi screen to identify kinases that affect the localization of FUS. A dual specificity protein kinase named CDC2-like kinase (CLK1) from the screen was found to be responsible for in post-translational modification of FUS and affects the localization of FUS in the nucleus. The identification of CLK1 as FUSmodifying kinase is consistent with roles ascribed to both in the binding and regulation of RNA.