Theses (Biochemistry and Microbiology)

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    Role of the PEST Domains in Proteasomal Degradation of Rett Protein: MeCP2
    (2024) Kalani, Ladan; Ausio, Juan
    Located on the X-chromosome is the gene encoding the nuclear protein Methyl CpG binding protein 2 (MeCP2). The instability of this protein causes pleiotropic neurological abnormalities, including the debilitating neurodevelopmental disease Rett syndrome (RTT). MeCP2, an epigenetic regulator abundant in neurons, is involved in pleiotropic molecular interaction. Many deleterious mutations of MeCP2 impact its mRNA or protein levels. Neuron maturation and dendritic arborization are compromised when MeCP2 levels are out of the homeostatic range. The mechanisms the cell uses to maintain MeCP2 levels within a tight range have yet to be fully understood. Several hypotheses addressed the homeostatic mechanisms of MeCP2, which involve miRNAs, N-terminal degradation signals or N-degrons, and the PEST domains that act as degradation switches upon post-translational modifications (PTMs). Our lab hypothesized the involvement of MeCP2 PEST-mediated degradation as a mechanism of its homeostatic regulation; however, this hypothesis has yet to be experimentally proven. I experimentally tested the PEST-mediated degradation of MeCP2 with Rett-causing mutations by integrating MeCP2 constructs that have an altered or deleted PEST domain and used microscopy, FRAP analysis and western blotting to characterize in vitro how these constructs behave relative to WT and mutated MeCP2. MeCP2 has Rett-causing mutations that cause lower protein levels, such as T158M; the PEST motif expedites its degradation as deleting it results in higher protein levels. Moreover, mutations that result in higher levels of MeCP2, such as R294X, show stronger DNA binding relative to WT, as assessed by NaCl fractionation. For the first time, we report that the Ct-PEST domain of MeCP2 plays a role in its degradation.
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    Investigation of thyroid hormone-dependent molecular changes in the bullfrog back skin with special emphasis on the innate immune system
    (2023-12-04) Corrie, Lorissa; Helbing, Caren C.
    As the aquatic tadpole undergoes metamorphosis to become a terrestrial juvenile frog, its innate immune system must adapt to its new environment. Skin is a primary line of defense in the innate immune system since it acts as an important physical, microbial, and chemical barrier that is constantly in contact with a microbially diverse environment throughout its life cycle. In amphibians, skin undergoes complex remodelling during postembryonic development. Metamorphosis is regulated by thyroid hormone (TH) initiated gene expression that leads to the start of the metamorphic programming. This can be induced by the addition of exogenous TH. While different tissues respond to TH in distinctive ways during metamorphosis, how these tissues respond to TH is poorly understood. Temperature modulation, which regulates metamorphic timing, is a unique way to uncover early TH-induced transcriptomic events related to a molecular memory. Using RNA-sequencing analysis, American bullfrog (Rana [Lithobates] catesbeiana) back skin transcripts were profiled during natural and temperature-modulated induced metamorphosis. During natural metamorphosis, significant differential expression was observed in over 6,500 transcripts. Premetamorphic tadpoles maintained at 5oC, a temperature that is non-permissive for inducing morphological changes, showed 83 differentially expressed transcripts within 48 h after TH administration. Of note is the induction of thibz that has previously been identified as a molecular memory component in other tissues. Over 3,600 differentially expressed transcripts were detected in TH-treated tadpoles compared to the controls at permissive temperature (24oC) after 48 h or when tadpoles were held at 5oC and then shifted to 24oC. We identified several innate immune system components: keratins, mucins, and antimicrobial peptides (AMPs) whose transcript levels changed during natural and TH-induced metamorphosis. A bioinformatics AMP identification pipeline, rAMPage, was applied to these RNA-seq data sets to identify 489 novel AMP candidates. AMPs have direct acting and immune stimulatory antimicrobial abilities and are promising antibiotic alternatives in public health and agricultural sectors. We synthesized and tested un-amidated and amidated C-terminal versions of 111 top-scoring novel AMP candidates for their antimicrobial activity and cell toxicity with a focus on agriculturally relevant pathogens (avian pathogenic Escherichia coli (APEC) and Salmonella enterica serovar Enteritidis (SE)). We found that 28 peptides were moderately or highly active (Minimum inhibitory concentration  32 μg/mL) against APEC and six against SE, most of which displayed low cell toxicity. Active AMPs were physicochemically classified as positively charged, alpha helices with amphipathic characteristics. These AMPs are candidates for further testing as therapeutics to prevent or treat disease in poultry. While the present work is focused on the identification of amphibian AMPs, it has far-reaching applications, and the approach can be used to identify AMPs as an antibiotic alternative from any genomic resource.
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    Engineering a Tp0751-based Vaccine Scaffold: Applying Iterative Protein Design to Optimize Antigen Presentation
    (2023-08-31) Thompson, Lexie; Boulanger, Martin J.; Cameron, Caroline E.
    Treponema pallidum subspecies pallidum is the causative organism of syphilis, a widespread sexually transmitted infection and re-emerging public health threat. The continued resurgence of syphilis, despite the discovery of effective penicillin treatment, highlights the need for preventative and affordable syphilis control. Previous efforts to develop a protective vaccine candidate have been hindered due to the limited discovery of protective antigens and the paucity of outer membrane proteins on the bacterial surface. A majority of the currently identified vaccine candidates are integral outer membrane proteins that, due to their highly insoluble nature, cannot be recombinantly produced in a properly folded and soluble state. Our focus is expressing the immunogenic epitopes from these large integral membrane proteins in a more sophisticated expression platform, allowing us to bypass the expression of these full-length proteins. Our designs focus on using Tp0751, a T. pallidum adhesin protein, as a scaffold on which to build our recombinant vaccine candidate. These studies aimed to engraft epitopes from a select few of these integral outer membrane proteins into the flexible loop and termini regions of Tp0751 to yield a stable and soluble candidate capable of recapitulating the immune response raised against each separate protein. Here we have successfully shown that not only is Tp0751 amenable to this engineering with up to four separate epitope engraftments, but that a lead chimeric candidate offers partial protection in a rabbit model. Altogether, the body of work summarized in this thesis highlights the success of Tp0751 as a vaccine platform and opens new avenues for future recombinant vaccine production.
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    Investigating the Functional Roles of the Sialidase NanH and Glycopeptidase Amuc_1438 in the Enzymatic Degradation of Mucin
    (2023-08-31) Medley, Brendon; Boraston, Alisdair B.
    The mucosal layer within the gastrointestinal tracts of animals and humans serves a critical function in safeguarding the epithelial layer against pathogens, including Clostridium perfringens. This layer consists of mucin-based glycoproteins, forming a dual-layered structure: an inner layer firmly attached to the epithelial cells, acting as a protective barrier, and an outer layer that fosters a favourable environment for commensal organisms. Both commensal and pathogenic bacterial species possess an array of enzymes designed for mucin degradation, fulfilling two main purposes: utilizing the abundant carbohydrate network as an energy and carbon source, and breaking down the mucus layer during pathogenic invasion. However, our understanding of these enzymatic tools employed by the gut microbiota remains incomplete, leaving gaps in our knowledge concerning pathogenic invasion and host-microbe interactions. This thesis presents a comprehensive analysis of two enzymes involved in mucin degradation: the intracellular sialidase NanH from Clostridium perfringens and the previously uncharacterized Amuc_1438 from Akkermansia muciniphila. The thesis investigates the structure of NanH, comparing it to its extracellular counterpart NanI, and provides structural insight into the specificity for sialic acids linked to glycans with α(2,3) over α(2,6) linkages. Additionally, through structural and functional investigations, Amuc_1438 is revealed to possess glycopeptidase activity, targeting specifically glycopeptides containing a Tn-antigen on a serine or threonine residue. Intriguingly, this work outlines that Amuc_1438 likely belongs to an uncharacterized family of glycopeptidases. The primary aim of this thesis is to demonstrate how these two distinct enzymes fit into the enzymatic pathway of mucin degradation observed in both commensal and pathogenic gut bacteria. By shedding light on the structural characteristics and functional roles of NanH and Amuc_1438, this research contributes to a deeper understanding of the intricate enzymatic processes involved in mucin degradation, thus enhancing our knowledge of pathogenic mucosal-invasion strategies and host-microbe interactions.
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    Modulation of T Cell Antitumor Immunity Through Acetylcholine Signaling
    (2023-08-22) Guagliano, Ryan; Lum, Julian J.
    Immunotherapies such as chimeric antigen receptor T cells (CAR-T cells) have shown promising results in many cancer patients but are still limited in solid cancers. Solid cancers contain immunosuppressive factors in the tumor microenvironment (TME), such as hypoxia and glucose deprivation. Antigen heterogeneity reduces therapy effectiveness in many cancers as CAR-T receptors need to recognize specific antigen that may be absent. This thesis investigates the function of a prominent TME metabolite: the neurotransmitter acetylcholine (ACh). Primary data from our lab shows that T cells infiltrating the TME have elevated ACh. Recent publications show ACh signaling influences mouse T cells to express a transcription factor FoxP3, a marker for regulatory T cells (Tregs) that contribute to the suppressive TME. However, there is little progress in testing the impact of these ACh-stimulated T cells’ anticancer functions. Data from my thesis indicates that folate receptor alpha (FRα) CAR-T cell antitumor effector function is enhanced, rather than suppressed by ACh. ACh promotes transient expression of human FOXP3 in activated proinflammatory effector T cells (Teffs) expressing interferon gamma (IFN-γ). My results identify the alpha 7 nicotinic acetylcholine receptor (α7 nAChR) is involved in increasing FOXP3, but not IFN-γ expression in human T cells and the enzyme choline acetyltransferase (ChAT) which catalyzes the rate-limiting step in the synthesis of ACh, is required for self-regulation of FOXP3 and the activation marker CD25 following activation. Helios, a transcription factor and Treg stability marker is likewise transiently expressed by active Teffs and only FOXP3+/Helios-, IFN-γ+ Teffs proliferate throughout expansion. Taken together, my results indicate that ACh signaling in vitro enhances T cell activation and differentiation into antitumor Teffs, and this could be used for novel methods to increase the efficiency of current solid cancer therapies through manipulation of FOXP3 and/or IFN-γ.
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    Evolution in microorganisms: From proteins to whole genomes
    (2023-08-22) McGuire, Bailey; Nano, Francis E.; Boraston, Alisdair B.
    Evolution is a constant and ongoing process which touches our lives in many ways. For organisms with short generation times, especially microorganisms, evolution can occur quite rapidly. Microbial evolution to changing environmental niches such as warming climates or a host animal immune system are driven by genetic mechanisms that are unpredictable and often poorly understood. We can simulate evolution of microbes to complex stressors through adaptive laboratory evolution. There are a few published studies of adaptive laboratory evolution to high incubation temperatures performed in Escherichia coli, and in each study different genetic changes are responsible for the strains’ enhanced thermotolerance. Outside of the laboratory, the virus which causes COVID-19, SARS-CoV-2, has evolved rapidly throughout the pandemic, gaining higher infectivity and improved immune escape. In this thesis, I explore adaptive laboratory evolution of Escherichia coli to heat, the evolution of the SARS-CoV-2 spike protein and optimizing the production of SARS-CoV-2 protein fragments in E. coli. I rapidly adapted E. coli to increasing incubation temperatures, raising their maximum growth temperature on LB agar by 2 °C in five months. I obtained whole-genome sequencing data of the starting strain and four isolates taken at different time points, processed and analyzed the data and uncovered the line’s unique evolutionary route to higher thermotolerance. One previously published study adapting E. coli to heat lacked whole-genome sequencing data. I obtained the strains (named BM28 and BM28 ∆lysU) and determined and analyzed their genome sequences. Lastly, our research group optimized the inexpensive production of SARS-CoV-2 protein fragments in E. coli and identified and produced a particular spike protein fragment which is both immunodominant and relatively unchanged over the virus’ evolution. These findings improve our collective understanding of adaptation to heat in E. coli, clarify some misconceptions around heat adaptation from the BM28 study and provide genetic constructs and instructions for the inexpensive production of SARS-CoV-2 protein fragments in E. coli.
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    Antibiotic resistance, antibiotic tolerance and the stringent response
    (2023-08-22) Deventer, Ashley; Hobbs, Joanne K.; Boraston, Alisdair B.
    Bacterial infections are a major global cause of mortality, and antibiotics are critical to their treatment. However, effective antibiotic therapy is threatened by antibiotic resistance and antibiotic tolerance. Tolerance is distinct from resistance as tolerant bacteria are still susceptible to antibiotics, but the rate of killing is significantly reduced. While resistance is a well-understood antibiotic evasion strategy, tolerance is an underappreciated bacterial phenomenon that greatly impacts treatment outcomes. Unlike resistance (which is identified by routine testing in laboratories), tolerance is not tested for in clinical laboratories, in part because there is no simple test available. Consequently, the clinical prevalence and significance of tolerance is unknown. Recently, the stringent response (SR) has emerged as a clinically-relevant mechanism implicated in both resistance and tolerance. The SR is a universal bacterial stress response that acts as a master regulator of bacterial physiology and virulence. In most bacteria, activation of the SR is controlled by the protein Rel. In this study, the SR was used as a model system in Staphylococcus aureus to better understand the wider consequences of SR-activating mutations for antibiotic therapy, and the magnitude of the problem of tolerance. Here, I demonstrate that SR activation promotes conjugal transfer of multidrug resistance plasmids between strains of S. aureus via elevated plasmid copy number. SR activation increased the transmission of plasmids from diverse families, suggesting that the SR plays a significant role in the dissemination of resistance. SR-activated mutants were also used in the development and validation of a simple screen to detect genotypic tolerance. This screen uses ATP as a proxy for viability and eliminates the need for enumeration of colony-forming units. In a pilot study, I applied the screen to a library of 39 S. aureus isolates from cystic fibrosis lung infections and detected tolerance in 8% of isolates. This study demonstrates that, while Rel mutations may arise during infection and confer tolerance, they have a secondary, coincidental consequence of promoting the dissemination of multidrug resistance plasmids. Furthermore, it demonstrates that the SR is a useful and much-needed model system for studying antibiotic evasion strategies in general. Ultimately, this study highlights the multifaceted implications of clinical Rel mutations and demonstrates that SR activation has significant consequences for antibiotic therapy.
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    Understanding the molecular basis of regulation of class I PI3Ks by activation signals, oncogenic mutations, and post-translational modifications
    (2022-12-22) Ranga Prasad, Harish; Burke, John
    Class I Phosphoinostide-3 Kinases (PI3K) generate Phosphatidylinositol-3, 4, 5 Trisphosphate (PIP3), which regulates important cellular tasks such as proliferation, cell growth, survival and metabolism through membrane recruitment and activation of downstream targets that bear PIP3 recognizing domains. Misregulation of class I PI3K signaling is found in several human diseases, such as cancer, immunological disorders, neurological disorders, diabetes, localized tissue overgrowth, and cardiovascular disease. Due to the essential roles of class I PI3Ks, their activity is held under tight control through molecular interactions with various activating partners and post- translational modifications (PTM). The aim of my thesis is to study the regulation of class I PI3Ks by activation signals, post-translational modifications (PTM), and oncogenic mutations. To this end, we have utilized a combination of cutting-edge biophysical and biochemical techniques like Hydrogen- Deuterium Exchange Mass Spectrometry (HDX-MS), lipid kinase assays and protein- lipid Fluorescence Resonance Energy Transfer (FRET) assays. Our results provide insights into novel aspects of regulation of PI3Kα by oncogenic mutations and the role of PTMs in modulating the activity of PI3Kγ. This work provides an excellent framework for understanding how PI3Ks are involved in human diseases.
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    The influence of thyroid hormone and temperature on the transcriptomic response of Rana [Lithobates] catesbeiana tadpole cultured back skin
    (2022-09-02) Evans, Ellis; Helbing, Caren C.
    Thyroid hormones (THs) are essential signaling molecules for the postembryonic development of all vertebrates. THs are capable of initiating a diverse set of developmental programs across multiple tissues. The role of TH in regulating gene expression is well-known, but the initiation of TH signaling is still not fully understood. In amphibians, THs are the sole hormones required for the metamorphosis from tadpole to juvenile froglet. Amphibians are a useful model for studying TH signaling, as they undergo extensive, tissue-specific response programs in response to exogenous TH. The metamorphosis of the American bullfrog, Rana [Lithobates] catesbeiana is temperature sensitive. R. catesbeiana tadpoles do not undergo metamorphosis at cold temperatures (4-5 °C) even in the presence of THs that should otherwise prompt it. However, tadpoles undergo metamorphosis at an accelerated rate when returned to warm temperatures (24-25 °C) forty days after their initial TH exposure. R. catesbeiana tadpoles possess a “molecular memory” of TH exposure which establishes the TH signal at cold temperatures and prompts accelerated metamorphosis after a return to warmer temperatures. The mechanisms of the molecular memory which allow it to uncouple the initiation of TH signaling from the execution of the TH response program are not fully understood. Previous research has established that transcripts encoding transcription factors are a substantial component of the TH-dependent transcriptomic response of cultured tailfin (C-Fin) at cold temperatures. However, not all of these putative transcripts encoding transcription factors required active transcription and translation for their induction, which suggests that the initiation of a TH signal involves mechanisms other than regulating gene expression. Herein, we used quantitative polymerase chain reaction (qPCR) and RNA-Sequencing (RNA-Seq) to investigate the TH-dependent transcriptomic response of the back skin, a tissue that undergoes extensive remodeling during metamorphosis. Cultured back skin (C-Skin) was TH-responsive in warm, cold and temperature shift conditions. Forty-four transcripts underwent significant changes in abundance in response to TH in cold temperatures under which the molecular memory is established. Seven of these transcripts encoded putative transcription factors. Surprisingly, the only TH-responsive transcript significantly changed at 4 °C in both the C-Skin and the previously studied C-Fin was thyroid hormone-induced basic leucine zipper-containing protein (thibz). Thibz has been found to be TH-responsive at cold temperatures in the liver, lung, liver, brain, tailfin and back skin of whole animals, which suggests it may be an important regulator of initiating TH signaling. The lack of overlap in the transcriptomic responses of C-Skin and C-Fin may suggest that even the early initiation of TH signaling has tissue-specificity. Alternately, the molecular memory may include mechanisms that do not require active transcription and translation. Transcripts associated with epigenetic modifications and post-transcriptional changes to mRNA stability were also significantly expressed at 4 °C within the C-Skin. Previous investigation of the putative transcription factors in C-Fin revealed that active transcription and translation was not always required for changes in transcript abundance. Multiple mechanisms may be at play in the TH response at different temperatures. In cold temperatures, TH may modulate mRNA stability to influence transcript abundance as a part of initiating TH signaling without executing metamorphosis. Further research is needed to explore potential alternative mechanisms of establishing the molecular memory and the accelerated metamorphic response. The temperature sensitivity of R. catesbeiana’s TH response is incredibly valuable in investigating mechanisms of early TH signaling during postembryonic vertebrate development.
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    The Development and Application of Mass Spectrometry-based Structural Proteomic Approaches to Study Protein Structure and Interactions
    (2022-08-26) Makepeace, Karl A.T.; Borchers, Christoph H.; Howard, Perry L.
    Proteins and their intricate network of interactions are fundamental to many molecular processes that govern life. Mass spectrometry-based structural proteomics represents a powerful set of techniques for characterizing protein structures and interactions. The last decade has witnessed a large-scale adoption in the application of these techniques toward solving a variety of biological questions. Addressing these questions has often been coincident with the further development of these techniques. Insight into the structures of individual proteins and their interactions with other proteins in a proteome-wide context has been made possible by recent developments in the relatively new field of chemical crosslinking combined with mass spectrometry. In these experiments crosslinking reagents are used to capture protein-protein interactions by forming covalent linkages between proximal amino acid residues. The crosslinked proteins are then enzymatically digested into peptides, and the covalently-coupled crosslinked peptides are identified by mass spectrometry. These identified crosslinked peptides thus provide evidence of interacting regions within or between proteins. In this dissertation the development of tools and methods that facilitate this powerful technique are described. The primary arc of this work follows the development and application of mass spectrometry-based approaches for the identification of protein crosslinks ranging from those which exist endogenously to those which are introduced synthetically. Firstly, the development of a novel strategy for comprehensive determination of naturally occurring protein crosslinks in the form of disulfide bonds is described. Secondly, the application of crosslinking reagents to create synthetic crosslinks in proteins coupled with molecular dynamics simulations is explored in order to structurally characterize the intrinsically disordered tau protein. Thirdly, improvements to a crosslinking-mass spectrometry method for defining a protein-protein interactome in a complex sample is developed. Altogether, these described approaches represent a toolset to allow researchers to access information about protein structure and interactions.
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    Characterization of ReNCell for studying chromatin associated proteins MeCP2 and histone H1
    (2022-08-05) Kim, Bo Hyun "Cindy"; Ausió, Juan
    Methyl-CpG binding protein 2 (MeCP2) and histone H1 are important chromatin associated proteins. Both exhibit their own extent of complexity as MeCP2 is an intrinsically disordered protein (IDP) that interacts with many different partners involved in several cellular processes and histone H1 consists of 11 different subtypes each of them associated with different posttranslational modifications (PTMs). An interesting avenue for the study of these proteins is in neurons where MeCP2 is very abundant and histone H1 level is half that observed in other somatic tissues. Several reports in the past have proposed that this lower level of histone H1 is due to the abundance of MeCP2 which displaces histone H1. However, this hypothesis has been debated and there is no clear consensus. In an attempt to study this controversy, a cell model system ReNCell WT and MeCP2-KO was used that can be induced to differentiate into neurons. The protein levels, transcript levels and localization of histone H1 subtypes in these cells were analyzed using HPLC, RT-qPCR and immunofluorescence, respectively. The results show that ReNCell WT and MeCP2-KO do not exhibit significant differences in their relative amount of histone H1 protein and transcript level neither at the proliferative nor at the later differentiated stages. However, HPLC analyses show that the histone H1 subtypes of these two cell types exhibit significant elution differences probably resulting from differences in their PTM content. Immunofluorescence analyses show that WT ReNCell differentiation as determined by extension of dendritic or axonic processes can be seen to occur over the course of one week and there is a significant difference in the nuclear area of these two cells at 8 DIV. This study provides important preliminary data for future research in MeCP2 and histone H1 using this cell model system and show that MeCP2 may have a bearing on histone H1 PTMs.
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    Cytoplasmic switch of ARS2 isoforms promotes nonsense-mediated mRNA decay and arsenic sensitivity
    (2022-04-27) Perez, M.M.; Howard, Perry L.
    The life of RNA polymerase II (RNAPII) transcripts is shaped by the dynamic formation of mutually exclusive ribonucleoprotein complexes (RNPs) that direct transcript biogenesis and turnover. A key regulator of RNA metabolism in the nucleus is the scaffold protein ARS2 (arsenic resistance protein 2), that binds to the cap binding complex (CBC) and regulates processing, degradation, and export of RNAPII transcripts. We report here that alternative splicing of ARS2’s intron 5, generates cytoplasmic isoforms that lack 270 amino acids from the N-terminal of the protein and are functionally distinct from nuclear ARS2. ARS2 isoforms distinctive roles are evidenced under physiological conditions and stress. Under physiological conditions, ARS2 isoforms differentially regulate transcript degradation through nonsense mediated decay (NMD). Switching of ARS2 isoforms within the CBC in the cytoplasm has dramatic functional consequences, changing ARS2 from a NMD inhibitor to a NMD promoter that enhances the binding of UPF1 to CBP80 and ERF1, favouring SURF complex formation, SMG7 recruitment and transcript degradation. ARS2 isoform exchange is also relevant during arsenic stress. Cytoplasmic ARS2 is specifically induced during arsenic exposure. It is crucial for arsenic sensitivity, and promotes a global response to arsenic in a CBC independent manner. We propose that ARS2 isoform switching promotes the proper recruitment of RNP complexes during NMD and the cellular response to arsenic stress. The existence of non-redundant ARS2 isoforms is relevant for cell homeostasis, stress response and cancer treatment.
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    Immune regulatory metabolites in the tumor microenvironment suppress T cell function in ovarian cancer
    (2022-04-26) Kilgour, Marisa; Lum, Julian J.
    Immune regulatory metabolites are key features of the tumor microenvironment (TME), yet with a few exceptions, their identities remain largely unknown. Here, we profiled tumor and T cells from tumor and ascites of patients with high-grade serous carcinoma (HGSC) to uncover the metabolomes of these distinct TME compartments. Cells within the ascites and tumor had pervasive metabolite differences, with a striking enrichment in 1-methylnicotinamide (MNA) in T cells infiltrating the tumor compared to ascites. Despite the elevated levels of MNA in T cells, the expression of nicotinamide N-methyltransferase, the enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine to nicotinamide, was restricted to fibroblasts and tumor cells. Functionally, MNA induces T cells to secrete the tumor promoting cytokine tumor necrosis factor alpha. Thus, TME-derived MNA contributes to the immune modulation of T cells and represents a potential immunotherapy target to treat human cancer.
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    Advancing understanding of secondary cell wall polymer binding and synthesis in S-layers of Gram-Positive bacteria
    (2022-04-21) Legg, Max; Evans, S. V.
    Self-assembling protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and many additional processes, which makes them appealing targets for therapeutics and biotechnological applications, including live vaccines, liposome drug delivery and biosensors. Unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of specialized S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Characterization of this interaction in the Gram-positive model organism Paenibacillus alvei CCM 2051T reveals that, remarkably, binding-site switches can occur between two distinct SLH-domain SCWP receptor-site grooves in the S-layer protein SpaA, possibly as part of a mechanism to alleviate strain in the S-layer. To date, however, analysis of this novel mechanism has been limited to the terminal SCWP monosaccharide and the internal SCWP repeat disaccharide ligand analogues, leaving open the role of subsequent SCWP sugar residues in binding, as well as whether the two receptor sites are also suited to accommodate longer SCWP ligands that better approximate the biological target at the surface of P. alvei. To address this, the objective of this work aims to uncover and characterize the details of the SpaA SLH-domain (SpaASLH¬) SCWP-interaction by determining the co-crystal structures of SpaASLH¬, and single (SpaASLH/G109A) and the corresponding double (SpaASLH/G46A/G109A) mutants in complex with synthetic terminal disaccharide and trisaccharide analogues of the P. alvei CCM 2051T SCWP target. These structural characterizations have been supplemented with disaccharide and trisaccharide binding data, which was obtained through thermodynamic ITC analyses carried out by collaborators. The co-crystal structures of P. alvei SpaASLH with synthetic, terminal SCWP disaccharide and trisaccharide analogues, together with previously published monosaccharide-bound SpaASLH structures, reveal that while the SLH trimer accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly-exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations for longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division. In addition, to further clarify the steps involved in SCWP biosynthesis, we present a crystal structure of the unliganded UDP-GlcNAc 2-epimerase enzyme MnaA, which catalyzes the interconversion of UDP-GlcNAc into UDP-ManNAc—an essential building block of the P. alvei SCWP target. The P. alvei MnaA epimerase adopts a GT-B fold that is consistent with the architecture of previously published structures of other bacterial non-hydrolyzing UDP-GlcNAc 2-epimerase enzymes for which substrate binding is observed in the cleft located between the two domains. Characterization of this structure, coupled with an analysis of the sequence of the MnaA protein, reveals the presence of conserved residues that define the catalytic and allosteric sites in homologous enzymes from different organisms. These residues are positioned to accommodate substrate within the MnaA binding cleft in much the same manner as the published enzyme homologues, suggesting that allosteric regulation as a mechanism for enzyme regulation is conserved in P. alvei MnaA. These investigations are part of a greater effort toward understanding SLH domain-mediated SCWP-interactions in Gram-positive organisms, and provide insight into the structure and putative function of this SCWP biosynthetic enzyme. By understanding these processes, this knowledge may contribute to providing a platform for the rational design of Gram-positive inhibitors. Such inhibitors could selectively target, for example, the bacterial S-layer SCWP-binding interaction, or perhaps the essential biosynthetic enzymes involved in producing the exclusive targets that these S-layer proteins recognize and bind, and would thus represent a new class of antimicrobial therapeutics.
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    Deciphering the immunosuppressive landscape of high-grade serous ovarian cancer
    (2022-01-19) Smazynski, Julian; Nelson, Brad H.; Webb, John R.
    High-grade serous ovarian cancer (HGSC) remains the most common and lethal subtype of ovarian cancer with a 5-year survival rate of ~30%, highlighting an urgent need for new treatments. Cancer immunotherapy has emerged as an efficacious strategy aimed at harnessing the exquisite capabilities of our immune system to destroy malignant cells. However, the development of more effective immunotherapies is hampered by our limited understanding of the phenotype of bona fide tumor-reactive T cells versus irrelevant bystanders. Further, T cells that exhibit tumor specificity appear to encompass a tissue resident memory (TRM) phenotype but combat a harsh immunosuppressive tumor microenvironment, often leading to an exhausted phenotypic state and evasion of immune-mediated destruction. These insights have led to rapid clinical implementation of so-called “checkpoint blockade” therapies that re-invigorate T cell-mediated tumor destruction by blocking surface inhibitory receptors or ligands. Thus, by identifying the phenotype of prognostically favourable TRM T cells and the immunosuppressive networks they face, my thesis work tackles a critical challenge in designing the next generation of therapeutic interventions for this disease. To address this challenge, I hypothesized that (1) the TRM phenotype could be modulated for improving adoptive T cell therapy; (2) TRM TIL characterized by the co-expression of CD103, PD-1, and CD39 in HGSC provide improved prognostic benefit indicative of enriched tumor reactivity; (3) the TIGIT/CD155 signalling axis plays a crucial role in shaping the immunosuppressive landscape impeding TRM T cells in HGSC. Firstly, I developed methods for modulating the TRM phenotype on expanded human and murine T cells for adoptive cell therapy and assessed the therapeutic impact of these phenotypes. Secondly, we applied high-dimensional flow cytometry, single-cell sequencing, and multiplexed immunofluorescence to primary human HGSC specimens to explore the single-cell phenotypic profiles and prognostic significance of tumor-infiltrating T cells co-expressing three putative markers of tumor reactivity: CD39, CD103, and PD-1. These ‘triple-positive’ T cells exhibited a highly activated/exhausted phenotype and superior prognostic value relative to all other T-cell subsets, suggesting these markers enrich for tumor-reactive clones. Furthermore, these triple-positive cells exhibited heightened expression of the inhibitory checkpoint TIGIT, which plays a prominent role in tumor-mediated immune suppression. Finally, to explore the therapeutic implications of this finding, we investigated the relationship between the TIGIT signaling axis on TIL and prognosis in HGSC. Once again utilizing high-dimensional flow cytometry, multi-color histological imaging, and gene expression profiling we found T cells from HGSC frequently express TIGIT ex vivo and post-clinical expansion. Further, CD155, the dominant ligand for TIGIT, was largely expressed on malignant epithelium in HGSC and showed a negative association with immune infiltration. Thus, TRM T cells represents a compelling immunotherapeutic immune subset in HGSC and one that could be bolstered by immune checkpoint inhibition of the TIGIT/CD155 axis.
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    The effects of a helminth-altered gut metabolome and deworming on host immunity
    (2021-12-22) Brosschot, Tara Pauline; Reynolds, Lisa A.
    Helminths are parasitic worms that can establish long-lived infections by modulating host immune responses. Helminth infection has been associated with a reduced prevalence of allergic disease in human populations, and impaired immunity to co-infecting pathogens. Several human and mouse studies suggest that helminths may impair host responses to concurrent bacterial infection. In this thesis, we study the extent to which helminth infection affects Salmonella colonization and how anthelmintic treatment (deworming) impacts immunity to Salmonella in a mouse model of co-infection. We find that helminth co-infection allows Salmonella to establish in the lumen of the small intestine. Further, we find that deworming prior to bacterial infection restores impaired immunity to Salmonella in the small intestine, however, deworming after Salmonella has established during helminth co-infection does not revert elevated bacterial burdens. To ensure their longevity in the host, helminths release immunomodulatory molecules, and modulate immunity through changes in the gut microbiota. The microbiota is known to influence mucosal immunity through the production of metabolites, but metabolites have not received much attention in the context of helminth modification of immune responses. This thesis uncovers the impact of helminth infection on levels of short-chain fatty acids (SCFAs) and bile acids, two groups of metabolites with immunomodulatory potential. We found that helminth infection increases small intestinal levels of the branched-chain SCFA isovalerate and lowers the small intestinal bile acid concentration. We next explored the consequences of these metabolite shifts on susceptibility to bacterial infection, helminth fitness and regulatory T cells. Collectively, these results contribute to the understanding of host-pathogen interactions in a co-infection scenario, which ultimately, will help to inform strategies for disease control in helminth-endemic areas. Further, our data contributes to the characterization of the helminth-modified intestinal metabolome, which future work can build on to reveal novel immunomodulatory pathways that can be targeted to relieve symptoms in inflammatory diseases such as allergic asthma.
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    Understanding the molecular basis of Rab-GEF activity and specificity of the mammalian TRAPP complexes
    (2021-09-23) Harris, Noah James; Burke, John Edward
    Rab GTPases are among the most important families of proteins involved in regulating the trafficking and delivery of cellular cargo to their proper locations. The multi-subunit Transport Protein Particle complexes, TRAPPII and TRAPPIII, activate Rabs by catalyzing GDP/GTP nucleotide exchange. These two distinct complexes share seven subunits, yet they differ in their complex-specific subunits. Even though the TRAPP complexes are similar in subunit composition, they can activate different Rabs and therefore regulate distinct trafficking pathways. Intriguingly, the mechanism underlying the specificity of TRAPPII/III for different Rabs is poorly understood. This thesis is centered around understanding the molecular basis of TRAPP-Rab specificity in the mammalian TRAPPII/III complexes. To address this, we used a combination of different powerful techniques including biochemical nucleotide exchange assays, Hydrogen Deuterium eXchange Mass Spectrometry (HDX-MS), and electron microscopy to understand this specificity both in solution and on membranes. Biochemical assays against 20 different Rab GTPases revealed that TRAPPIII only has activity on Rab1 and Rab43. HDX-MS experiments comparing the TRAPPII and TRAPPIII complexes showed that there are extensive differences existing near the Rab binding site, thus highlighting the critical role that the complex specific subunits serve in modifying either complex’s Rab binding interface. TRAPPII and TRAPPIII showed increased activity in the presence of lipid membranes and HDX-MS revealed large dynamic changes occurring in both TRAPP complexes in the presence of membranes. Altogether, the work summarized in this thesis provides novel insight into the unique functions of the two TRAPP complexes and it reveals how the complex specific subunits can rearrange the Rab binding site which leads to a possible mechanism for Rab specificity.
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    The chilling tail of temperature’s influence on thyroid hormone signalling in the post-embryonic developmental response of Rana catesbeiana cultured tail fin
    (2021-09-14) Koide, Emily; Helbing, Caren C.
    Thyroid hormone (TH) is a critical signalling molecule for all vertebrate organisms, playing an especially crucial role in postembryonic development. Given its importance, many studies have focused on further elucidating the initial TH signal response and its method of transduction. Although the primary mechanism of TH response is genomic signalling, alternative mechanisms of early TH signal transduction have been relatively poorly studied. The North American bullfrog, Rana catesbeiana, is a useful model to study these early responses as tadpole post-embryonic development, or metamorphosis, can be experimentally induced through exposure to TH. The experimental induction of the TH signalling program leads to similar morphological endpoints as seen in natural metamorphosis in the transition of a tadpole to a juvenile froglet, such as regression of the tail. This TH-induced developmental program can also be manipulated through temperature where, as temperatures lower, developmental rate is delayed and at 5°C metamorphosis is completely stalled. Interestingly, when tadpoles exposed to TH at 5°C are introduced to permissive temperatures (24°C), an accelerated developmental program ensues, even when no more endogenous TH signal remains. Previous research has shown that this phenomenon can also be seen on the molecular level where only a select few transcripts have been shown to be responsive to TH at 5°C. However, the characteristic, if not augmented, TH response program is seen on the transcriptomic level when tadpoles are shifted to 24°C. This indicates that there is a molecular memory where the TH signal is induced in cold temperatures but not executed until more permissive temperatures arise. The extent and regulation of the transcriptomic program involved in this TH-induced molecular memory has yet to be understood. Herein we use the broader probing technique of RNA-seq analysis to identify potential components of the molecular memory. Eighty-one gene transcripts were TH-responsive at 5°C in cultured R. catesbeiana tail fin indicating that the molecular memory is more complex than previously thought. A number of these transcripts encoded regulators of transcription. Closer examination of select transcripts including a novel krüppel-like factor family member, klfX, at 5oC indicated that not all of the candidate molecular memory transcripts are regulated through active transcription and active translation is not required. When moved into 24°C an accelerated transcriptomic response occurred even when no additional TH is added, suggesting that a priming event occurs by TH exposure at 5°C allowing an accelerated metamorphosis at permissive temperatures. The molecular memory may be used as a means to isolate the initiating TH signalling response and the regulation of this program to allow further elucidation of early TH signalling in post-embryonic development.
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    Development of automated iMALDI assays for the robust quantitation of cell signalling proteins in the PI3K pathway to improve guided cancer treatment
    (2021-08-30) Frohlich, Bjorn Christian; Helbing, Caren C.; Borchers, Christoph H.
    The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody cross-reactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. IMALDI, which combines immuno-enrichment with analysis using a benchtop MALDI-Time-of-Flight (TOF) mass spectrometer, is an especially well-suited method for translating mass-spectrometry based assays into the clinical lab. We systematically optimized the iMALDI workflow regarding sensitivity, robustness, and throughput while developing highly flexible automation protocols using a Bravo 96LT liquid handling robot. We further developed custom R scripts to improve data visualization and analysis. One hour digestion using a protein to trypsin ratio of 1:2, followed by direct immuno-enrichment for 1 h yielded high and consistent peptide recoveries. We demonstrated that the PTEN and p110α iMALDI assays can be multiplexed using both simultaneous and sequential enrichment, reducing the amount of required sample material as well as simplifying the workflow. The PTEN+p110α iMALDI assay was validated and demonstrated high accuracy for both target proteins (90-112% recovery of known spiked-in concentrations) as well as high precision and 5-day reproducibility (overall CVs of 9%) across the linear range of the assay (0.6 to 20 fmol). Lower limits of quantitation below 1 fmol were achieved. Endogenous PTEN and p110α were quantified in cell lines as well as fresh-frozen tumor tissue samples. A novel two-point internal calibration strategy (2-PIC) was developed, based on spiking two peptide isotopologues into the sample as internal standards, avoiding the need for an external calibration. We quantified endogenous PTEN in a Colo-205 cell line using the PTEN iMALDI assay, as well an orthogonal PTEN immuno-multiple reaction monitoring (immuno-MRM) method to demonstrate this technique. Excellent agreement was shown between both calibration approaches (residual standard deviation between 2-PIC and external calibration of 1.6-5.8%), as well as high correlation between PTEN iMALDI and PTEN immuno-MRM (R²= 0.9966) and good agreement between quantified amounts (0.48±0.01 and 0.29±0.02 fmol/µg of total protein). Finally, we analysed a set of patient samples from a AKT inhibitor AZD5363 drug trial using a multi-site workflow combining the developed PTEN+p110α assay with established AKT1+AKT2 iMALDI assays and untargeted proteomics. We demonstrated how the combination of targeted and untargeted proteomics approaches may be used to gain novel insights into the tumor biology of patient tissue samples. Further, we showed that the PTEN iMALDI assay has good correlation with a comparable immunohistochemistry method (R²=0.86), and that our assays can be further multiplexed, reducing the required amount sample material. Thus, we showed that iMALDI is promising tool for biomarker quantitation.
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    Dissecting the role of bifunctional histone chaperone Fpr4 in transcription and recombination
    (2021-08-25) Kirlikaya, Baran; Nelson, Chris
    Eukaryotic DNA is wrapped around nucleosomes and assembled into chromatin, which is the substrate for DNA-centric processes including transcription and recombination. Histone chaperones are central players in these processes as they mediate nucleosome dynamics and modulate chromatin structure. Fpr4 is a yeast histone chaperone with separate nucleoplasmin- like and FK506-binding protein (FKBP) proline isomerase domains. In vitro, these domains facilitate nucleosome assembly and chromatin remodelling activities, respectively. However, the importance of each domain in chromatin regulation in vivo remains unclear. Here I demonstrate that yeast cells lacking Fpr4 have an abnormally high number of ribosomal DNA (rDNA) copies in the nucleolus, which is a hallmark of hyper-recombination. Deletion mutant analysis shows that rDNA copy number maintenance is mediated by the C-terminal FKBP domain. Fpr4 also takes part in silencing the expression of a set of nuclear genes. By contrast, I show that Fpr4 regulation of transcription is dependent on the nucleoplasmin-like domain at some genes, and on the FKBP domain at others. These results demonstrate that Fpr4 is likely to regulate different chromatin loci through distinct mechanisms. The results presented here also identify a new participant in chromatin regulation in the nucleolus and pave the way for future studies to help us better understand rDNA copy number fluctuations, transcriptional regulation and nucleolar organisation in the eukaryotic cell.