Browsing by Author "Schriemer, David"

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    Advances in Hydrogen Exchange Mass Spectrometry to Study Microtubules and MAPs
    (2015-05-27) Burns, Kyle; Schriemer, David
    Microtubules are a fundamental component to the cellular cytoskeleton and are responsible for cell structure, motility, intracellular transport, and formation of the mitotic spindle. The ability to serve the diverse functions requires constant flux between polymerization and depolymerization. The regulation of microtubule dynamics is accomplished by microtubule associated proteins that can interact with microtubules, or its tubulin dimer, to induce polymerization or depolymerization. The depolymerization mechanism induced by mitotic centromere-associated kinesin (MCAK) was studied in detail. Studying a large protein system such as the regulation of microtubule dynamics at the molecular level requires an integrative structural biology approach. Hydrogen exchange mass spectrometry (HX-MS) is a vital technique for studying the protein dynamics and was optimized for the analysis of large protein complexes. Two HX-MS platforms consisting of a FT-MS and a high-resolution QTOF mass spectrometer were evaluated by comparing the figures-of-merit for a typical bottom-up HX-MS experiment: peptide identification, deuterium measurement accuracy, and deuterium measurement precision. The Orbitrap Velos identified 64% more peptides than the TripleTOF 5600, independent of protein size. Precision in deuterium measurements using the Orbitrap marginally exceeded that of the TripleTOF, depending on the Orbitrap resolution setting; however, the unique nature of FT-MS data generates situations where deuteration measurements can be inaccurate. The findings presented support the use of the TripleTOF 5600 for further development of hydrogen exchange methods. A data-independent acquisition approach was developed that combines peptide fragmentation data and a new peptide scoring algorithm (WUF, Weighted Unique Fragment) to provide MS/MS data for HX measurements while reducing manual validation. The scoring incorporates elements of the validation process and preserves high peptide identification accuracy. When compared to a conventional Mascot-driven HX-MS method, HX-MS2 produces two-fold higher tubulin sequence depth at a peptide utilization rate of 74%. The HX-MS2 method was applied to study the microtubule depolymerization process induced by MCAK. In the described model, the N terminus is responsible for the lateral separation in conjunction with the outward curvature induced by the motor domain. The C terminus is responsible for regulating the microtubule interactions.
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    Age-induced errors in DNA replication and repair in Saccharomyces cerevisiae
    (2023-01-17) Mair, Nicola; Cobb, Jennifer; Goodarzi, Aaron; Williams, Gareth; Schriemer, David
    Aging is a multifactor process that leads to a widespread decrease in cellular function. The single most significant risk factor for developing cancer is age. Cancer is a disease of genome instability, and much of this genomic damage accumulates during DNA replication and inefficient repair. Although DNA replication and repair mechanisms have been extensively studied, little is known about whether these processes are altered with age. Accordingly, my thesis aims to define age-related differences in DNA replication and repair in the model organism budding yeast (Saccharomyces cerevisiae). By employing budding yeast as a model system, I have developed a protocol to isolate large quantities of precisely aged cells, thus allowing molecular comparisons between young and old cells. The results indicate that during cellular aging, there is a reduction in the efficiency of replication initiation. There is a reduction in the recruitment of polymerases and MCM helicases to the origins of replication. This coincides with a decrease in origin firing and DNA synthesis. I have also shown that double-strand break (DSB) repair becomes highly mutagenic as yeast cells age. The efficiency of homologous recombination and non-homologous end joining decreases, and the incidence of repair through alternative end-joining increases. Aged cells have an overall lower level of repair, and the recruitment of repair proteins to DSBs is impaired. Altogether, my work has identified mechanisms of DNA replication and repair that become increasingly dysregulated with age.
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    An Analysis of Alpha Particle Radiation Exposure from the Population Level to the Biological Consequences
    (2021-05-11) Pearson, Dustin Daniel; Goodarzi, Aaron; Lees-Miller, Susan; Schriemer, David; Peters, Cheryl; Masson, Jean-Yves
    Lung cancer accounts for 38% of North American cancer related deaths and is the 7th leading cause of cancer related deaths for never-smokers. Radon (222Rn) gas inhalation is the 2nd leading cause of lung cancer in Canada and is the primary source of ionizing radiation (IR) exposure for most humans globally. Radioactive radon is an odorless, tasteless and colourless gas generated by the decay series of soil radionuclides including radium, thorium and uranium. Decaying 222Rn emits alpha particles, a type of high Linear Energy Transfer (LET) IR that, when contacting biological material, will induce complex and clustered DNA damage that is extremely difficult for human cells to repair accurately or quickly, increasing the risk of cancer-causing genetic mutations. The ability to study alpha particles has been limited and therefore it is still unclear as to how a cell repairs high LET-induced DNA damage. Radon is often captured, contained, and concentrated to hazardous levels within our built environment and, importantly, exposure to radon is increasing in many regions. Indeed, 21st-century North American residential properties contain historic radon highs, although the reasons for this are unclear. To address this, I analyzed 250,000+ real-time radon data points and 20,000+ long-term radon readings linked to residential property metrics. I conclude that alpha particle exposure from radon is occurring at doses and dose rates of serious risk to humans, and which correspond to verified cancer-causing radon exposures in mammals. Further to this, Canadians most at risk for high radon exposure are i) younger, ii) more likely to live in newer residential properties and iii) more likely to be starting parenthood. I find that Canadian properties most associated with high radon were single detached, large (>1750 sq. ft.), newer properties, with newer, more glazed windows and were home to people who do not open their windows regularly. Using multivariate analysis, I present new models useful to predict high and low radon in the Canadian residential environment and better understand particle IR exposure at population level. To understand the biological consequences of radon, I have helped to develop a new, high-throughput alpha particle irradiation system. Using this system, I identify functional interactions between the SNM1A and SNM1C/Artemis nucleases during repair of particle-induced complex DNA damage lesions. Using biology and biochemistry, I present evidence that supports the hypothesis that SNM1C functions to resolve DNA interstrand crosslink clustered together with DNA double strand breaks. My work is transdisciplinary: I have generated mechanistic insight in particle radiobiology, developed new technology that resolves long-standing logistical barriers, and I have built population-level data with implications to public health in terms of identifying at risk populations for radon exposure, and building practices relevant to engineering out radon exposure in the future.
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    Bayesian Approaches to Integrative Structural Biology Using Sparse and Ambiguous Data
    (2022-01) Gaalswyk, Kari; MacCallum, Justin; Fraser, Marie; Schriemer, David
    Protein structure determination plays an important role in understanding the biological activity of proteins and protein complexes. While traditional experimental approaches such as x-ray crystallography and nuclear magnetic resonance (NMR) have been extremely successful, they are not applicable to all systems. Computational modelling can explore the structure and dynamics of proteins. However, these methods often struggle to converge for large, complex systems. Instead, we can turn to integrative structural biology, an emerging field combining experimental and computational approaches thereby overcoming some of these limitations. One of the main challenges these methods face is how to account for noise and ambiguity in experimental data, which can make it difficult to interpret and correctly incorporate the data into a model. In this thesis, an integrative approach was applied to this problem by incorporating experimental data into physical modelling through Bayesian inference. We first applied this method to the small, well-defined protein GB1 using solid-state paramagnetic NMR restraints. We were able to determine the structure to < 1 Å RMSD in the limit of a sparse dataset. We then expanded our approach to the larger protein calmodulin in complex with a peptide. The introduction of a Bayesian technique to include additional, complementary paramagnetic NMR data enabled the identification of dominant conformations within 3 Å of a reference structure. Next, we addressed a long-standing limitation in NMR experimental methods and introduced a technique to quantitatively determine the accuracy of experimental data. This methodology allowed us to solve for the structure of the protein while simultaneously addressing the ambiguity in the experimental data. We were able to accurately determine the structures of five small proteins within ~2 Å RMSD of reference structures. Finally, we approached the challenge of modelling protein-protein complexes using inherently ambiguous cross-linking mass spectrometry (XL-MS) data. We were able to accurately dock transferrin with a transferrin binding protein and explore the interfacial region of the complex. The work in this thesis expands on existing Bayesian approaches to structure determination by enabling the inclusion of sparse and ambiguous experimental data, furthering the scope of integrative methods.
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    Biochemical and Structural Approaches Toward Determining the Role of the DEK Protein in Homologous Recombination Repair
    (2023-01-16) La, Justin; Williams, Gareth; Goodarzi, Aaron; Schriemer, David
    The DEK proto-oncogene is a nuclear protein important in regulating many cellular processes including DNA double-strand break repair and chromatin biology, through protein-protein and protein-nucleic acid interactions. Mutations or altered expression of DEK result in the development of various human diseases including acute myeloid leukemia and many other types of cancer. However, despite its importance in biology, there is a lack in knowledge on the mechanistic and structural basis for how DEK interactions regulate the numerous cellular processes it is involved in. Here, I developed purification protocols for full-length and subdomains of DEK, and developed biochemical assays to investigate DEK-DNA interactions and role of DEK in regulating homologous recombination repair. The DNA binding results showed that DEK can bind a wide variety of DNA substrates with a range of affinities between ⁓130-1300 nM. Phosphorylation of full-length DEK blocked DNA binding, consistent with and extending published work. The preliminary homologous recombination repair results showed that DEK directly binds to the RAD51 recombinase in vitro and may promote RAD51-mediated double-stranded DNA displacement during strand invasion. The results and protocols developed from this thesis provide a platform for future efforts to further characterize DEK’s role in HRR, to test the effects of disease-associated mutations, and to determine structures of DEK complexes with DNA, RAD51, and other interacting proteins including the nucleosome. This future work will address the gap in knowledge regarding the mechanistic basis for DEK’s role in homologous recombination repair and may provide new insights into its functions in human diseases.
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    Characterization of Staufenl ribonucleoproteins by mass spectrometry and biochemical analyses reveal the presence of diverse host proteins associated with human immunodeficiency virus type 1
    (Frontiers in Microbiology, 2012-10-25) Milev, Miroslav; Ravichandran, Mukunthan; Khan, Morgan; Schriemer, David; Mouland, Andrew
    The human immunodeficiency virus type 1 (HIV-1) unspliced, 9 kb genomic RNA (vRNA) is exported from the nucleus for the synthesis of viral structural proteins and enzymes (Gag and Gag/Pol) and is then transported to sites of virus assembly where it is packaged into progeny virions. vRNA co-exists in the cytoplasm in the context of the HIV-1 ribonucleoprotein (RNP) that is currently defined by the presence of Gag and several host proteins including the double-stranded RNA-binding protein, Staufen1. In this study we isolated Staufen1 RNP complexes derived from HIV-1-expressing cells using tandem affinity purification and have identified multiple host protein components by mass spectrometry. Four viral proteins, including Gag, Gag/Pol, Env and Nef as well as >200 host proteins were identified in these RNPs. Moreover, HIV-1 induces both qualitative and quantitative differences in host protein content in these RNPs. 22% of Staufen1-associated factors are virion-associated suggesting that the RNP could be a vehicle to achieve this. In addition, we provide evidence on how HIV-1 modulates the composition of cytoplasmic Staufen1 RNPs. Biochemical fractionation by density gradient analyses revealed new facets on the assembly of Staufen1 RNPs. The assembly of dense Staufen1 RNPs that contain Gag and several host proteins were found to be entirely RNA-dependent but their assembly appeared to be independent of Gag expression. Gag-containing complexes fractionated into a lighter and another, more dense pool. Lastly, Staufen1 depletion studies demonstrated that the previously characterized Staufen1 HIV-1-dependent RNPs are most likely aggregates of smaller RNPs that accumulate at juxtanuclear domains. The molecular characterization of Staufen1 HIV-1 RNPs will offer important information on virus-host cell interactions and on the elucidation of the function of these RNPs for the transport of Gag and the fate of the unspliced vRNA in HIV-1-producing cells.
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    Efficient Clearance of Inhaled Nanoparticles Depends on Strong Adhesion to the Epithelium: The Role of Hydrophilicity, Coating, Size, and Shape
    (2023-09-14) Bogari, Nawaf Nasir; Amrein, Matthias; Green, Francis; Yates, Robin; Shi, Yan; Schriemer, David; Hubbs, Ann
    Health alerts regarding high levels of fine particles in ambient air are increasingly common, reflecting a significant worldwide crisis. These particles contribute substantially to premature death, a problem only expected to grow. Among these, nanoparticles pose a particular threat, linked to respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and cancer as well as extrapulmonary effects. The health implications of nanoparticles differ, rendering simple particle concentration nearly meaningless. In this thesis, we focused on understanding the accumulation of nanoparticles in the lung, which is a critical aspect of their toxicity. We developed in-vitro assays to predict whether particles remain in the alveolar lumen to be cleared through alveolar and airway pathways, and which particles traverse the alveolar epithelium to induce interstitial lung disease or enter the blood and lymphatic vessels to cause systemic effects. We hypothesized that the fate of inhaled nanoparticles in the alveolar lung primarily depends on their adhesion strength to alveolar epithelium and the physical/chemical characteristics of the particle. To test this, we employed atomic force microscopy (AFM) for adhesion force measurement, transmission electron microscopy (TEM) to correlate adhesion strength to nanoparticle uptake, and confocal laser scanning microscopy (CLSM) to study nanoparticle translocation. Characteristics of nanoparticles, including protein corona coating, alveolar surfactant coating, hydrophobicity/hydrophilicity, size and geometry were studied in the presence or absence of pharmacological blockers to determine endocytic mechanism(s) involved in the interaction with the epithelium. Results showed that the adhesion of nanoparticles to the epithelial cells was an active process, and the strength of adhesion to the epithelium correlated directly to their uptake and transcytosis. Amorphous silica nanoparticles (ASN) with 15 nm in diameter were found to adhere strongly and translocate across the epithelium, whereas nanocarbon black particles 15 nm in diameter (nCB15) exhibited weak adhesion and remained in the alveolar lumen. Interestingly, commonly studied zeta potential had no influence on the interaction, whereas particle coating with surfactant increased their potential to accumulate in the alveolar lumen. Rendering ASN particles hydrophobic reduced their adhesion to the epithelium. The size of nanoparticles was linked to how cells perceived nanoparticles, with particles larger than 150 nm being endocytosed in a clathrin-enhanced mechanism, while particles less than 150 nm were taken up in a caveolin-enhanced mechanism. The epithelium did not show a preferred endocytic mechanism with respect to clathrin or caveolin for silica nanorods (SNR) yet showed a cytotoxic response to these elongated particles. This work contributes to the development of an effective framework for assessing the potential risks of inhaled nanoparticles, and our novel approach of categorization can support public health policies aiming to reduce exposure to nanoparticles in various environments.
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    Engineering Streptavidin with Switchable Ligand Binding Affinity Using Disulfide Bonds at the Biotin Entry Gateway
    (2020-07-22) Marangoni, Jesse M; Wong, Sui-Lam; Ng, Kenneth; Evans, Stephen; Schriemer, David; Ro, Dae-Kyun; Gedamu, Lashitew
    Streptavidin is widely used in biotechnological applications for its specific, high-affinity interaction with its natural ligand biotin, a small vitamin that can be easily conjugated to biomolecules of interest. However, its utility is often limited since many applications require reversibility in binding whereas others require higher affinity than that offered by wild-type streptavidin. For some applications, it would even be beneficial to allow extremely tight binding and subsequent ligand release within a single protocol. To combine both extremely tight and reversible binding into a single protein, two streptavidin muteins, designated M88 and M112, were engineered to each contain a distinct disulfide on opposite sides of a flexible loop critical for ligand binding. Each disulfide bond has markedly different effects on protein structure and binding kinetics. While the disulfide in M112 caused a detrimental conformational change which decreased biotin binding affinity, oxidized M88 showed a ~250-fold decrease in off-rate constant at 21°C and increased thermostability when compared to wild-type streptavidin. Furthermore, reduction of the disulfide bond increased the off-rate constant ~19,000-fold compared to the oxidized form, reducing the half-life for dissociation from 50 years to 1 day. Increasing the temperature to 50°C allows ligand release from the reduced form with a half-life of 9 minutes. M88 thus displays redox and temperature dependent ligand binding, both of which can be used to switch between high- and low-affinity states. M88 coupled to a matrix can be used to capture and release biotinylated biomolecules. For applications where increased temperature is not viable, further engineering of M88 has been used to reduce ligand binding affinity. The relative ease of controlling protein disulfide bond redox state with mild chemical agents allows switchable affinity of M88 towards biotin.
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    Functional Genomics and Metabolite Profiling as Tools for Alkaloid Biosynthetic Gene Discovery
    (2015-12-16) Dinsmore, Donald Reed; Facchini, Peter James; Moorhead, Gregory; Schriemer, David; Derksen, Darren
    The benzylisoquinoline alkaloids (BIAs) are diverse group of plant specialized metabolites found in the families Papaveracea, Ranunculaceae, Berberidaceae and Menispermaceae. Plants remain the only commercial source for BIAs and their biosynthesis is poorly understood. O-methyltransferases (OMTs) are wide spread in BIA biosynthesis. Putative OMTs were found in stem and root Next-Generation Sequencing transcriptomic databases. Putative OMT cDNAs were isolated from Papaver somniferum and commercially synthesized. Recombinant protoberberine 2-O-methyltransferase (2OMT) was heterologously expressed in Escherichia coli and assayed. 2OMT demonstrated the 2-O-methylation of protoberberine alkaloids and the 7-O-methylation of simple BIAs. The substrate range and tissue specific expression of 2OMT suggest its in vivo role is converting (S)-cheilanthifoline to (S)-sinactine. A LC-MS based targeted alkaloid profiling of twenty BIA producing species from the families Papaveracea, Ranunculaceae, Berberidaceae and Menispermaceae was conducted.
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    Next-Generation Sequencing of vlsE Recombinational Switching in the Lyme Spirochete
    (2017) Verhey, Theodore; Chaconas, George; Schriemer, David; Samuels, Scott; Zimmerly, Steven; DeVinney, Rebekah; de Koning, Jason; McGhee, James
    Borrelia burgdorferi and other spirochetes that cause Lyme disease effectively evade the acquired immune response through antigenic variation. The VlsE antigen is expressed on the spirochete surface during mammalian infection. Virtually unlimited numbers of variants are generated through segmental gene conversion events at the vlsE gene from a series of nearby silent cassette sequences that are homologous to the variable region of vlsE. In contrast to other antigenic variation systems, the molecular mechanism for this switching is unknown. Switching at vlsE is dependent on mammalian host factors and the only known requirement is the RuvAB branch migrase; RecA and many of the other homologous recombination proteins in B. burgdorferi are not required. In this study, we developed a new assay for switching at vlsE based on PacBio sequencing and an analytical pipeline that allows the analysis of tens of thousands of full-length variants. We developed the first fully-automated and unbiased method to accurately identify switch events and non-templated mutations from sequence data. This software also contains a large suite of dataset management and analysis tools to quantify many aspects of switch events and the switching process in the dataset. Following a time-course of B. burgdorferi infection in immunocompetent and immunocompromised mice in a variety of tissues, we uncover a series of new insights into recombinational switching at vlsE. We demonstrate that although switching requires mammalian host factors, the rate of vlsE switching is unaffected by the presence or absence of the required immune system. We identify residues that undergo diversifying and stabilizing selection in the VlsE protein in the presence of acquired immunity. We also report accurate rates of recombination and nontemplated mutation, the size and origin of switch events, a role for local sequence homology in promoting switching, and that switch events accumulate in a clustered rather than uniform fashion. We also quantify a secondary mechanism of sequence variation by demonstrating that polymerase slippage generates in-frame, surface-localized insertions and deletions that contribute to VlsE variability.
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    Plant Defense Responses in Opium Poppy Cell Cultures Revealed by Liquid Chromatography-Tandem Mass Spectrometry Proteomics
    (Molecular & Cellular Proteomics, 2008-08-05) Zulak, Katherine G.; Khan, Morgan F.; Alcantara, Joenel; Schriemer, David; Facchini, Peter J.
    Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids, including the narcotic analgesic morphine and the antimicrobial agent sanguinarine. In contrast to the plant, cell cultures of opium poppy do not accumulate alkaloids constitutively but produce sanguinarine in response to treatment with certain fungal-derived elicitors. The induction of sanguinarine biosynthesis provides a model platform to characterize the regulation of benzylisoquinoline alkaloid pathways and other defense responses. Proteome analysis of elicitor-treated opium poppy cell cultures by two-dimensional denaturing-polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry facilitated the identification of 219 of 340 protein spots based on peptide fragment fingerprint searches of a combination of databases. Of the 219 hits, 129 were identified through pre-existing plant proteome databases, 63 were identified by matching predicted translation products in opium poppy-expressed sequence tag databases, and the remainder shared evidence from both databases. Metabolic enzymes represented the largest category of proteins and included S-adenosylmethionine synthetase, several glycolytic, and a nearly complete set of tricarboxylic acid cycle enzymes, one alkaloid, and several other secondary metabolic enzymes. The abundance of chaperones, heat shock proteins, protein degradation factors, and pathogenesis-related proteins provided a comprehensive proteomics view on the coordination of plant defense responses. Qualitative comparison of protein abundance in control and elicitor-treated cell cultures allowed the separation of induced and constitutive or suppressed proteins. DNA microarrays were used to corroborate increases in protein abundance with a corresponding induction in cognate transcript levels.
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    Screening and Characterization of Antimicrobial Compounds and Material against Vibrio cholerae
    (2023-10-12) Kim, Haeun (Sara); MacDonald, Justin A.; Dong, Tao G.; DeVinney, Rebekah; Schriemer, David
    Antibiotic resistance (ABR) is a multisectoral global threat. With an increasing number of critical pathogens acquiring resistance against multiple classes of antibiotics, we face the terror of the post-antibiotic era. Vibrio cholerae, the cholera-causing pathogen, is no exception to the menace of multi-drug resistance. Strains from worldwide outbreaks have shown resistance to all antibiotics used to treat cholera, including ciprofloxacin. With cholera being prevalent in developing countries and regions affected by natural and anthropogenic disasters, an increase in fatalities is a rising concern in these areas with the number of effective antibiotics rapidly decreasing. This thesis explores the alternative approaches to treating cholera and controlling the spread of ABR pathogens in the environment. First, a library of 400 chemical compounds with antimicrobial activities, the Pathogen Box, is screened for compounds that exhibit killing or inhibiting activities against V. cholerae. Two compounds, MMV687807 and MMV675968, demonstrated bactericidal and bacteriostatic activity against the pathogen with high efficacy, respectively. RNA-seq analyses of V. cholerae treated with each compound revealed that their impact on multiple cellular functions, including carbon metabolism and iron homeostasis. Whole genome sequencing of spontaneous resistance mutants uncovered that activation of an efflux system via mutation to their regulator confers resistance to MMV687807. MMV675968 was identified to function similar to trimethoprim, targeting the dihydrofolate reductase of V. cholerae. This compound has the potential to be developed as a trimethoprim replacement with a minimum inhibitory concentration lower by 14-fold. Next, the antibacterial property of a newly synthesized cellulose aerogel cross-linked with different concentrations of silver/lignin nanoparticles (lignin only, 1Ag/L, 2Ag/L, and 3Ag/L) was studied in collaboration with Xiao He. Each aerogel was incubated with Gram-positive or Gram-negative pathogens, and surviving colonies were counted. Increase in killing efficiency was observed with increasing concentration of Ag/L nanoparticles. The cellulose aerogel with 3 Ag/L exhibited exceptional killing across all tested species: E. coli: > 99.99%, P. aeruginosa: > 99.9%, V. cholera: > 99.99%, S. aureus: > 99.99%, B. subtilis: > 97.4%. Through screening small molecules and exploring synthetic material cross-linked with nanoparticles, this thesis demonstrates the significance of developing new tools to inhibit bacterial pathogens and fight the spread of ABR.
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    The impact of clinical irradiation on tumour cells and the microenvironment of undifferentiated pleomorphic sarcoma
    (2023-07) Brown, Samuel; Morrissy, Sorana; Monument, Michael; Jirik, Frank; Schriemer, David
    Introduction: Immunotherapy resistance and tumour heterogeneity in undifferentiated pleomorphic sarcoma (UPS) are driven by an immune-cold tumour microenvironment (TME) with abundant pro-tumour macrophages and low lymphocyte infiltration. While external beam radiation (EBR) can stimulate an immune-hot phenotype in certain contexts, the effects of EBR in UPS remain poorly defined. In Alberta, neoadjuvant protocols can differ between patients. One group undergoes high-dose EBR (50 Gray EBR or low-dose doxorubicin combined with 30 Gray EBR), while another group will not receive neoadjuvant therapy. Pre-treating intends to locally control disease, however, consequences on the TME remain unknown. Here, I quantify the effects of neoadjuvant EBR on both tumour and immune cells using clinical samples. Approach: We profiled transcriptomes from 19 UPS patients, treated with either surgery only, the Edmonton, or the Calgary neoadjuvant protocols. To improve coverage of intra-tumoural TME heterogeneity, we separately sampled multiple regions within each tumour. Immune and tumour cell transcriptomes were collected individually using the NanoString GeoMx digital spatial profiling (DSP) platform. To support these results we further generated global proteomics data from the same samples and integrated these with a large external bulk RNAseq cohort. Results: I systematically evaluated the impact of high-dose radiotherapy on surgical UPS samples with high spatial resolution across histologically-distinct tumour regions. In Aim 1, differential expression analysis identified radiation-associated genes in both tumour and TME cells with significant changes most dramatic in the tumour margin. In Aim 2, I identified gene expression programs and their usage across samples in the 2 treatment groups. Expression programs altered by radiation represented activities related to enhanced antigen presentation and upregulation of cytokine secretion. In Aim 3, data integration revealed that radiation is positively associated with a phenotype that improves patient survival. Conclusion: This study broadens our understanding of how radiation impacts UPS biology. I identify prognostic expression programs that cells alter in response to high-dose radiation and show that the tumour margin is a key region in need of additional research. This work paves the way toward the identification of combination therapies that synergize with radiation to optimize the immune response in UPS.
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    The Role of CHD6 in the DNA Damage Response
    (2024-04-26) Provencher, Luc; Goodarzi, Aaron; Williams, Gareth; Schriemer, David; Corcoran, Jennifer; van Attikum, Haico
    Cells can often experience oxidative stress from events or processes such as dysfunctional metabolism, acute or chronic inflammation, hyper proliferation, and/or exposure to oxidizing or ionizing agents. Oxidative damage to DNA is one of the most common forms of DNA damage, thus cells have evolved mechanisms to cope with and repair lesions to prevent mutation accumulation and promote cell survival. One of the earliest responses to oxidative DNA damage is the activation of poly(ADP-ribose) (PAR) polymerase, resulting in the formation of negatively charged polymers on proteins, chromatin, and DNA, thereby recruiting repair factors and chromatin remodeling enzymes to reshape chromatin at damage sites. Remodeling of chromatin is critical for cellular resilience to oxidative stress, due to the refractory effect that nucleosome arrays can have on DNA damage signalling and repair. The Chromodomain Helicase DNA-binding (CHD) 6 (CHD6) chromatin remodeling enzyme is recruited to sites of oxidative DNA damage in a PAR-dependent manner and is known to be important for preserving cell health during oxidative stress conditions; however, its exact role in repair and DNA repair outcomes is not fully understood. To discover the molecular mechanism by which CHD6 is regulated by and functions within the oxidative DNA damage response, I carried out biochemical, molecular, and cellular studies investigating how individual protein domains of CHD6 contribute to its recruitment and retention at DNA damage sites induced by laser micro-irradiation, and what happens to cells when CHD6 function is lost. I identified CHD6 as capable of directly binding PAR, and that both a functional PAR interacting motif and previously undescribed DNA binding domain are required for effective localization and retention of CHD6 at DNA damage sites. Investigating the biological outcomes of CHD6 loss, I discovered that CHD6-/- normal human tissue-derived cells are sensitive to PARP-trapping small molecule inhibitors and other causes of DNA replication stress, identifying a mechanism of synthetic lethality between CHD6 function and PARP-trapping drugs that does not depend on defective homologous recombination repair. Through molecular biology and mass spectrometry-based techniques, I have identified CHD6 as a base excision repair accessory factor, whose loss causes the accumulation of abasic sites in the genome. Consequently, treatment with PARP-trapping inhibitors results in overwhelming replication stress in CHD6-/- cells that proves lethal to cells. My work has compelling implications for cancer therapy development, where identification of novel ways to increase the effectiveness of PARP inhibitors or overcome acquired resistance to these drugs can positively impact patient outcomes.
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    The Role of LbpB in Iron Acquisition and Cellular Defense in Neisseria meningitidis
    (2016) Ostan, Nicholas; Schryvers, Anthony; Schriemer, David; Armstrong, Glen; DeVinney, Rebekah
    Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in many Gram-negative pathogens. Recent studies have demonstrated that the LbpB protein expressed by Neisseria meningitidis - a causative agent and major contributor to bacterial meningitis - plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homologue, transferrin-binding protein (TbpB), there is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. Nevertheless, there have been several published structural models on the LbpB-Lf interaction that are incompatible. In this study I will present comparative experimental data regarding the LbpB-Lf interaction and TbpB-Tf interaction. Using biophysical data to determine tertiary structure information, I have constructed a new model of LbpB that forms the basis for a new LbpB:Lf interaction. Our data suggests a 1:1 complex of LbpB:hLf is consistent with an LbpB-N/hLf-C binding mode, consistent with a role in iron acquisition. A 2:1 complex of LbpB:hLf:LbpB contains both an LbpB-N/hLf-C interaction as well as an LbpB-C/hLf-N interaction directly with the anti-microbial peptide lactoferricin. We thus show a novel way in which the LbpB from N. meningitidis may sequester anti-microbial peptides, or hinder proteolytic derivation of lactoferricin from lactoferrin by forming large cross-linked complexes of lactoferrin.
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    To define the interactome of neuronal primary cilia
    (2021-09-17) Whitmore, Brandon Alexander; Guo, Jiami; Dufour, Antoine; Huang, Peng; Schriemer, David; Mains, Paul
    Primary cilia are projections of the plasma membrane on almost all mammalian cells in the body, including neurons. The purpose of primary cilia is to act as a cellular antenna due to the unique membrane composition of the primary cilia which is abundant in different receptors, like GPCRs and RTKs, and ion channels. Dysfunction of cilia signaling gives rise to a class of diseases known as ciliopathies. Ciliopathies present with brain structural and functional deficits and have been implicated in intellectual disabilities, Autism Spectrum Disorder, and Schizophrenia. While a number of ciliopathies have been determined, there is a gap in the knowledge of the mechanisms of some ciliopathies due to an incomplete understanding of the primary cilia proteome, especially within the brain. Understanding the primary cilia proteome may determine potential gene candidates that have been implicated in ciliopathies. The gap in the knowledge of the neuronal ciliary proteome arises from the difficulty in purifying mammalian primary cilia. Different proteomics techniques have been used to begin to study the primary cilia composition within kidney cells; however no techniques have been applied to neurons to study the proteome of neuronal primary cilia. The brain and kidneys respond to different extracellular cues and have different environments, indicating that neuronal primary cilia should have a unique membrane composition and respond to distinct extracellular cues compared to primary cilia of other cells. To identify the unique membrane composition of neuronal primary cilia I developed an in vitro proximity labeling method to isolate ciliary proteins from primary neuron cultures. From this work, I was able to create the first list of potential neuronal primary cilia proteins. As an alternative method to understand the membrane composition, I identified a number of signaling molecules that affect primary cilia morphology and impact signaling within cilia. These results will be used to begin constructing an interactome of neuronal primary cilia signaling.
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    Towards a Biochemical Reconstitution of Nepenthes Pitcher Fluid for the Treatment of Celiac Disease
    (2014-09-12) Yang, Menglin; Schriemer, David
    Celiac disease (CD) is an autoimmune disorder that is triggered by the incomplete digestion of gliadins in dietary gluten due to the abundance of P and Q residues in their protein sequence(s). This thesis provides an initial assessment of the proteolytic activity of Nepenthes plant extracts, which is attributed to the aspartic proteases, nepenthesin I and II, potential as an oral protease therapeutic for CD. To this end, nepenthesin I and II were produced recombinantly and characterized. The recombinant nepenthesins were able to reconstitute the proteolytic activity of Nepenthes extracts except for cleavage after P, which was attributed to a previously unidentified protease. Nevertheless, the Nepenthes extracts and recombinant nepenthesin I/II were assessed for their capacity to detoxify gliadins. Although the recombinant nepenthesins alone did not appear sufficient, the Nepenthes plant extracts appeared to efficiently detoxify gliadin, which supports the proposed formulations potential as an effective oral therapeutic for CD.
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    Towards Probing Structure and Function Relationships of Proteins in Complex Sample Types Using HX-MS
    (2017) Sheff, Joey; Schriemer, David; Thurbide, Kevin; Heyne, Belinda
    The functional protein state is a dynamic one, and this behaviour should be accurately reported by our biophysical toolset. Hydrogen-exchange mass spectrometry (HX-MS) is a powerful means of probing changes in the conformational ensemble of interacting proteins, and helps shape our understanding of structure and function relationships. This dissertation describes the development of novel tools for HX-MS, geared towards interacting with biologically relevant systems that are prohibitively large and complex for current structural approaches. First, we introduced a set of standards to correct for dispensing during sample workup. This improved both systematic and random error, and increased the statistical power in differential experiments. Next, we scaled HX-compatible digestion strategies to determine how the analysis of complexes is limited. Digestion with traditional proteases was efficient, with modest coverage of a > 500 kDa sample. Our results suggested that the remaining limiting factors in the analysis of larger systems were related to chromatographic performance. We demonstrated the potential of specific prolyl-endoproteases to mitigate sample complexity. However, we discovered that peptide mapping was inadequate in all proteolytic approaches, and should be resolved if complete HX-MS datasets are desired. A proteomics-inspired nanoHX-MS system was next described. Resolution was improved by eliminating post-column band-broadening with our in-source configuration and a 50-fold improvement in sensitivity was achieved. We then investigated the validity of using overlapping peptides to increase structural resolution. Induction of secondary structure, and charge effects upon interaction with the chromatographic stationary phase perturbed exchange behaviour. Therefore, the fundamental assumption that residue exchange rates are independent of their parent peptide is invalid. These effects must be accounted for to obtain accurate modelling of site-resolved exchange with any high-resolution strategy. Finally, a multivariate strategy was tailored for large scale HX-MS screens. HX-MS readings were complemented by functional data (IC50) and used to characterize a panel of 18 compounds against Eg5, a mitotic kinesin. Canonical mechanisms were confirmed and roughly classified based on inhibitory strength. A modified binding mode and novel allosteric mechanism was discovered for Terpendole E, an inhibitor with activity in clinically relevant resistant Eg5 mutants.