Browsing by Author "MacDonald, Justin"
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- ItemOpen AccessA Tale of a ‘Tail’ – Understanding the Role of Ku80 C-terminal Region in Non-Homologous End Joining(2015-12-14) Radhakrishnan, Sarvan Kumar; Lees-Miller, Susan; Cobb, Jennifer; MacDonald, JustinNon-homologous end joining (NHEJ) is the major DNA double strand (DSB) break repair pathway in mammalian cells. The first step in NHEJ is recognition of DSBs by the Ku heterodimer and subsequent recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a serine/threonine protein kinase, to form the DNA-PK complex. This complex aligns DSB ends, activates Artemis endonuclease activity and finally recruits XRCC4-DNA ligase IV, which ligates DNA ends. The Ku heterodimer consists of 70 and 80 kDa subunits and is conserved throughout evolution. It has been suggested that the extreme C-terminal 14 amino acids of Ku80 is required for DNA-PKcs recruitment and activation. However, another study demonstrated that deletion of the Ku80 C-terminal region (CTR) does not abolish DNA-PKcs activation. Thus, there is considerable ambiguity regarding the role of the Ku80 CTR in DNA-PKcs recruitment and activation. The aim of this study is to understand the role of Ku80 CTR in NHEJ with focus on its ability to recruit and activate DNA-PKcs kinase activity. Using clonogenic cell survival assays, I confirmed that hamster cells expressing Ku80 CTR deletions are radiosensitive and also showed sensitivity to other DSB inducing agents such as doxorubicin and neocarzinostatin. I then generated Ku80 C-terminal deletions (Ku80 residues 1-718 and 1-569), cloned them into baculovirus vectors and expressed and purified the corresponding Ku heterodimers from insect cells. In vitro autophosphorylation reactions, in presence of calf-thymus DNA, using purified proteins showed that Ku heterodimer with Ku80 residues 1-718 showed only a slight defect in DNA-PKcs autophosphorylation, whereas heterodimer with Ku80 residues 1-569 had significant defects in multiple DNA-PKcs autophosphorylation sites. Surprising results were observed when defined DNA structures such as 25 base pair (bp) blunt ended double stranded (ds) DNA was used. Deletion of the entire Ku80 CTR (residues 570-732) lead to abrogation of DNA-PKcs kinase activity and inability to interact with DNA-PKcs protein. On the other hand, deletion of extreme C-terminal 14 amino acids of Ku80 did not affect DNA-PKcs kinase activity but showed defects in its ability to interact with DNA-PKcs. These defects may underlie the radiation and chemosensitivity of Ku80 CTR deletion mutants.
- ItemOpen AccessArabidopsis thaliana D group MPKs interactions with RLPH2 and PP1(2021-01-08) Toth, Ryan B.; Moorhead, Greg; Samuel, Marcus; Ng, Kenneth; MacDonald, Justin; Moorhead, GregMitogen activated protein kinases (MPK) are key members of the MPK cascade, a pathway that results in an immediate response from (external) stimuli such as infection, cold, or drought. The activation of an MPK relies its activation loop being phosphorylated on a threonine and tyrosine residue. A unique bacteria-like phosphatase Rhizobiales-like phosphatase 2 (RLPH2), which we bioinformatically characterized as a serine/threonine phosphatase, has been discovered to act as a tyrosine phosphatase. In this thesis, data monitoring RLPH2s dephosphorylation of different peptides derived from MPK activation loops would reveal a preference for the dephosphorylation of D group MPKs over non-D group MPKs, and indicate that the central residue in the TXY motif plays a key role in RLPH2 substrate determination. Additionally, RLPH2 would show a preference for the in vitro dephosphorylation of MPK9 (a D group MPK) over MPK3 (a non-D group MPK). An engaging observation is that right next to the TxY motif in D group MPKs is a putative Protein Phosphatase 1 (PP1) regulatory protein binding RVxF motif. This motif is not present in any other characterized MPK, suggesting an interaction between PP1 and D group MPKs. In this thesis, PP1 protein binding assays with MPK9 WT and an MPK9 RASA mutant would show binding occurs between MPK9 and PP1 through the RVXF motif in vitro. Peptide and protein substrate dephosphorylation assays with PP1 would show threonine dephosphorylation on MPK9 and the effect the RVXF motif plays on this interaction. In summary, the specificity of RLPH2 as a D group MPK regulator was examined along with the interaction between PP1 and the D group MPKs via the RVXF motif, with mention of a potential relationship between these three interactions.
- ItemOpen AccessClostridium difficile infection susceptibility is controlled by alterations to the gut microbiota before and after antibiotic exposure(2014-07-30) Schenck, Louis Patrick; Beck, Paul; MacDonald, JustinClostridium difficile (Cdif) infections (CDI) cause devastating diarrheal disease. Small clinical trials have generated excitement for the use of fecal microbial transplants as a therapeutic option; however, the exact components of the microbiota needed for protection against CDI have remained elusive. C57Bl/6 mice from two vendors, Jackson (JAX) and Charles River (CRV), were exposed to antibiotics before gavage with Cdif spores. JAX mice developed severe CDI as evidenced by weight loss, histological damage, and increased pro-inflammatory cytokines, whereas CRV mice were resistant to CDI. There were significant differences in the gut microbiota between the mice, but CRV mice retained a high abundance of Parabacteroides after antibiotics. Following cohousing, JAX mice had less severe CDI and CRV mice had more severe CDI. Transfer of P. distasonis, a CRV commensal bacteria, was able to dampen effects of CDI in JAX mice. This study may lead to more targeted bacteriotherapy for the treatment and prevention of CDI.
- ItemOpen AccessDeath-associated Protein Kinase 3 (DAPK3), Identified as a Potential Key Factor for the Progression of Ulcerative Colitis (UC), Regulates Intestinal Epithelial Repair(2021-12-14) Chen, Tina Huey-Miin; MacDonald, Justin; Yates, Robin; Humberto, JijonUlcerative colitis (UC) is a progressive disorder that elevates the risk of cancer development through a colitis-dysplasia-carcinoma sequence. Recent evidence demonstrates the necessity of Yes-associated protein (YAP) signaling, interceded by cytoskeletal remodeling, for intestinal regeneration. Death-associated protein kinase 3 (DAPK3) is a regulator of actin-cytoskeleton reorganization that controls proliferation and apoptosis. In this thesis, DAPK3 was identified as a candidate gene involved in UC progression. Gene ontology (GO) enrichment analysis revealed a shift in transcriptome landscape as UC progressed from left-sided colitis to pancolitis to colitis-associated dysplasia (CAD), from being immune-centric to being cytoskeleton-dependent. Molecular interaction network analysis of genes differentially expressed in left-sided colitis, pancolitis, and CAD revealed one pairwise line or edge that was topologically important to the network structure. This edge was found to be highly enriched in actin-based processes, and DAPK3 was the sole protein kinase associated with this edge. Pharmacological inhibition of DAPK3 in Caco-2 human intestinal epithelial cells (IECs) with the HS38 compound augmented cell proliferation to enhance wound closure. This phenotype corresponded with the increased colocalization of YAP with F-actin, which is indicative of YAP activation. In mice recovering from dextran sodium sulfate (DSS-) induced UC, pharmacological inhibition of DAPK3 increased YAP nuclear localization in IECs, another indicator of YAP activation. However, IEC proliferation was repressed, and mice exhibited increased disease severity when DAPK3 was inhibited with the HS38 compound. It is yet unclear if the incongruous phenotype between the cell and animal studies was the consequence of DAPK3 function on other cell types (e.g., macrophages). In summary, this thesis established DAPK3 as a key factor in intestinal epithelial regeneration and UC progression by way of YAP signaling. Nevertheless, the role that DAPK3 play in different cell types will need further investigation to decipher the full consequence of DAPK3 inhibition on UC progression.
- ItemOpen AccessElucidating the Regulatory Mechanisms that Govern Proneural Gene Function in the Developing Neocortex(2016) Wilkinson, Grey; Schuurmans, Carol; Berube, Nathalie; Hansen, David; MacDonald, Justin; Biernaskie, JeffThe neocortex is the site of higher order cognitive functioning and sensory processing. It is an essential region of the brain as a reduction in neocortical mass at birth due to reduced neuronal number is often associated with intellectual deficits and behavioural abnormalities. It is thus important that we decipher how neurogenesis is regulated during neocortical development. My research has focused on determining how neocortical progenitors decide to proliferate or differentiate; a cell fate choice that dictates how many neurons are born during development. Specifically, I have studied the basic helix-loop-helix transcription factors encoded by the proneural genes Neurogenin 2 (Neurog2) and Achaete scute-like 1 (Ascl1). Neurog2 and Ascl1 are expressed in neural progenitor cells and give rise to distinct neuronal and glial cell types. In the developing telencephalon, Neurog2 promotes the differentiation of excitatory projection neurons while Ascl1 promotes the differentiation of interneurons and glioblasts. My general hypothesis was that Neurog2-Ascl1 form a genetic switch, and that the extrinsic/intrinsic cues that control this genetic switch lie at the crux of cortical progenitor cell fate decisions, ensuring that cortical cells differentiate in sequence and on time. Data that supports this hypothesis, demonstrates that Neurog2-Ascl1 do indeed form a cross-repressive genetic switch, acting together to regulate the timing of laminar fate transitions, and conferring a metastable stem cell state onto a subpool of cortical progenitors. Furthermore, I made inroads into understanding how Neurog2 proneural activity is regulated, demonstrating that Mbt1, a polycomb group protein, regulates Neurog2 function. In sum, through my PhD work, I have made significant inroads into deciphering the molecular mechanisms that control the balance between neocortical progenitor cell self-renewal and differentiation.
- ItemOpen AccessHsp90 Regulates the NLRP3 Inflammasome via the NF-kB Signaling Pathway(2020-04-17) Sparksman, Steven; Beck, Paul; Muruve, Daniel; MacDonald, Justin; McKay, Derek; Braun, JaniceAn over-reactive inflammatory response can lead to chronic inflammation and auto-immune disorders such as Crohn’s disease, ulcerative colitis or cancer. At the heart of the host’s inflammatory response is an immune cell intracellular sensor protein known as NLRP3 that regulates the cellular response to a wide range of PAMPS/DAMPS. NLRP3 has been characterized primarily as an inflammasome-forming protein in response to infection and injury. The inflammasome regulates IL-1β and IL-18 maturation leading to their subsequent secretion from the immune cell. Secretion of these cytokines recruits other immune cells and factors that leads to the resolution of the initiating infection or injury. Hsp90, with its co-chaperone SGT1, was shown to be required for NLRP3 inflammasome activation via a direct protein-protein interaction. An Hsp90-SGT1 interaction was suggested to stabilize NLRP3 prior to inflammasome activation allowing the sensing of PAMPS/DAMPS; however, the mechanism, timing and sequence of events of this interaction have yet to be shown experimentally. Thus, the central hypothesis of this thesis is that Hsp90 regulates the activation of the NLRP3 inflammasome by stabilizing NLRP3 via direct protein-protein interactions. Treatment with DMAG, an Hsp90 inhibitor, blocked canonical NLRP3 function in differentiated THP-1 immune cells. However, we found no evidence that Hsp90-SGT1 was involved in protein-protein interactions with NLRP3. Instead, experiments revealed that DMAG attenuated IL1β gene transcription but did not interfere with translocation of the transcription factor, NF-kB to the nucleus. This suggests Hsp90 regulates the NLRP3 inflammasome by regulating transcription of NLRP3 inflammasome component genes. This project has revealed new insights for Hsp90 in the inflammatory response and suggests Hsp90 as a credible target for chronic inflammatory disorders.
- ItemOpen AccessMechanics of Amphibian Skeletal Muscle at Long Lengths(2017) Hisey, Brandon; Herzog, Walter; Biewener, Andrew; Syme, Douglas; Stefanyshyn, Darren; MacIntosh, Brian; MacDonald, JustinLittle is known about the mechanical behavior of skeletal muscle at long lengths, or how these behaviors scale to different hierarchical levels. The purpose of this thesis is to examine the mechanical behavior of skeletal muscle leading up to, and at the point of failure. Failure was defined as a compromise in the muscles’ ability to produce force, as indicated by a decrease in force, during a steady stretch. Stretches were performed in active (i.e. contracting) and passive muscles in three different preparations from the semitendinosus muscle of the frog Rana pipiens – myofibrils, permeabilized fibres, and whole muscles. Stress in active myofibrils was significantly greater than in passive ones with the progression of stretch to failure, and was persistent despite being stretched to lengths beyond overlap of actin and myosin filaments (who’s interactions are responsible for the production of contractile force). This divergence in stress was reduced at the cellular level, and was abolished in whole muscles. It is suggested that higher active compared to passive stress is a result of an increased contribution by the large molecular spring, titin. As higher hierarchical levels are examined, the introduction of other passive elements and connections may break during stretch and mask the divergent behavior observed in myofibrils. Histology and electron microscopy showed complete loss of regular striation patterns in both active and passive fibres stretched to sarcomere lengths of approximately 5 um. Whole muscles showed indicators of damage as well, but to a much lesser extent, and with active muscles showing more evidence of damage. Mechanical data and histology suggest that failure in whole muscles occurs outside of the cell, perhaps in the extracellular matrix or at the myotendinous junction. Finally, it was demonstrated that whole muscles possessing a low passive compliance (in this case, the tibialis anterior from R. pipiens) were more prone to failure at short lengths than muscles having a higher compliance (i.e. semitendinosus). It is suggested that differing muscle compliances represent adaptive strategies to prevent damage according to functional demands of the specific muscle.
- ItemOpen AccessMolecular Network Analyses Implicate Death-Associated Protein Kinase 3 (DAPK3) as a Key Factor in Colitis-Associated Dysplasia Progression(Oxford University Press, 2022-05-23) Chen, Huey-Miin; MacDonald, JustinBackground: Ulcerative colitis (UC) is a progressive disorder that elevates the risk of colon cancer development through a colitis-dysplasia-carcinoma sequence. Gene expression profiling of colitis-associated lesions obtained from patients with varied extents of UC can be mined to define molecular panels associated with colon cancer development. Methods: Differential gene expression profiles of 3 UC clinical subtypes and healthy controls were developed for the GSE47908 microarray data set of healthy controls, left-sided colitis, pancolitis, and colitis-associated dysplasia (CAD) using limma R. Results: A gene ontology enrichment analysis of differentially expressed genes (DEGs) revealed a shift in the transcriptome landscape as UC progressed from left-sided colitis to pancolitis to CAD, from being immune-centric to being cytoskeleton-dependent. Hippo signaling (via Yes-associated protein [YAP]) and Ephrin receptor signaling were the top canonical pathways progressively altered in concert with the pathogenic progression of UC. A molecular interaction network analysis of DEGs in left-sided colitis, pancolitis, and CAD revealed 1 pairwise line, or edge, that was topologically important to the network structure. This edge was found to be highly enriched in actin-based processes, and death-associated protein kinase 3 (DAPK3) was a critical member and sole protein kinase member of this network. Death-associated protein kinase 3 is a regulator of actin-cytoskeleton reorganization that controls proliferation and apoptosis. Differential correlation analyses revealed a negative correlation for DAPK3-YAP in healthy controls that flipped to positive in left-sided colitis. With UC progression to CAD, the DAPK3-YAP correlation grew progressively more positive. Conclusion: In summary, DAPK3 was identified as a candidate gene involved in UC progression to dysplasia.
- ItemOpen AccessPhysiological and molecular mechanisms contribute to altered smooth muscle contractility during dextran sodium sulfate-induced colitis in mice(2008) Bonilla, Alexandra Valderrama; MacDonald, Justin
- ItemOpen AccessProtecting the gut against Clostridium difficile: A role for Keratinocyte growth factor(2014-07-18) Alhassan, Basmah Faris; Beck, Paul; MacDonald, JustinClostridium difficle (Cdiff) infection (CDI) causes severe colitis via its toxins: toxin A and toxin B (TcdAB), inducing barrier disruption, inflammation and cell death. Current treatments are failing and the need to search for new targets is urgent. Several host factors have shown to modulate CDI in animals and patients. Intestinal growth factors are a major part of the mucosal host response in the gut. Among them, keratinocyte growth factor (KGF) has been shown to be protective in many colitis models. In this thesis, the protective role of KGF was demonstrated against Cdiff toxin injury. In vitro, KGF protected Caco-2 cells from barrier disruption and cell death induced by TcdAB. Exogenous KGF administration protected mice from acute intestinal toxin damage. Interestingly, KGF deletion did not impact the acute toxin-induced colitis in mice; however, endogenous KGF was essential for normal recovery from TcdAB-induced colitis as KGF−/− mice demonstrated impaired recovery after 24-48 hours post TcdAB exposure. Findings from this study may lead to identifying a cause for the variability in clinical response among patients with CDI as well as new therapeutic targets for this devastating disease.
- ItemOpen AccessRegulation of Vascular Smooth Muscle Cell Motility by Protein Kinases: A Focus on Zipper-Interacting Protein Kinase(2017) Al-Ghabkari, Abdulhameed; MacDonald, Justin; Walsh, Michael; Moorhead, GregoryZipper-interacting protein kinase (ZIPK) is a protein serine/threonine kinase that mediates a variety of cellular functions. ZIPK is a key regulator of vascular smooth muscle (VSM) cell contractility and motility. Further investigations for the upstream regulators, activation signals, downstream target(s), regulatory mechanism(s) and the role of ZIPK in different signaling modules are required. Two major strategies were utilized in this study to examine ZIPK signaling in VSM cells; chemical genetics and application of small molecule inhibitors. For the chemical genetics strategy, ZIPK was engineered in the ATP-binding pocket by replacing a bulky amino acid (Leu) with a small one (Gly). This ZIPK-L93G kinase was introduced into VSM cells and then the NM-PPI inhibitor (selective for L93G-ZIPK, not for WT-ZIPK) was applied, which demonstrated an inhibitory effect on myosin phosphorylation. This strategy provides another approach to study ZIPK signaling, especially in the absence of specific inhibitors for ZIPK. Recent investigations led to the development of two small molecule inhibitors for ZIPK; DI and HS-38. The selectivity of the DI compound was assessed by; (i) in vitro studies; (ii) in situ analysis; and (iii) in silico molecular modelling. Our studies revealed that the DI compound was not selective for ZIPK; DI had effects on the cellular cytoskeleton and motility that were associated with diminution of ROCKII signaling pathways. The HS-38 compound was advantageous to delineate a novel signaling relationship between ZIPK and focal adhesion kinase (FAK). ZIPK inhibition by HS-38 or ZIPK knockdown with siRNA resulted in suppression of pY397-FAK phosphorylation and remodeling of the cytoskeletal architecture. Additional molecular details of this signaling mechanism implicate a member of the dual specificity phosphatase family (CDC14A) in a putative partnership with ZIPK to regulate focal adhesion dynamics and FAK phosphorylation.
- ItemOpen AccessStructural Dissection and Catalytic Properties of the NLRP (Nucleotide-Binding Domain and Leucine-Rich Repeat-Containing Gene Family, Pyrin Domain Containing) Family of Inflammatory Proteins(2022-09-19) Sandall, Christina F.; MacDonald, Justin; Muruve, Daniel; Lees-Miller, SusanInflammasomes are high molecular weight hetero-oligomeric protein complexes nucleated by innate immune cytosolic pattern recognition receptors (PRRs) including the NOD-like receptors (NLRs). A subset of NLR proteins include those with N-terminal Pyrin domains (NLRPs) which play critical roles in the detection and response to both endogenous and exogenous danger signals. The NLRP3 inflammasome is the most highly studied and contributes to a multitude of inflammatory and autoimmune conditions. Thus, this work provides a more detailed understanding of NLRP3 activation mechanisms that will be essential for the rationalised development of future pharmacological interventions. First, the impact of orientation and linkage on NLRP3 capture with immobilised ATP demonstrates ATP binds this protein with an exposed phosphate tail and buried adenine ring. Decreased competitive recovery with free ATP indicated NLRP3 underwent a conformational change upon ATP binding. Additionally, P-linked ATP Sepharose provides a strategy for capture of the entire NLRP family and enrichment of NLRP3 containing samples for mass spectrometry analyses. Next, the ATP hydrolysis kinetics of NLRP proteins and hyperactive NLRP3 disease mutant R262W were evaluated with GFP nano-trap beads and a bioluminescent ATPase assay. NLRP proteins displayed distinct ATPase kinetic profiles, suggesting variable ATP sensitivity and kinetics of assembly for some NLRP proteins. Classical Michaelis-Menten kinetics were observed for NLRP1, 3 and 12, and positive Hill cooperativity was revealed for NLRP3R262W as well as NLRP6 and 7. Furthermore, NLRP3 inhibitors targeting ATPase activity demonstrated promising results in blocking inflammasome activation. Next, the impacts of NLRP3 phosphorylation at Ser295 on protein structure, ATP binding & inflammasome activation were evaluated in silico. These results suggest that modifications to the NACHT domain are conveyed globally and result in variable nucleotide hydrolysis and inflammasome activation. Finally, unique mechanisms of ATP and ADP binding and the detailed structural impacts were illuminated by molecular dynamics simulations with NLRP3. NLRP3-ADP simulations indicate high protein stability and few global rearrangements, while NLRP3-ATP binding was thermodynamically favourable, induced protein flexibility, and resulted in global structural rearrangements that were initiated from the NACHT domain. To conclude, a detailed mechanism of ATP induced structural changes provided a basis for rationale design of NLRP3 inhibitors, and a region of NLRP3 in a cleft between the HD2 and LRR domains was proposed for pharmacological targeting.
- ItemOpen AccessVitamin D induces the release of IL-1b via activation of the NLRP3 inflammasome(2012-07-30) Tulk, Sarah; MacDonald, Justin; Beck, PaulThe Crohn’s disease pathogenesis has been proposed to involve a hypoactive innate immune response and impaired acute inflammatory response. Recently, vitamin D3 has been shown to induce expression of the innate immune receptor, NOD2. Herein, PMA-differentiated THP-1 cells were treated with the active form of vitamin D3, 1,25(OH)2D3, and assessed for release of the pro-inflammatory cytokine IL-1β. We present novel findings that 1,25(OH)2D3 induces the release of IL-1β from PMA-differentiated THP-1 cells. Further, circulating vitamin D3, 25(OH)D3, was also able to induce the IL-1β response. Both 1,25(OH)2D3-induced and 25(OH)D3-induced IL-1β release were found to be dependent on the NLRP3 inflammasome. These novel findings support a beneficial role for vitamin D3 in Crohn’s disease; by boosting basal IL-1β secretion from macrophages, vitamin D3 could help counteract the impaired innate immune and acute inflammatory responses seen in this disease.