Browsing by Author "MacDonald, Justin Anthony"
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Item Open Access Application of immobilized ATP to the study of NLRP inflammasomes(2019-01-11) Liao, Kuo Chieh; Sandall, Christina F.; Carlson, David A.; Ulke-Lemée, Annegret; Platnich, Jaye; Hughes, Philip Floyd; Muruve, Daniel A.; Haystead, Timothy Arthur James; MacDonald, Justin AnthonyThe NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well-studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.Item Open Access Effects of phosphorylation on the NLRP3 inflammasome(2019-03-05) Sandall, Christina F.; MacDonald, Justin AnthonyThe pyrin domain containing Nod-like receptors (NLRPs) are a family of pattern recognition receptors known to regulate an array of immune signaling pathways. Emergent studies demonstrate the potential for regulatory control of inflammasome assembly by phosphorylation, notably NLRP3. Over a dozen phosphorylation sites have been identified for NLRP3 with many more suggested by phosphoproteomic studies of the NLRP family. Well-characterized NLRP3 phosphorylation events include Ser198 by c-Jun terminal kinase (JNK), Ser295 by protein kinase D (PKD) and/or protein kinase A (PKA), and Tyr861 by an unknown kinase but is dephosphorylated by protein tyrosine phosphatase non-receptor 22 (PTPN22). Since the PKA- and PKD-dependent phosphorylation of NLRP3 at Ser295 is best characterized, we provide detailed review of this aspect of NLRP3 regulation. Phosphorylation of Ser295 can attenuate ATPase activity as compared to its dephosphorylated counterpart, and this event is likely unique to NLRP3. In silico modeling of NLRP3 is useful in predicting how Ser295 phosphorylation might impact upon the structural topology of the ATP-binding domain to influence catalytic activity. It is important to gain as complete understanding as possible of the complex phosphorylation-mediated mechanisms of regulation for NLRP3 in part because of its involvement in many pathological processes.Item Open Access Examining the Role of Non-Canonical NOD-like Receptors and Inflammasomes in Inflammation and Disease(2018-03-21) Platnich, Jaye Matthew; Muruve, Daniel A.; MacDonald, Justin Anthony; Power, Christopher; Duff, Henry J.; Peters, Nathan C.The NOD-like Receptors (NLRs) are a family of pattern recognition receptors known to regulate a variety of immune signaling pathways. A substantial portion of NLR research focuses on the pyrin domain-containing NLRP subfamily. The canonical NLRPs are inflammasome-forming proteins responsible for the activation of caspase-1 and the maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18. In contrast, the non-canonical inflammasome-independent NLRPs regulate a variety of other pathways, including MAPK and NF-κB, through the formation of non-inflammasome complexes. Interestingly, not all inflammasomes are nucleated by NLRPs. The recently characterized non-canonical caspase-4 (caspase-11 in mice) inflammasome is known to be a key driver of the innate immune response to intracellular pathogens (and the molecules associated with them), by triggering both inflammatory cell death and the activation of canonical inflammasomes. At the outset of this PhD work, the understanding of both non-inflammasome-forming NLRPs and the non-canonical caspase-4 inflammasome was poor and the studies were sparse. It was the goal of this thesis to characterize the expression, gene regulation, and function of the non-inflammasome-forming NLR protein NLRP6, both at the cellular and biochemical level. Furthermore, using a pathogen-associated molecular pattern (PAMP)-driven model of inflammation, we sought to elucidate the function of the non-canonical caspase-4 inflammasome, particularly as it pertains to the regulation of the canonical inflammasome and cell death. By studying the fundamental biology underlying these lesser-known mediators of the innate immune system, we hoped to better understand their contribution to the early immune response and their role in driving inflammatory disease with a view to, one day, ameliorating the condition of patients suffering from these afflictions through the development of targeted therapeutics.Item Open Access Sex dimorphism in the cardiometabolic effects of the smoothelin-like 1 protein deletion in an ageing mouse model(2023-04-14) Murali, Megha; MacDonald, Justin Anthony; Cole, William; Howlett, Susan; Thompson, Jennifer; Patel, Vaibhav; Childs, SarahThe smoothelin-like 1 (SMTNL1) protein is a regulator of vascular homeostasis, wherein it modulates smooth muscle contraction, myogenic response, endothelial barrier function, and inflammatory responses. Vascular function is known to reciprocally influence cardiac function, with impaired diastole and left ventricular (LV) stiffness causally linked to endothelial dysfunction and coronary microvascular inflammation. SMTNL1, also highly expressed in skeletal muscle, can impact the energy metabolism and insulin sensitivity of female mice during pregnancy. Therefore, I investigated the metabolic and cardiac outcome in a mouse model deficient in SMTNL1 with advancing age. The cardiac function of SMTNL1 global knockout (KO) mice (3-, 12- and 18-month-old, male and female) was assessed by echocardiography and pressure-volume loop, and the metabolic measurements of middle-aged animals (12-month-old, male and female) were carried out in the comprehensive lab animal monitoring system (CLAMS). The cardiac hemodynamic profiling showed that young male SMTNL1 KO mice developed hypertension and diastolic dysfunction as evidenced by increased systolic blood pressure, end diastolic pressure, LV relaxation time constant (tau), and steeper end diastolic pressure-volume relationship. Unlike young males, the 3-month-old female KO mice did not develop diastolic dysfunction. However, ageing elicited progressive impairment of diastolic function in female KO mice with reduced LV diastolic compliance and elevated filling pressures in comparison to their wild-type counterparts, along with a preserved systolic function in all age groups. Furthermore, flow-mediated dilation, an index of endothelial function was impaired in both male and female KO cohorts at 12 months age, the endocrinal equivalent of human menopausal age. Ageing was also associated with altered cardiac morphology and fibrotic levels along with energy imbalance and glucose intolerance with the loss of SMTNL1. Collectively, the study identified pathophysiological effects of SMTNL1 deletion on metabolic, vascular, and diastolic function with increasing age in a sex-dependent manner. The SMTNL1 KO model for the first time recapitulates the sex dimorphic onset of diastolic dysfunction that is central to heart failure with preserved ejection fraction (HFpEF) condition and represents a novel preclinical model with comorbidities to study the etiology of the HFpEF clinical phenotype.Item Open Access Targeting Pim kinases and DAPK3 to control hypertension(2018-07-04) Carlson, David A.; Singer, Miriam R.; Sutherland, Cindy; Redondo, Clara; Alexander, Leila T.; Hughes, Philip Floyd; Knapp, Stefan; Gurley, Susan B.; Sparks, Matthew A.; MacDonald, Justin Anthony; Haystead, Timothy Arthur JamesSustained vascular smooth muscle hypercontractility promotes hypertension and cardiovascular disease. The etiology of hypercontractility is not completely understood. New therapeutic targets remain vitally important for drug discovery. Here we report that Pim kinases, in combination with DAPK3, regulate contractility and control hypertension. Using a co-crystal structure of lead molecule (HS38) in complex with DAPK3, a dual Pim/DAPK3 inhibitor (HS56) and selective DAPK3 inhibitors (HS94 and HS148) were developed to provide mechanistic insight into the polypharmacology of hypertension. In vitro and ex vivo studies indicated that Pim kinases directly phosphorylate smooth muscle targets and that Pim/DAPK3 inhibition, unlike selective DAPK3 inhibition, significantly reduces contractility. In vivo, HS56 decreased blood pressure in spontaneously hypertensive mice in a dose-dependent manner without affecting heart rate. These findings suggest including Pim kinase inhibition within a multi-target engagement strategy for hypertension management. HS56 represents a significant step in the development of molecularly targeted antihypertensive medications.