Browsing Cumming School of Medicine by Department "Biochemistry & Molecular Biology"
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- ItemOpen AccessAnalysis of recombinational switching at the antigenic variation locus of the Lyme spirochete using a novel PacBio sequencing pipeline(Wiley, 2018-01) Verhey, Theodore B; Castellanos, Mildred; Chaconas, GeorgeThe Lyme disease spirochete evades the host immune system by combinatorial variation of VlsE, a surface antigen. Antigenic variation occurs via segmental gene conversion from contiguous silent cassettes into the vlsE locus. Because of the high degree of similarity between switch variants and the size of vlsE, short-read NGS technologies have been unsuitable for sequencing vlsE populations. Here we use PacBio sequencing technology coupled with the first fully-automated software pipeline (VAST) to accurately process NGS data by minimizing error frequency, eliminating heteroduplex errors and accurately aligning switch variants. We extend earlier studies by showing use of almost all of the vlsE SNP repertoire. In different tissues of the same mouse, 99.6% of the variants were unique, suggesting that dissemination of Borrelia burgdorferi is predominantly unidirectional with little tissue-to-tissue hematogenous dissemination. We also observed a similar number of variants in SCID and wild-type mice, a heatmap of location and frequency of amino acid changes on the 3D structure and note differences observed in SCID versus wild type mice that hint at possible amino acid function. Our observed selection against diversification of residues at the dimer interface in wild-type mice strongly suggests that dimerization is required for in vivo functionality of vlsE.
- ItemOpen AccessAntigenic variation in the Lyme spirochete: detailed functional assessment of recombinational switching at vlsE in the JD1 strain of Borrelia burgdorferi(Wiley, 2019-01) Verhey, Theodore B; Castellanos, Mildred; Chaconas, GeorgeBorrelia burgdorferi is a causative agent of Lyme disease and establishes long-term infection in mammalian hosts. Persistence is promoted by the VlsE antigenic variation system, which generates combinatorial diversity of VlsE through unidirectional, segmental gene conversion from an array of silent cassettes. Here we explore the variants generated by the vls system of strain JD1, which has divergent sequence and structural elements from the type strain B31, the only B. burgdorferi strain in which recombinational switching at vlsE has been studied in detail. We first completed the sequencing of the vls region in JD1, uncovering a previously unreported 114 bp inverted repeat sequence upstream of vlsE. A five-week infection of WT and SCID mice was used for PacBio long read sequencing along with our recently developed VAST pipeline to analyze recombinational switching at vlsE from 40,000 sequences comprising 226,000 inferred recombination events. We show that antigenic variation in B31 and JD1 is highly similar, despite the lack of 17 bp direct repeats in JD1, a somewhat different arrangement of the silent cassettes, divergent inverted repeat sequences and general divergence in the vls sequences. We also present data that strongly suggest that dimerization is required for in vivo functionality of VlsE.
- ItemOpen AccessApplication 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.
- ItemOpen AccessA Borrelia burgdorferi mini-vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat(Wilery, 2018-09) Castellanos, Mildred; Verhey, Theodore B; Chaconas, GeorgeBorrelia burgdorferi evades the host immune system by switching the surface antigen. VlsE, in a process known as antigenic variation. The DNA mechanisms and genetic elements present on the vls locus that participate in the switching process remain to be elucidated. Manipulating the vls locus has been difficult due to its instability on Escherichia coli plasmids. In this study, we generated for the first time a mini-vls system composed of a single silent vlsE variable region (silent cassette 2) through the vlsE gene by performing some cloning steps directly in a highly transformable B. burgdorferi strain. Variants of the mini system were constructed with or without the long inverted repeat (IR) located upstream of vlsE and on both circular and linear plasmids to investigate the importance of the IR and plasmid topology on recombinational switching at vlsE. Amplicon sequencing using PacBio long read technology and analysis of the data with our recently reported pipeline and VAST software showed that the system undergoes switching in mice in both linear and circular versions and that the presence of the hairpin does not seem to be crucial in the linear version, however it is required when the topology is circular.
- ItemOpen AccessCSM murray award lecture - functional studies of the Lyme disease spirochete - from molecules to mice(Canadian Science Publishing, 2012-02-17) Chaconas, GeorgeLyme borreliosis, also known as Lyme disease, is now the most common vector transmitted disease in the northern hemisphere. It is caused by the spirochete Borrelia burgdorferi and related species. In addition to their clinical importance, these organisms are fascinating to study because of the wide variety of unusual features they possess. Ongoing work in the laboratory in several areas will be described. (1) The segmented genomes contain up to two dozen genetic elements, the majority of which are linear with covalently closed hairpin ends. These linear DNAs also display a very high degree of ongoing genetic rearrangement. Mechanisms for these processes will be described. (2) Persistent infection by Borrelia species requires antigenic variation through a complex DNA rearrangement process at the vlsE locus on the linear plasmid lp28-1. Novel features of this recombination process will be presented. (3) Evidence for a new global regulatory pathway of B. burgdorferi gene expression that is required for pathogenicity will be described. The DEAH box RNA helicase HrpA is involved in this pathway, which may be relevant in other bacteria. (4) The mechanism of B. burgdorferi to effectively disseminate throughout its host is being studied in real time by high resolution intravital imaging in live mice. Recent work will be presented.
- ItemOpen AccessEffects 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.
- ItemOpen AccessEfficient isolation of highly purified tonsil B lymphocytes using RosetteSep with allogeneic human red blood cells(BioMed Central, 2009-05) Zuccolo, Jonathan; Unruh, Tammy L.; Deans, Julie P.
- ItemOpen AccessGoing (Reo)viral: factors promoting successful revival oncolytics infection(MDPI, 2018-08-11) Bourhill, Tarryn; Mori, Yoshinori; Rancourt, Derrick Emile; Shmulevitz, Maya; Johnston, Randal N.Oncolytic viruses show intriguing potential as cancer therapeutic agents. These viruses are capable of selectively targeting and killing cancerous cells while leaving healthy cells largely unaffected. The use of oncolytic viruses for cancer treatments in selected circumstances has recently been approved by the Food and Drug Administration (FDA) of the US and work is progressing on engineering viral vectors for enhanced selectivity, efficacy and safety. However, a better fundamental understanding of tumour and viral biology is essential for the continued advancement of the oncolytic field. This knowledge will not only help to engineer more potent and effective viruses but may also contribute to the identification of biomarkers that can determine which patients will benefit most from this treatment. A mechanistic understanding of the overlapping activity of viral and standard chemotherapeutics will enable the development of better combinational approaches to improve patient outcomes. In this review, we will examine each of the factors that contribute to productive viral infections in cancerous cells versus healthy cells. Special attention will be paid to reovirus as it is a well-studied virus and the only wild-type virus to have received orphan drug designation by the FDA. Although considerable insight into reoviral biology exists, there remain numerous deficiencies in our understanding of the factors regulating its successful oncolytic infection. Here we will discuss what is known to regulate infection as well as speculate about potential new mechanisms that may enhance successful replication. A joint appreciation of both tumour and viral biology will drive innovation for the next generation of reoviral mediated oncolytic therapy.
- ItemOpen AccessHairpin Telomere Resolvases(American Society for Microbiology, 2014-11-24) Kobryn, Kerri; Chaconas, GeorgeCovalently closed hairpin ends, also known as hairpin telomeres, provide an unusual solution to the end replication problem. The hairpin telomeres are generated from replication intermediates by a process known as telomere resolution. This is a DNA breakage and reunion reaction promoted by hairpin telomere resolvases (also referred to as protelomerases) found in a limited number of phage and bacteria. The reaction promoted by these enzymes is a chemically isoenergetic two-step transesterification without a requirement for divalent metal ions or high-energy cofactors and uses an active site and mechanism similar to that for type IB topoisomerases and tyrosine recombinases. The small number of unrelated telomere resolvases characterized to date all contain a central, catalytic core domain with the active site, but in addition carry variable C- and N-terminal domains with different functions. Similarities and differences in the structure and function of the telomere resolvases are discussed. Of particular interest are the properties of the Borrelia telomere resolvases, which have been studied most extensively at the biochemical level and appear to play a role in shaping the unusual segmented genomes in these organisms and, perhaps, to play a role in recombinational events.
- ItemOpen AccessIntegration of transcriptional inputs at promoters of the arabinose catabolic pathway(BioMed Central, 2010-06-02) Davidson, Carla J.; Narang, Atul; Surette, Michael G.
- ItemOpen AccessThe Lyme disease spirochete can hijack the host immune system for extravasation from the microvasculature(Wiley, 2021-04-23) Tan, Xi; Petri, Björn; DeVinney, Rebekah; Jenne, Craig N; Chaconas, GeorgeLyme disease is the most common tick-transmitted disease in the northern hemisphere and is caused by the spirochete Borrelia burgdorferi and related Borrelia species. The constellation of symptoms attributable to this malady result from vascular dissemination of B. burgdorferi throughout the body to invade various tissue types. However, little is known about the mechanism by which the spirochetes can breach the blood vessel wall to reach distant tissues. We have studied this process by direct observation of spirochetes in the microvasculature of living mice using multilaser spinning-disk intravital microscopy. Our results show that in our experimental system, instead of phagocytizing B. burgdorferi, host neutrophils are involved in the production of specific cytokines that activate the endothelium and potentiate B. burgdorferi escape into the surrounding tissue. Spirochete escape is not induced by paracellular permeability and appears to occur via a transcellular pathway. Neutrophil repurposing to promote bacterial extravasation represents a new and innovative pathogenic strategy.
- 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 AccessThe Pace of Prostatic Intraepithelial Neoplasia Development Is Determined by the Timing of Pten Tumor Suppressor Gene Excision(Public Library of Science, 2009) Luchman, H. Artee; Benediktsson, Hallgrimur; Villemaire, Michelle L.; Peterson, Alan C.; Jirik, Frank R.
- ItemOpen AccessQuantum dot interactions and flow effects in angiogenic zebrafish (Danio rerio) vessels and human endothelial cells(Elsevier, 2017-01) Jiang, Xiao-Yu; Sarsons, Christopher D; Gomez-Garcia, M Juliana; Cramb, David T; Rinker, Kristina D; Childs, Sarah JNanoparticle (NP) interactions with biological tissues are affected by the size, shape and surface chemistry of the NPs. Here we use in vivo (zebrafish) and in vitro (HUVEC) models to investigate association of quantum dots (QDs) with endothelial cells and the effect of fluid flow. After injection into the developing zebrafish, circulating QDs associate with endothelium and penetrate surrounding tissue parenchyma over time. Amino-functionalized QDs cluster, interact with cells, and clear more rapidly than carboxy-functionalized QDs in vivo, highlighting charge influences. QDs show stronger accumulation in slow-flowing, small caliber venous vessels than in fast-flowing high caliber arterial vessels. Parallel-plate flow experiments with HUVEC support these findings, showing reduced QD-EC association with increasing flow. In vivo, flow arrest after nanoparticle injection still results in venous accumulation at 18 h. Overall our results suggest that both QD charge and blood flow modulate particle-endothelial cell interactions.
- ItemOpen AccessSimultaneous binding of the N- and C-terminal cytoplasmic domains of aquaporin 4 to calmodulin(Elsevier, 2022-01) Ishida, Hiroaki; Vogel, Hans J; Conner, Alex C; Kitchen, Philip; Bill, Roslyn M; MacDonald, Justin AAquaporin 4 (AQP4) is a water transporting, transmembrane channel protein that has important regulatory roles in maintaining cellular water homeostasis. Several other AQP proteins exhibit calmodulin (CaM)-binding properties, and CaM has recently been implicated in the cell surface localization of AQP4. The objective of the present study was to assess the CaM-binding properties of AQP4 in detail. Inspection of AQP4 revealed two putative CaM-binding domains (CBDs) in the cytoplasmic N- and C-terminal regions, respectively. The Ca2+-dependent CaM-binding properties of AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional 1H, 15N-HSQC NMR with 15N-labeled CaM. The N-terminal CBD of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a Kd of 3.4 μM. The C-terminal AQP4 peptide interacted with both the C- and N-lobes of CaM (2:1 binding ratio; Kd1: 3.6 μM, Kd2: 113.6 μM, respectively). A recombinant AQP4 protein domain (recAQP4CT, containing the entire cytosolic C-terminal sequence) bound CaM in a 1:1 binding mode with a Kd of 6.1 μM. A ternary bridging complex could be generated with the N- and C-lobes of CaM interacting simultaneously with the N- and C-terminal CBD peptides. These data support a unique adapter protein binding mode for CaM with AQP4.
- ItemOpen AccessStructure, function, and evolution of linear replicons in Borrelia(Annual Reviews, 2010-06-10) Chaconas, George; Kobryn, KerriSpirochetes of the genus Borrelia include important human pathogens that cause Lyme borreliosis and relapsing fever. The genomes of Borrelia species can be composed of up to 24 DNA molecules, most of which are linear. The plasmid content and linear replicon sequence arrangement vary widely between isolates. The linear replicons are terminated by covalently closed DNA hairpins or hairpin telomeres. Replication of these elements involves a unique reaction, called telomere resolution, to produce hairpin telomeres from replicative intermediates. The telomere resolvase, ResT, is thought to contribute to the genetic flux of the linear molecules by promoting stabilized telomere fusions. Telomere resolvases are related to the tyrosine recombinases and ResT can generate the crucial reaction intermediate of this class of enzyme, the Holliday junction. This observation has led to the proposal that telomere resolvases evolved from tyrosine recombinases inducing DNA linearization in the genomes that acquired them.
- ItemOpen AccessTargeting 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.