UVM Theses and Dissertations
Format:
Print
Author:
Tilley, Lucas D.
Dept./Program:
Microbiology and Molecular Genetics
Year:
2014
Degree:
PhD
Abstract:
Chemical genetics refers to a set of experimental approaches which use small organic molecules to produce cellular phenotypes typically by modulation of protein function. Conceptually, these approaches have many characteristics in common with molecular genetic approaches, particularly in that chemical genetics strives to link a phenotype to a specific gene product. Target identification and validation are the most challenging aspects of the forward chemical genetic approach. The techniques for target identification and validation continue to increase in number and sophistication. However, the most straightforward approach for target validation is genetic ablation of the suspected target and characterization of the mutant's sensitivity to the compound. Once a small molecule target is validated, the small molecule can be used to explore the physiological relevance of the target.
During an effort to discover the biologically relevant target of a small molecule inhibitor (Conoidin A) of T. gondii invasion, we discovered a novel cytoskeletal protein that we have named TgCBAP. To begin elucidating the function of TgCBAP, we disrupted the TgCBAP gene by homologous recombination. Parasites lacking TgCBAP are as sensitive to the effects of Conoidin A as wild-type parasites, demonstrating that TgCBAP is not the biologically relevant target of Conoidin A. However, [Delta]TgCBAP parasites are significantly smaller than wild type parasites and display a growth defect in culture. Furthermore, TgCBAP shows an unusual subcellular localization, forming small rings at the apical and basal ends of the parasite and punctate, ring-like structures around the parasite periphery. These data identify a new marker of the apical and basal subcompartments of T. gondii, reveal a potentially novel compartment along the parasite periphery, and identify TgCBAP as a determinant of parasite size that is required for a maximally efficient lytic cycle.
In T. gondii, a cyclic GMP-dependent protein kinase (TgPKG) has been experimentally validated as the biological relevant target of the small molecule inhibitor 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-y1]pyridine (Compound 1). At submicromolar concentrations, Compound 1 blocks extracellular parasite motility and host cell invasion, yet has a minimal effect on intracellular replication. Validation of TgPKG as the bona fide target of Compound 1 has strongly implicated the substrates of TgPKG in these same cellular processes. To date, no substrate of TgPKG has been identified. Identification of the TgPKG substrates involved in these processes would further our understanding of these essential events in the lytic cycle and may lead to the identification of new drug targets. We employed biochemical and quantitative phosphoproteomic analysis to identifY components of the TgPKG signaling pathway using Compound 2, a more potent structural analog of Compound 1. We discovered that Compound 2 blocks calcium and cGMP-mediated increase in TgMyoA phosphorylation in wildtype parasites, but not in parasites resistant to Compound 2. In our quantitative proteomics analysis, we also discovered a 12 candidate TgPKG substrates. These results· identify potential downstream effectors of TgPKG activity and provide a direction for future investigations into TgPKG signaling.
During an effort to discover the biologically relevant target of a small molecule inhibitor (Conoidin A) of T. gondii invasion, we discovered a novel cytoskeletal protein that we have named TgCBAP. To begin elucidating the function of TgCBAP, we disrupted the TgCBAP gene by homologous recombination. Parasites lacking TgCBAP are as sensitive to the effects of Conoidin A as wild-type parasites, demonstrating that TgCBAP is not the biologically relevant target of Conoidin A. However, [Delta]TgCBAP parasites are significantly smaller than wild type parasites and display a growth defect in culture. Furthermore, TgCBAP shows an unusual subcellular localization, forming small rings at the apical and basal ends of the parasite and punctate, ring-like structures around the parasite periphery. These data identify a new marker of the apical and basal subcompartments of T. gondii, reveal a potentially novel compartment along the parasite periphery, and identify TgCBAP as a determinant of parasite size that is required for a maximally efficient lytic cycle.
In T. gondii, a cyclic GMP-dependent protein kinase (TgPKG) has been experimentally validated as the biological relevant target of the small molecule inhibitor 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-y1]pyridine (Compound 1). At submicromolar concentrations, Compound 1 blocks extracellular parasite motility and host cell invasion, yet has a minimal effect on intracellular replication. Validation of TgPKG as the bona fide target of Compound 1 has strongly implicated the substrates of TgPKG in these same cellular processes. To date, no substrate of TgPKG has been identified. Identification of the TgPKG substrates involved in these processes would further our understanding of these essential events in the lytic cycle and may lead to the identification of new drug targets. We employed biochemical and quantitative phosphoproteomic analysis to identifY components of the TgPKG signaling pathway using Compound 2, a more potent structural analog of Compound 1. We discovered that Compound 2 blocks calcium and cGMP-mediated increase in TgMyoA phosphorylation in wildtype parasites, but not in parasites resistant to Compound 2. In our quantitative proteomics analysis, we also discovered a 12 candidate TgPKG substrates. These results· identify potential downstream effectors of TgPKG activity and provide a direction for future investigations into TgPKG signaling.