Investigating the Mechanism and Mode of Action of Golgi Toxins

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dc.contributor.advisorLiszczak, Glenen
dc.contributor.committeeMemberPosner, Bruce A.en
dc.contributor.committeeMemberWang, Feien
dc.contributor.committeeMemberNijhawan, Deepaken
dc.creatorCervantes, Christopher Luisen
dc.creator.orcid0000-0001-7351-6954
dc.date.accessioned2024-01-11T20:20:38Z
dc.date.available2024-01-11T20:20:38Z
dc.date.created2023-12
dc.date.issuedDecember 2023
dc.date.submittedDecember 2023
dc.date.updated2024-01-11T20:20:39Z
dc.description.abstractAuxin-inducible forward genetics uncovered point mutations within Golgi Brefeldin A Resistant Guanine Nucleotide Exchange Factor 1 or GBF1 following lethal dose selection with a synthetic disubstituted pyrimidine toxin called Golgitox (GTOX). Resistant clones were also cross-resistant to the fungal toxin, Brefeldin A (BFA), and synthetic GBF1 inhibitor, Golgicide A (GCA). Like BFA and GCA, GTOX triggered Golgi disassembly via GBF1. Given that BFA is a reported molecular glue, we profiled Gbf1-Arf interactions in 293T cell lysates pre-treated with Golgi toxin. Both GTOX and GCA promoted GBF1-dependent interactions with Arfs 4 and 5, whereas BFA also interacted with Arf1. GBF1 domain mapping revealed that the HUS-SEC7-HDS1 domains were sufficient for promoting GTOX-dependent engagement with Arfs 4 and 5. Meanwhile, structural activity relationship studies showed that modifying the methyl group on the benzimidazole ring preserved GTOX activity and interactions between Gbf1 and Arfs 4 and 5. To assess which Arfs regulate BFA and GTOX cytotoxicities, genome-wide CRISPR/Cas9 compound enrichment screens were carried out, which identified ARF4 as being the most enriched hit. Next, we validated that ARF4 loss-of-function partially confers resistance to BFA and GTOX. Next, we asked whether GTOX preferentially interacts with ARF4-GDP versus ARF4-GTP. We found that exogenous ARF4 T31N (GDP-locked mutant) sensitized HCT116 scramble control cells 4-fold to GTOX, interacted with Gbf1 just as well as WT Arf4, but failed to rescue Arf4-mediated BiP retrieval. Collectively, these results suggest that the Gbf1-GTOX-Arf4-GDP complex is functionally inactive but deleterious to cell viability. Taken together, GTOX may act as a molecular glue to suppress GBF1 functions through downstream effector substrates like Arfs 4 and 5.en
dc.format.mimetypeapplication/pdfen
dc.identifier.oclc1417098722
dc.identifier.urihttps://hdl.handle.net/2152.5/10240
dc.language.isoenen
dc.subjectADP-Ribosylation Factorsen
dc.subjectGolgi Apparatusen
dc.subjectGuanine Nucleotide Exchange Factorsen
dc.titleInvestigating the Mechanism and Mode of Action of Golgi Toxinsen
dc.typeThesisen
dc.type.materialtexten
thesis.degree.departmentGraduate School of Biomedical Sciencesen
thesis.degree.disciplineBiological Chemistryen
thesis.degree.grantorUT Southwestern Medical Centeren
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen

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