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dc.contributor.advisorCelia A. Schiffer, PhD
dc.contributor.authorSilvas, Tania V.
dc.date2022-08-11T08:08:46.000
dc.date.accessioned2022-08-23T16:07:54Z
dc.date.available2022-08-23T16:07:54Z
dc.date.issued2018-01-31
dc.date.submitted2018-02-27
dc.identifier.doi10.13028/M2KD6T
dc.identifier.urihttp://hdl.handle.net/20.500.14038/32340
dc.description.abstractAnalyzing protein tertiary structure is an effective method to understanding protein function. In my thesis study, I aimed to understand how surface features of protein can affect the stability and specificity of enzymes. I focus on 2 proteins that are involved in human disease, Profilin (PFN1) and APOBEC3A (A3A). When these proteins are functioning correctly, PFN1 modulates actin dynamics and A3A inhibits retroviral replication. However, mutations in PFN1 are associated with amyotrophic lateral sclerosis (ALS) while the over expression of A3A are associated with the development of cancer. Currently, the pathological mechanism of PFN1 in this fatal disease is unknown and although it is known that the sequence context for mutating DNA vary among A3s, the mechanism for substrate sequence specificity is not well understood. To understand how the mutations in Profilin could lead to ALS, I solved the structure of WT and 2 ALS-related mutants of PFN1. Our collaborators demonstrated that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization was illuminated by my X-ray crystal structures of several PFN1 proteins. I found an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis. To characterize A3A’s substrate specificity, we solved the structure of apo and bound A3A. I then used a systematic approach to quantify affinity for substrate as a function of sequence context, pH and substrate secondary structure. I found that A3A preferred ssDNA binding motif is T/CTCA/G, and that A3A can bind RNA in a sequence specific manner. The affinity for substrate increased with a decrease in pH. Furthermore, A3A binds tighter to its substrate binding motif when in the loop region of folded nucleic acid compared to a linear sequence. This result suggests that the structure of DNA, and not just its chemical identity, modulates A3 affinity and specificity for substrate.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectAPOBEC3 deoxycytidine deaminases
dc.subjectprofilin
dc.subjectamyotrophic lateral sclerosis
dc.subjectALS
dc.subjectprotein tertiary structure
dc.subjectBiochemistry
dc.subjectEnzymes and Coenzymes
dc.subjectMedicinal-Pharmaceutical Chemistry
dc.subjectNervous System Diseases
dc.subjectStructural Biology
dc.titleInvestigating the Structural Basis for Human Disease: APOBEC3A and Profilin
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1962&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/955
dc.legacy.embargo2020-02-27T00:00:00-08:00
dc.identifier.contextkey11661955
refterms.dateFOA2022-08-24T06:25:13Z
html.description.abstract<p>Analyzing protein tertiary structure is an effective method to understanding protein function. In my thesis study, I aimed to understand how surface features of protein can affect the stability and specificity of enzymes. I focus on 2 proteins that are involved in human disease, Profilin (PFN1) and APOBEC3A (A3A). When these proteins are functioning correctly, PFN1 modulates actin dynamics and A3A inhibits retroviral replication. However, mutations in PFN1 are associated with amyotrophic lateral sclerosis (ALS) while the over expression of A3A are associated with the development of cancer. Currently, the pathological mechanism of PFN1 in this fatal disease is unknown and although it is known that the sequence context for mutating DNA vary among A3s, the mechanism for substrate sequence specificity is not well understood.</p> <p>To understand how the mutations in Profilin could lead to ALS, I solved the structure of WT and 2 ALS-related mutants of PFN1. Our collaborators demonstrated that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization was illuminated by my X-ray crystal structures of several PFN1 proteins. I found an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.</p> <p>To characterize A3A’s substrate specificity, we solved the structure of apo and bound A3A. I then used a systematic approach to quantify affinity for substrate as a function of sequence context, pH and substrate secondary structure. I found that A3A preferred ssDNA binding motif is T/CTCA/G, and that A3A can bind RNA in a sequence specific manner. The affinity for substrate increased with a decrease in pH. Furthermore, A3A binds tighter to its substrate binding motif when in the loop region of folded nucleic acid compared to a linear sequence. This result suggests that the structure of DNA, and not just its chemical identity, modulates A3 affinity and specificity for substrate.</p>
dc.identifier.submissionpathgsbs_diss/955
dc.contributor.departmentBiochemistry and Molecular Pharmacology
dc.description.thesisprogramBiochemistry and Molecular Pharmacology
dc.identifier.orcid0000-0002-8174-2633


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