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UBC Theses and Dissertations

Assaying the function of human variants found in SMAD4 and BMPR1A using Drosophila melanogaster Pisio, Amanda

Abstract

This thesis describes two projects that examined protein function in neural plasticity and cancer development. The first project created assays that tested the pathogenicity of human variants identified in SMAD4 and BMPR1A, genes that are associated with juvenile polyposis syndrome. As exome sequencing becomes easier and more cost-effective, many human variants are being identified. However, for most variants, their impact on protein function and their ability to cause disease are unknown. Drosophila melanogaster offers an efficient system for testing human variant protein functionality in a panel of assays to screen through many variants. I have used simple overexpression assays in Drosophila to test human SMAD4 and BMPR1A variants. I developed two assays in which wildtype SMAD4, but not loss of function variants, caused either lethality or wing vein defects. I screened through seven human SMAD4 variants implicated in disease to assess their relative function and identified four that exhibit functional differences to wildtype. I also tested human BMPR1A but found that overexpression of this gene in Drosophila had no effect. I postulated this is due to a lack of ligand binding. Therefore, I created reagents for alternative methods to screen BMPR1A variant function. First, I generated mimetic mutations in the orthologous tkv gene. Second, I created a chimeric gene comprising the extracellular domain of Tkv and the intracellular domain of BMPR1A. I postulate that this chimera should bind Drosophila BMP ligands and activate canonical BMP signaling, allowing for assays of BMPR1A variants in the intracellular domain. These reagents and assays are important for experimentally determining ariant activity and for improving our understanding of structure/function relationships for SMAD4 and BMPR1A. Going forward, functionally testing large numbers of variants will inform personalized medicine approaches and improve computer models for projecting pathogenicity of human variants. In the second project, I created CalpA and CalpB double mutants to test whether a reduction of calpain activity could stimulate de novo neurite formation. Also, I overexpressed a proteolytic target of Calpain, Cortactin, and created a Calpain proteolysis resistant version of Cortactin. Surprisingly, I was unable to identify any phenotype in the nervous system.

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