Orsolya Mózner’s PhD thesis investigates how genetic variants influence the molecular regulation and function of clinically relevant membrane proteins. The candidate applied molecular and cell biology tools to study how mutations and polymorphisms impact the protein expression, localization, and activity of two human membrane proteins, ABCG2 and PMCA4b, and the SARS-CoV-2 spike protein.
The first part of the thesis focuses on the ABCG2 multidrug transporter. The candidate examined the effects of the naturally occurring K360del variant, located in an unstructured cytoplasmic loop of ABCG2 by overexpressing and studying ABCG2 variants in HEK293, HeLa and MDCKII cells. Functional assays showed that this variant does not impair transporter activity or membrane localization and may even enhance trafficking and cell surface expression.
The second project investigated the PMCA4b calcium pump, encoded by the ATP2B4 gene, in the context of a malaria-associated genetic haplotype. To evaluate erythroid-specific regulatory effects, the candidate used K562 and HEL92 erythroid cell lines alongside HEK293 as a non-erythroid control. Dual-luciferase assays demonstrated that SNPs within the haplotype reduce promoter activity specifically in erythroid cells, likely through the disrupted binding of the GATA-1 transcription factor. These findings provide a molecular explanation for reduced PMCA4b expression in red blood cells linked to the malaria-associated haplotype.
The third project addresses the SARS-CoV-2 spike protein RBD (receptor binding domain) variants. The candidate generated stable suspension HEK293 cell lines to express the SARS-CoV-2 spike RBD corresponding to different viral variants. These products were purified and used in an ELISA assay to detect anti-RBD antibody levels in the sera of vaccinated individuals and COVID-19 patients. The results showed high inter-individual variability, declining antibody levels over time, and cross-reactivity with Omicron variants, demonstrating the assay’s potential for monitoring immune responses against emerging variants.
These studies apply a shared molecular and cell biology toolkit to investigate molecular mechanisms regulating membrane proteins and their variants, with implications in pharmacogenetics, infectious disease biology and the development of diagnostic assays. The insights gained contribute to a deeper understanding of how clinically relevant molecular variations can shape protein regulation and function.
