Monitoring of cellular trafficking of prostate membrane antigen through its biolabeling with non-canonical amino acids and protein cellular trafficking synchronization techniques.

• Grant number: 25/13036-0
• Support Opportunities: Scholarships in Brazil - Master
• Start date: November 01, 2025
• End date: October 31, 2027
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Grantee: Beatriz Clini de Souza
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 23/09167-7 - Engineering antibodies and bacteria for tumor diagnosis and therapeutics., AP.R

Abstract

Prostate-specific membrane antigen (PSMA) is a transmembrane protein frequently expressed in prostate cancer cells and used as a target in diagnostic and therapeutic (theranotic) strategies. PSMA is believed to be internalized by clathrin-mediated endocytosis, a characteristic that makes it a promising target for the selective delivery of therapeutic agents. The effect that different PSMA ligands (inhibitors and antibodies, for example) have on this cellular internalization event is unknown, and the main objective of this project is precisely to study the cellular trafficking of this protein in the absence or presence of anti-PSMA antibodies (nanobodies), focusing on the study of the average residence time that this molecule remains in the cytoplasmic membrane. In an attempt to study this cellular trafficking through fluorescence microscopy assays, we modified cultured cells to express PSMA fused to a fluorescent protein (eGFP). However, we found that this PSMA-eGFP fusion protein is retained in the endoplasmic reticulum (ER), probably due to the size of the resulting fusion protein. We now propose replacing this eGFP with a much smaller fluorescent probe, thus minimizing the chances of interference in normal PSMA trafficking. This probe will be a non-canonical amino acid residue that will be bioincorporated into PSMA during its biosynthesis within the cells used in the study. We also intend to improve the quality of this study by synchronizing PSMA trafficking using the RUSH (Retention Using Selective Hooks) system, which allows the controlled release of a population of protein molecules from the ER (labeled PSMA in our case) by adding biotin to the culture medium. Additionally, we will evaluate whether specific PSMA ligands, such as the NB7 nanobody that we have been studying as a drug carrier for PSMA+ cells, modulate their permanence in the plasma membrane. The analyses will be conducted in genetically modified HeLa cells, analyzed by fluorescence microscopy. This study integrates synthetic biology and protein engineering tools, with potential application in targeted therapies against prostate cancer.