Production of capturing agents to study ubiquitination during epithelial to mesenchymal transition

• Grant number: 21/03575-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2021
• End date: December 31, 2022
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Vitor Marcel Faça
• Grantee: Izadora Archiolli
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Cancer is a set of multifactorial diseases that generate disordered cell growth, forming malignant tumors that can invade adjacent tissues or distant organs, through the process of metastasis, which is responsible for the lethality of the disease. One of the molecular mechanisms that occurs during metastasis is the epithelial-mesenchymal transition (EMT). EMT is controlled by multiple factors that trigger the cells' ability to detach from a primary tumor mass, fall into the circulation and create secondary sites of the disease. Some studies indicate that post-translational modification of proteins ubiquitination is closely related to EMT, favoring or preventing tumor progression. For all these reasons, we intend to use a specific capture strategy for ubiquitinated proteins through the production of an antibody for immunoaffinity purification. In order to accomplish this goal, we will synthesize the peptide with 13 amino acids ESTLHLVLRLRGG, which represents the C-terminal of ubiquitin and use it as an antigen to initially obtain polyclonal antibodies as well as synthetic antibodies obtained by the "phage-dysplay" technology. These affinity agents will be used to explore two in vitro cell models of EMT, derived from mammary and ovarian glands adenocarcinoma. The cell models will be induced to the EMT process, with monitoring of the process biomarkers by western blotting assays and functional cell migration assays. Proteins extracted from these models will pass through the isolation stage of ubiquitinated targets due to immunoaffinity and, finally, they will be undergone a large-scale proteomic analysis and also directed proteomics using the technique of monitoring multiple reactions. Therefore, it will be possible to identify proteins different involved in the EMT process and eventually the specific enzymes that control such ubiquitination. This study may reveal new targets for detecting tumor progression and potentially new targets for pharmacological inhibition of EMT or for tumor progression.