Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Author(s)

dos Passos, Patricia M. S. ; Spagnol, Valentine ; de Correia, Camila R. S. T. B. ; Teixeira, Felipe R.

• Document type: Journal article
• Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 183, p. 11-pg., 2022-05-01.

Abstract

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitinconjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases. (AU)

• FAPESP's process: 20/15771-6 - Functional characterization of the CRLs (Cullin RING ligases) E3 ubiquitin-ligases in Leishmania infantum
• Grantee: Felipe Roberti Teixeira
• Support Opportunities: Regular Research Grants
• FAPESP's process: 16/20258-0 - Visceral leishmaniasis: genomics approaches for integrated molecular analysis of host and parasite
• Grantee: Sandra Regina Costa Maruyama
• Support Opportunities: Research Grants - Young Investigators Grants