Study on ubiquitination of proteins of the MRPS18 family in vitro

Abstract

Aim. It is known that in cancerous cells of childhood tumors the pathological changes often include inactivation of the TP53 and RB-E2F1 cellular pathways. One of the proteins controlling the latter pathway is MRPS18-2, that belongs to a family of mitochondrial ribosomal proteins MRPS18. It is important, to study the stability of proteins of this family and their ubiquitination, that might help to conclude about the functional properties of these proteins and their role in cell transformation. Methods. Cloning of cDNA in FLAG vector for expression of fusion proteins, transfection of human tumor cells MCF7, study on cellular localization of MRPS18 family proteins and their ubiquitination by fluorescence microscopy, using specific antibodies. Results. The FLAG-MRPS18-1 and FLAG-MRPS18-3 fusion proteins are partially co-localizing with the HA-Ub fusion protein in the cytoplasm of MCF7 cells. The FLAG-MRPS18-2 protein is localized also in the nucleus. Conclusions. Nuclear localization of the FLAG-MRPS18-2 protein may indicate its additional functions in the cell: due to the interaction with the RB protein and the positive effect on mono-ubiquitination of histone H2B, the MRPS18-2 protein may be involved in the regulation of chromatin structure.