Ubiquitin specific proteases 1 (USP1) – potential partner of Bcr-Abl oncoprotein: bioiformatic analysis and recombinant constructs creation

  • С. В. Антоненко Institute of Molecular Biology and Genetics of NAS of Ukraine Ukraine, 03680, Kyiv, Akad. Zabolotnogo str., 150
  • І. В. Кравчук Institute of Molecular Biology and Genetics of NAS of Ukraine Ukraine, 03680, Kyiv, Akad. Zabolotnogo str., 150
  • Д. С. Гур'янов Institute of Molecular Biology and Genetics of NAS of Ukraine Ukraine, 03680, Kyiv, Akad. Zabolotnogo str., 150
  • Г. Д. Телегєєв Institute of Molecular Biology and Genetics of NAS of Ukraine Ukraine, 03680, Kyiv, Akad. Zabolotnogo str., 150

Abstract

Aims. The result of translocation between chromosomes 9 and 22 is Bcr-Abl oncoprotein which causes the development of chronic myeloid leukemia (CML). According to preliminary research with the use of mass-spectrometry USP1 protein was identified as potential candidate for interaction with Bcr-Abl oncoprotein. The aim of this work is bioinformatic analysis of USP1 protein as a potential partner of Bcr-Abl; development of genetic constructs for studying of protein-protein interactions between USP1 and Bcr-Abl proteins, Bcr-Abl deubiquitination and USP1 localization. Methods. Bioinformatic analysis (using programs Estimation of protein Expression and Solubility, NetPhos2.0 Server, Disphos and KinasePhos), PCR, ligation, restriction, isolation and purification of DNA. Results. The 68% of protein solubility of USP1 was determined.  Conserved and disordered region were identified, secondary structure of protein was predicted. We found that phosphorylation of USP1 protein may occur at Ser, Thr, Tyr residues. We developed pUC18-USP, pECFP-C3-USP1 and pCMV-HA-USP1 vectors. Conclusions. The 68 % solubility of the protein is a good prediction of outcome for futher experiments. Availability of phosphorylation sites (Ser, Thr, Tyr) may confirm the hypothesis of activation of USP1 by Bcr-Abl. pECFP-C3-USP1 vector will be used for detection of USP1 in mammalian cells. Moreover, pCMV-HA-USP1 vector was developed for detection of protein-protein interactions between USP1 and Bcr-Abl and for estimation of deubiquitination state of Bcr-Abl oncoprotein.

Key words: chronic myeloid leukemia (CML), Bcr-Abl protein, USP1 protein, deubiquitination phosphorylation.