Direct Molecular Fishing of Protein Partners for Proteins Encoded by Genes of Human Chromosome 18 in HepG2 Cell Lysate

Abstract

The aim of this work was to identify the spectrum of possible protein-protein interactions (PPI) for six target proteins (CYB5A, RAB27B, SMAD4, CXXC1, RNMT, ТТR) encoded by genes of human chromosome 18, which have a certain medical significance. For this purpose, a comprehensive approach based on the combined application of three technologies, direct molecular fishing based on affinity chromatography, protein mass spectrometry and SPR (surface plasmon resonance) analysis, was used. As a result, from 1 to 11 potential protein partners were isolated from the lysate of the human hepatocellular carcinoma HepG2 cell line for each target protein. Using the Biacore 3000 SPR biosensor, 10 potential PPIs, for which preparations of recombinant proteins were available, were validated. Positive results, which confirm direct interaction of the identified protein partners with target proteins, were obtained for five protein pairs (СYB5A/CPR, СYB5A/CYP2C9, СYB5A/CYCS, CPR/СYP2C9 and CXXC1/CYCS). The values of equilibrium dissociation constants of protein complexes (K_d values ranged from 10^–7 M to 10^–5 M) were determined for these PPIs. To evaluate the specificity of identified PPIs, 16 additional different PPIs of the target proteins were tested. The effectiveness of direct molecular fishing was evaluated using cytochrome b₅ (CYB5A) as the most studied target protein in the context of structural and functional relationships with its protein partners. The new data extend our knowledge in the field of interactomics of proteins encoded by human chromosome 18 genes and reduces the number of unverified bioinformatic predictions of possible PPIs presented in accessible Internet resources.