No abstract

There is increasing evidence that proteins function in the cell as integrated stable or temporally formed protein complexes, interactomes. Previously, using model systems we demonstrated applicability of direct molecular fishing on paramagnetic particles for protein interactomics (Ershov et al. Proteomics, 2012, 12, 3295). In the present study, we have used a combination of affinity-based molecular fishing and subsequent MS for investigation of human liver proteins involved in interactions with immobilized microsomal cytochrome b5 (CYB5A), and also transthyretin and BSA as alternative affinity ligands (baits). The LC-MS/MS identification of prey proteins fished on these baits revealed three sets of proteins: 98, 120, and 220, respectively. Comparison analysis of these sets revealed only three proteins common for all the baits. In the case of paired analysis, the number of common proteins varied from 2 to 9. The binding capacity of some identified proteins has been validated by a SPR-based biosensor. All the investigated proteins effectively interacted with the immobilized CYB5A (Kd values ranged from 0.07 to 1.1 μM). Results of this study suggest that direct molecular fishing is applicable for analysis of protein-protein interactions (PPI) under normal and pathological conditions, in which altered PPIs are especially important.

No abstract

To understand the role of the structural elements of cytochrome b5 in its interaction with cytochrome P450 and the catalysis performed by this heme protein, we carried out comparative structural and functional analysis of the two major mammalian forms of membrane-bound cytochrome b5 - microsomal and mitochondrial, designed chimeric forms of the heme proteins in which the hydrophilic domain of one heme protein is replaced by the hydrophilic domain of another one, and investigated the effect of the highly purified native and chimeric heme proteins on the enzymatic activity of recombinant cytochromes P4503A4 and P45017A1 (CYP3A4 and CYP17A1). We show that the presence of a hydrophobic domain in the structure of cytochrome b5 is necessary for its effective interaction with its redox partners, while the nature of the hydrophobic domain has no significant effect on the ability of cytochrome b5 to stimulate the activity of cytochrome P450-catalyzed reactions. Thus, the functional properties of cytochrome b5 are mainly determined by the structure of the heme-binding domain.