Abstract
Protein-protein interactions critically regulate many biological systems, but quantifying functional assembly of multipass membrane complexes in their native context is still challenging. Here, we combined modeling-assisted protein modification and information from human disease variants with a minimal-size fusion tag, split-luciferase-based approach to probe assembly of the NADPH oxidase 4 (NOX4)-p22phox enzyme, an integral membrane complex with unresolved structure, which is required for electron transfer and generation of reactive oxygen species (ROS). Integrated analyses of heterodimerization, trafficking, and catalytic activity identified determinants for the NOX4-p22phox interaction, such as heme incorporation into NOX4 and hot spot residues in transmembrane domains 1 and 4 in p22phox Moreover, their effect on NOX4 maturation and ROS generation was analyzed. We propose that this reversible and quantitative protein-protein interaction technique with its small split-fragment approach will provide a protein engineering and discovery tool not only for NOX research, but also for other intricate membrane protein complexes, and may thereby facilitate new drug discovery strategies for managing NOX-associated diseases.
Highlights
Protein–protein interactions (PPIs)4 are vital for the regulation of biological systems
The ability to transfer electrons from NADPH across the membrane to molecular oxygen resides in several distinct features of the catalytic NOX domain but requires in all NADPH oxidases heterodimerization with specific integral membrane proteins (p22phox, DUOXA1–2)
The partner protein p22phox can be modified at the C terminus without loss of expression or its ability to heterodimerize with NOX2 [8]
Summary
Understanding NOX4 heterodimerization is hindered by lack of structural information. We and others have attached tags at the enzyme’s N or C termini. The model suggests that C-terminal fusions, such as the combination NOX4LB1/p22-SB1, will form a complex This interaction was confirmed, albeit, as expected from the earlier GFP studies (Fig. 1), with markedly reduced catalytic activity, protein expression and cell-surface localization were comparable with catalytically active SB1-NOX4/p22-LB2 (Fig. 2, G–J). In contrast to N-terminal GFP-NOX4 [7], introduction of LB at the NOX4 N terminus (LB2-NOX4/p22-SB1) resulted in significantly reduced PPI and H2O2 generation (Fig. 2, K and L), indicating that SB versus LB orientation and linker length as well as the choice of fusion protein are critical aspects for NanoBiT association and NOX4 activity. Treatment of SB1-NOX4/p22-LB2– expressing cells with the heme synthesis inhibitor succinyl acetone reduced luminescence and H2O2 generation (not shown) These results indicate that NOX4 follows the NOX2 paradigm, where heme incorporation is a prerequisite for heterodimerization [21]. The p22phox TMH4 penultimate amino acid residues discriminate between NOX isoforms and affect selectively heterodimerization with p22phox, whereas changes in the p22phox Gly-24 region (TMH1) influence heterodimerization with NOX4, NOX2, and probably NOX1/3
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