Interaction Studies Between Human Nedd4 and the Human Epithelial Sodium Channel: and Identification of Novel Binding Partners of Nedd4

Abstract

The activity of the epithelial Na+ channel (ENaC) is required for the maintenance of salt and water balance in the body. Regulation of channel activity by the ubiquitin-protein ligase Nedd4, which interacts with the channel via its WW domains, has previously been shown in rodent studies. Mutations in channel subunits that disrupt this interaction cause Liddle’s syndrome, a severe inherited form of hypertension. The interaction between human Nedd4 and human ENaC subunits has been confirmed via in vitro binding assays and characterised using surface plasmon resonance and molecular modelling studies. We show that WW domains 2, 3 and 4 of human Nedd4 bound to the human ENaC subunits, whereas WW domain 1 did not bind. The binding affinities of the hNedd4 WW domains for hENaC C-terminal peptides have also been determined. We show that WW domains 2, 3 and 4 bind with differing affinities to Na+ channel subunit peptides, with the unique human WW domain 3 hypothesised to contribute most of the binding. Further, a single amino acid change (Arg2l6Thr) in WW1 enables binding to the α subunit of the channel to occur, with an affinity comparable to that of WW domain 4. The differences found in binding affinities between the various WW domains and Na+ channel subunit peptides are explained based on quantitative structural modelling of the complexes and their isolated components. An understanding of this interaction is imperative in the comprehension of the control of human hypertension. Additional binding partners of human Nedd4 have been identified via the use of MALDI-TOF analysis of proteins bound to hNedd4 WW domain-fusion proteins. Confirmation of one of these binding proteins, Bc110, has been achieved via in vitro and in vivo binding assays, and functionality assays of this interaction, and investigation of the function of one of the other identified proteins. VDAC1, suggest a role of hNedd4 as an anti-apoptotic agent. This study demonstrates the importance of multiple experimental protocols in the study of protein-protein interactions, and validates the inclusion of SPR, molecular modelling, and MALDI-TOF analyses in this approach. It also illustrates the extensive role that hNedd4 plays in cellular regulation, and indicates that further investigation of hNedd4 function is of vital importance, not only in understanding human hypertension, but also in a multitude of other normal and disease states.