From the apparent non-overlapping phenotypes of the Nedd4 and Nedd4-2 gene KO mice it is clear that the main substrates of these E3s are distinct, despite the very close similarity between these two ubiquitin ligases. Although Nedd4 and Nedd4-2 show very similar binding affinities for the same substrates in vitro (e.g., both bind all three ENaC subunits in vitro with high affinity), mouse data suggest that in vivo their substrate binding may be more restricted than envisaged. Many of the channels, transporters, and other potential targets of Nedd4 and Nedd4-2 () would need further validation, perhaps by the use of cells and tissues derived from the KO mice. Given the perinatal lethality of Nedd4 KO animals, it may be necessary to generate conditional KO mice to enable the study of physiological targets of Nedd4. It is possible that in some cases the functions of Nedd4 and Nedd4-2 are redundant. Thus, it may be informative to generate double KO mice.
It has been shown that accessory and adaptor proteins contribute to the specificity, diversity, and overall function of both Nedd4 and Nedd4-2.13
Identification and biochemical characterization of the interactomes for both Nedd4 and Nedd4-2 would facilite our understanding of these E3s and their substrates. To that end, systemic approaches using the current proteomic and bioinformatic techniques will be needed to generate the much desired information. The challenges for these approaches will include the low abundance of endogenous Nedd4 or Nedd4-2 and the potential false interactions sometimes associated with overexpression systems. It may be advantageous to tag the endogenous Nedd4 or Nedd4-2 using techniques, such as the TAP (tamdem affinity purification) tag.87
Nedd4 protein is known to be cleaved by caspases during apoptosis.88
The cleavage of Nedd4 removes the N-terminal C2 domain from the rest of the protein without disrupting the WW domains and the HECT. The removal of the N-terminal region of Nedd4 was reported to make the protein unstable, leading to the degradation of the WW–HECT cleavage product.88
As the functional significance of Nedd4 cleavage and degradation during apoptosis remains unknown, it may be interesting to study the apoptotic response of Nedd4-deficient cells from the KO animal.
Despite some recent advances, the regulation of Nedd4 and Nedd4-2 is still not fully understood. For example, many kinases seem to phosphorylate Nedd4 and Nedd4-2, but in most cases physiological relevance and link to cellular signaling seem unclear. Several proteins, such as Ndfip1 and Ndfip2, can bind both Nedd4 and Nedd4-2. However, it is not known whether the binding occurs in vivo
and whether the adaptors are used by Nedd4 and/or Nedd4-2 to facilitate substrate recruitment and protein trafficking. In yeast S. cerevisiae
Rsp5, the only Nedd4-like protein, uses many adaptor proteins, in addition to the Ndfip-like protein, Bsd2.13,89
One recently identified group of proteins, arrestin-related proteins (ARTS), facilitates Rsp5 recruitment to plasma membrane proteins.90,91
Rsp5 then ubiquitylates specific targets at the membrane leading to their endocytosis. Further cellular and biochemical studies will be necessary to establish whether Nedd4 and Nedd4-2 also use ARTS in mammalian cells to recognize and bind their targets at the plasma membrane.