Deletion of Nedd4-2 results in progressive kidney disease in mice.

NEDD4-2 (NEDD4L), a ubiquitin protein ligase of the Nedd4 family, is a key regulator of cell surface expression and activity of the amiloride-sensitive epithelial Na+ channel (ENaC). While hypomorphic alleles of Nedd4-2 in mice show salt-sensitive hypertension, complete knockout results in pulmonary distress and perinatal lethality due to increased cell surface levels of ENaC. We now show that Nedd4-2 deficiency in mice also results in an unexpected progressive kidney injury phenotype associated with elevated ENaC and Na+Cl- cotransporter expression, increased Na+ reabsorption, hypertension and markedly reduced levels of aldosterone. The observed nephropathy is characterized by fibrosis, tubule epithelial cell apoptosis, dilated/cystic tubules, elevated expression of kidney injury markers and immune cell infiltration, characteristics reminiscent of human chronic kidney disease. Importantly, we demonstrate that the extent of kidney injury can be partially therapeutically ameliorated in mice with nephron-specific deletions of Nedd4-2 by blocking ENaC with amiloride. These results suggest that increased Na+ reabsorption via ENaC causes kidney injury and establish a novel role of NEDD4-2 in preventing Na+-induced nephropathy. Contrary to some recent reports, our data also indicate that ENaC is the primary in vivo target of NEDD4-2 and that Nedd4-2 deletion is associated with hypertension on a normal Na+ diet. These findings provide further insight into the critical function of NEDD4-2 in renal pathophysiology.

Placing a disrupted degradation motif at the C terminus of proteasome substrates attenuates degradation without impairing ubiquitylation.

Protein elimination by the ubiquitin-proteasome system requires the presence of a cis-acting degradation signal. Efforts to discern degradation signals of misfolded proteasome substrates thus far revealed a general mechanism whereby the exposure of cryptic hydrophobic motifs provides a degradation determinant. We have previously characterized such a determinant, employing the yeast kinetochore protein Ndc10 as a model substrate. Ndc10 is essentially a stable protein that is rapidly degraded upon exposure of a hydrophobic motif located at the C-terminal region. The degradation motif comprises two distinct and essential elements: DegA, encompassing two amphipathic helices, and DegB, a hydrophobic sequence within the loosely structured C-terminal tail of Ndc10. Here we show that the hydrophobic nature of DegB is irrelevant for the ubiquitylation of substrates containing the Ndc10 degradation motif, but is essential for proteasomal degradation. Mutant DegB, in which the hydrophobic sequence was disrupted, acted as a dominant degradation inhibitory element when expressed at the C-terminal regions of ubiquitin-dependent and -independent substrates of the 26S proteasome. This mutant stabilized substrates in both yeast and mammalian cells, indicative of a modular recognition moiety. The dominant function of the mutant DegB provides a powerful experimental tool for evaluating the physiological implications of stabilization of specific proteasome substrates in intact cells and for studying the associated pathological effects.

Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope.

Proper functioning of the protein-folding quality control network depends on the network's ability to discern diverse structural perturbations to the native states of its protein substrates. Despite the centrality of the detection of misfolded states to cell home-ostasis, very little is known about the exact sequence and structural features that mark a protein as being misfolded. To investigate these features, we studied the requirements for the degradation of the yeast kinetochore protein Ndc10p. Mutant Ndc10p is a substrate of a protein-folding quality control pathway mediated by the E3 ubiquitin (Ub) ligase Doa10p at the endoplasmic reticulum (ER)/nuclear envelope membrane. Analysis of Ndc10p mutant derivatives, employing a reverse genetics approach, identified an autonomous quality control-associated degradation motif near the C-terminus of the protein. This motif is composed of two indispensable hydrophobic elements: a hydrophobic surface of an amphipathic helix and a loosely structured hydrophobic C-terminal tail. Site-specific point mutations expose these elements, triggering ubiquitin-mediated and HSP70 chaperone-dependent degradation of Ndc10p. These findings substantiate the ability of the ER quality control system to recognize subtle perturbation(s) in the native structure of a nuclear protein.