SUMMARY HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process, and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site, and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.

1-channel (abgENaC) regulates kidney salt-transport and blood pressure. Each ENaC subunit contains a PY motif (PPxY) and its mutation in b/gENaC causes Liddle syndrome, a hereditary hypertension. These (extended) PY motifs (PP 616 xY 618 xxL 621 ) serve as binding sites for the ubiquitin ligase Nedd4-2, which decreases cell-surface expression of ENaC by unknown route(s). Using polarized kidney epithelia [Madin-Darby canine kidney I (MDCK-I)] cells stably expressing extracellularly myc-tagged wild type (WT) or PY-motif mutants of bENaC (P616A, Y618A or L621A, with WT-agENaC), and live-imaging plus enzyme-linked immunosorbent assay (ELISA)-type assays to analyze routes/rates of ENaC internalization/recycling, we show here that cell-surface half-life of all PY mutants was fourfold longer than WT-ENaC ($120 versus 30 minutes), reflecting primarily reduced channel internalization but also attenuated replenishment of cell-surface ENaC from a large subapical pool. The Y618A mutant revealed more severe internalization and replenishment defects than the other PY mutants. Internalized WT-ENaC was detected in sorting/ recycling and late endosomes/lysosomes, while the Y618A mutant accumulated in the former. Nedd4-2 ubiquitinated ENaC at the apical membrane causing channel internalization and degradation. Cyclic AMP (cAMP) accelerated mobilization of subapical ENaC to the cell surface and long-term ENaC recycling, but only mobilization, not recycling, was inhibited in the PY mutants. These results suggest that the ENaC PY motifs (and Nedd4-2) primarily regulate channel internalization but also affect cAMPdependent replenishment, providing important insight into the Liddle syndrome defects.

Cystic fibrosis is caused by impaired ion transport due to mutated cystic fibrosis transmembrane conductance regulator, accompanied by elevated activity of the amiloride-sensitive epithelial Na + channel (ENaC). Here we show that knockout of the ubiquitin ligase Nedd4L (Nedd4-2) specifically in lung epithelia (surfactant protein C-expressing type II and Clara cells) causes cystic fibrosis-like lung disease, with airway mucus obstruction, goblet cell hyperplasia, massive inflammation, fibrosis, and death by three weeks of age. These effects of Nedd4L loss are likely caused by enhanced ENaC function, as reflected by increased ENaC protein levels, increased lung dryness at birth, amiloride-sensitive dehydration of lung explants, and elevated ENaC currents in primary alveolar type II cells analyzed by patch clamp recordings. Moreover, the lung defects were rescued with administration of amiloride into the lungs of young knockout pups via nasal instillation. Our results therefore suggest that the ubiquitin ligase Nedd4L can suppress the onset of cystic fibrosis symptoms by inhibiting ENaC in lung epithelia.

Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung.

Cystic Fibrosis is caused by mutations in CFTR, with a deletion of a phenylalanine at position 508 (F508del-CFTR) representing the most common mutation. The F508del-CFTR protein exhibits a trafficking defect and is retained in the endoplasmic reticulum. Here we describe the development of a high-content screen based on a functional assay to identify proteins that correct the F508del-CFTR defect. Using a HEK293 MSR GripTite cell line that stably expresses F508del-CFTR, we individually co-expressed ϳ450 unique proteins fused to the Cl ؊ -sensitive YFP(H148Q/I152L) mutant. We then tested correction of F508del-CFTR function by the CI ؊ /l ؊ exchange method following stimulation with forskolin/IBMX/genistein, using quantitative recordings in multiple individual cells with a high-content (high-throughput) Cellomics KSR imaging system. Using this approach, we identified several known and novel proteins that corrected F508del-CFTR function, including STAT1, Endothelin 1, HspA4, SAPK substrate protein 1, AP2M1, LGALS3/galectin-3, Trkfused gene, Caveolin 2, PAP/REG3␣, and others. The ability of these correctors to rescue F508del-CFTR trafficking was then validated by demonstrating their enhancement of maturation (appearance of band C) and by cell surface expression of F508del-CFTR bearing HA tag at the ectodomain using confocal microscopy and flow cytometry. These data demonstrate the utility of highcontent analyses for identifying proteins that correct mutant CFTR and discover new proteins that stimulate this correction. This assay can also be utilized for RNAi screens to identify inhibitory proteins that block

Nedd4 (Nedd4-1) is a Hect domain E3 ubiquitin ligase that also contains a C2 domain and three WW domains. Despite numerous in vitro studies, its biological function in vivo is not well understood. Here we show that disruption of Nedd4-1 in mice (leaving Nedd4-2 intact) caused embryonic lethality at mid gestation, with pronounced heart defects (double-outlet right ventricle and atrioventricular cushion defects) and vasculature abnormalities. Quantitative mass spectrometry and immunoblot analyses of lysates from the wild type and knock-out mouse embryonic fibroblasts to identify Nedd4-1 in vivo targets revealed dramatically increased amounts of thrombospondin-1 (Tsp-1) in the knock-out mouse embryonic fibroblasts and embryos. Tsp-1 is an inhibitor of angiogenesis, and its elevated level was mediated primarily by enhanced transcription. Interestingly, the administration of aspirin (an inhibitor of Tsp-1) to the pregnant heterozygote mothers led to a reduction in Tsp-1 levels and a substantial rescue of the embryonic lethality. These results suggest that Nedd4-1 is a suppressor of Tsp1 and that increased levels of Tsp-1 in the Nedd4-1 knock-out mice may have contributed to the developmental defect observed in the embryos.Ubiquitination regulates stability of many cellular proteins (mainly cytosolic) by tagging them for degradation by the 26 S proteasome (1). It also controls other cellular fates such as endocytosis and vesicular sorting of transmembrane proteins (2-4). The ubiquitination reaction is carried out by the sequential activation of E1, 4 E2, and E3 enzymes (1) , and HECW2) (6, 7). The WW domains of Nedd4 proteins usually bind their substrates by recognizing a short sequence, the PY motif ((L/P)PXY) (8 -10). Nedd4-2 was shown to regulate endocytosis of the epithelial Na ϩ channel by ubiquitination (11-15), an effect impaired in Liddle syndrome patients in whom the PY motif of the epithelial Na ϩ channel (binding site for Nedd4-2) is mutated. Other ion channels are also negatively regulated by . Although Nedd4-1 is more widely expressed than Nedd4-2, less is known about its function. Nedd4-1 was reported to bind to and regulate stability or vesicular sorting of several proteins, including CNrasGEF (21); LAPTM5 (22); the viral proteins HTLV-1 Gag (23), Marburg/Ebola (24 -26), and MLV Gag (27, 28); AP-1 (27); APOBEC3G (29); Grb10 (30, 31); and c-Cbl (32). Also, the Drosophila homolog of Nedd4-1, dNedd4, was recently shown to regulate neuromuscular synaptogenesis in flies by binding to commissureless and promoting its endocytosis (33). Nedd4-1 was also shown to indirectly regulate the function of the endocytic protein Eps15 (34). The proposed role of Nedd4-1 in regulating PTEN stability and nuclear translocation (35, 36) was recently challenged (37).Despite the numerous reports published on Nedd4-1 over the past decade, its in vivo function(s) in mammals are less clear (see "Discussion"). Here we describe the knock-out of Nedd4-1 in mice. We show that these mice exhibit severe cardiac and some vascular defects...

mTORC1 regulates cellular growth and is activated by growth factors and by essential amino acids such as Leu. Leu enters cells via the Leu transporter LAT1-4F2hc (LAT1). Here we show that the Na + /K + / 2Cl À cotransporter NKCC1 (SLC12A2), a known regulator of cell volume, is present in complex with LAT1. We further show that NKCC1 depletion or deletion enhances LAT1 activity, as well as activation of Akt and Erk, leading to activation of mTORC1 in cells, colonic organoids, and mouse colon. Moreover, NKCC1 depletion reduces intracellular Na + concentration and cell volume (size) and mass and stimulates cell proliferation. NKCC1, therefore, suppresses mTORC1 by inhibiting its key activating signaling pathways. Importantly, by linking ion transport and cell volume regulation to mTORC1 function, NKCC1 provides a long-sought link connecting cell volume (size) to cell mass regulation.

No abstract