NEDD4

Abstract: Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.

Abstract: Ebola virus budding is mediated by the VP40 matrix protein. VP40 can bud from mammalian cells independent of other viral proteins, and efficient release of VP40 virus-like particles (VLPs) requires interactions with host proteins such as tsg101 and Nedd4, an E3 ubiquitin ligase. Ubiquitin itself is thought to be exploited by Ebola virus to facilitate efficient virus egress. Disruption of VP40 function and thus virus budding remains an attractive target for the development of novel antiviral therapies. Here, we investigate the effect of ISG15 protein on the release of Ebola VP40 VLPs. ISG15 is an IFN-inducible, ubiquitin-like protein expressed after bacterial or viral infection. Our results show that expression of free ISG15, or the ISGylation system (UbE1L and UbcH8), inhibits budding of Ebola virus VP40 VLPs. Addressing the molecular mechanism of this inhibition, we show that ISG15 interacts with Nedd4 ubiquitin ligase and inhibits ubiquitination of VP40. Furthermore, the L-domain deletion mutant of VP40 (ΔPT/PY), which does not interact with Nedd4, was insensitive to ISG15-mediated inhibition of VLP release. These data provide evidence of antiviral activity of ISG15 against Ebola virus and suggest a mechanism of action involving disruption of Nedd4 function and subsequent ubiquitination of VP40.

Abstract: α-Synuclein is an abundant brain protein that binds to lipid membranes and is involved in the recycling of presynaptic vesicles. In Parkinson disease, α-synuclein accumulates in intraneuronal inclusions often containing ubiquitin chains. Here we show that the ubiquitin ligase Nedd4, which functions in the endosomal–lysosomal pathway, robustly ubiquitinates α-synuclein, unlike ligases previously implicated in its degradation. Purified Nedd4 recognizes the carboxyl terminus of α-synuclein (residues 120–133) and attaches K63-linked ubiquitin chains. In human cells, Nedd4 overexpression enhances α-synuclein ubiquitination and clearance by a lysosomal process requiring components of the endosomal-sorting complex required for transport. Conversely, Nedd4 down-regulation increases α-synuclein content. In yeast, disruption of the Nedd4 ortholog Rsp5p decreases α-synuclein degradation and enhances inclusion formation and α-synuclein toxicity. In human brains, Nedd4 is present in pigmented neurons and is expressed especially strongly in neurons containing Lewy bodies. Thus, ubiquitination by Nedd4 targets α-synuclein to the endosomal–lysosomal pathway and, by reducing α-synuclein content, may help protect against the pathogenesis of Parkinson disease and other α-synucleinopathies.