Nedd4 and Itch are E3 ubiquitin ligases that, in vitro, ubiquitinate similar targets and thus are thought to function similarly. T cells lacking Itch show spontaneous activation and T helper type 2 (TH2) polarization. To test whether the loss of Nedd4 affects T cells in the same way, we generated Nedd4+/+ and Nedd4−/−fetal liver chimeras. Nedd4−/−T cells developed normally but proliferated less, produced less interleukin 2, and provided inadequate help to B cells. Nedd4−/−T cells contain increased amounts of Cbl-b protein, and Nedd4 was required for Cbl-b poly-ubquitination induced by CD28 co-stimulation. These data demonstrate that Nedd4 promotes the conversion of naive T cells into activated T cells. We propose that Nedd4 and Itch ubiquitinate distinct target proteins in vivo.

Background:The activity of Itch and related E3 ligases is restricted by autoinhibition. Results: Itch autoinhibition is maintained by an intramolecular interaction between its WW and HECT domains, and Ndfip1 relieves this interaction to allow trans-thiolation. Conclusion:The primary role of Ndfip is to relieve autoinhibition of Itch and related ligases. Significance: We describe a novel mechanism that regulates the activity of several catalytic E3 ligases.

Nedd4 family interacting protein 1 (Ndfip1) is an adaptor protein that regulates Itch, an E3 ubiquitin ligase that ubiquitylates JunB, thereby preventing interleukin (IL)-4 and IL-5 production. Mice lacking Ndfip1 or Itch develop T helper type 2 (TH2)-mediated inflammation in the skin and lungs and die prematurely. In this study we show that Ndfip1 − / − mice also develop inflammation in the gastrointestinal tract. Inflammation is characterized by infiltration of eosinophils and T cells and is accompanied by a failure to gain weight. T cells are both necessary and sufficient for eosinophil recruitment and inflammation. This is because Ndfip1 − / − T cells become activated and produce IL-5. Itch mutant mice develop much less severe gastrointestinal inflammation, suggesting that Ndfip1 regulation of Itch cannot entirely account for this phenotype and that Ndfip1 has both Itch-dependent and Itch-independent roles. Ndfip1 may also regulate human disease. We found single-nucleotide polymorphisms in the Ndfip1 locus that associate with inflammatory bowel disease. Taken together, our data support a role for Ndfip1 in gastrointestinal inflammation in both mice and humans.

Ndfip1 is an adaptor for the E3 ubiquitin ligase Itch. Both Ndfip1- and Itch-deficient T cells are biased towards TH2 cytokine production. Here we demonstrate that lungs from Ndfip1−/− mice showed increased numbers of neutrophils and TH17 cells. This was not because Ndfip1−/− T cells are biased towards Th17 differentiation. In fact, fewer Ndfip1−/− T cells differentiated into TH17 cells in vitro due to high IL-4 production. Rather, TH17 differentiation was increased in Ndfip1−/− mice due to increased numbers of IL-6 producing eosinophils. IL-6 levels in mice that lacked both Ndfip1 and IL-4 were similar to WT controls and these mice had fewer TH17 cells in their lungs. These results indicate that TH2 inflammation, such as that observed in Ndfip1−/− mice, can increase TH17 differentiation by recruiting IL-6 producing eosinophils into secondary lymphoid organs and tissues. This may explain why TH17 cells develop within an ongoing TH2 inflammatory response.

Programmed mutagenesis of the immunoglobulin locus of B lymphocytes during class switch recombination (CSR) and somatic hypermutation requires RNA polymerase II (polII) transcription complex-dependent targeting of the DNA mutator activation-induced cytidine deaminase (AID). AID deaminates cytidine residues on substrate sequences in the immunoglobulin (Ig) locus via a transcription-dependent mechanism, and this activity is stimulated by the RNA polII stalling cofactor Spt5 and the 11-subunit cellular noncoding RNA 39-59 exonucleolytic processing complex RNA exosome. The mechanism by which the RNA exosome recognizes immunoglobulin locus RNA substrates to stimulate AID DNA deamination activity on its in vivo substrate sequences is an important question. Here we report that E3-ubiquitin ligase Nedd4 destabilizes AID-associated RNA polII by a ubiquitination event, leading to generation of 39 end free RNA exosome RNA substrates at the Ig locus and other AID target sequences genome-wide. We found that lack of Nedd4 activity in B cells leads to accumulation of RNA exosome substrates at AID target genes and defective CSR. Taken together, our study links noncoding RNA processing following RNA polII pausing with regulation of the mutator AID protein. Our study also identifies Nedd4 as a regulator of noncoding RNAs that are generated by stalled RNA polII genome-wide.

Despite gathering evidence that ubiquitylation can direct non-degradative outcomes, most investigations of ubiquitylation in T cells have focused on degradation. Here, we integrated proteomic and transcriptomic datasets from primary mouse CD4 + T cells to establish a framework for predicting degradative or non-degradative outcomes of ubiquitylation. Di-glycine remnant profiling was used to reveal ubiquitylated proteins, which in combination with whole-cell proteomic and transcriptomic data allowed prediction of protein degradation. Analysis of ubiquitylated proteins identified by di-glycine remnant profiling indicated that activation of CD4 + T cells led to an increase in non-degradative ubiquitylation. This correlated with an increase in non-proteasome-targeted K29, K33 and K63 polyubiquitin chains. This study revealed over 1,200 proteins that were ubiquitylated in primary mouse CD4 + T cells and highlighted the relevance of non-proteasomally targeted ubiquitin chains in T cell signaling. Engagement of the T cell antigen receptor (TCR) and the costimulatory receptor CD28 initiates proliferation and effector T cell differentiation. Altering the abundance-via Reprints and permissions information is available at www.nature.com/reprints.

While the pathways that permit IL-2 production and the full activation of T cells upon antigen encounter are fairly well defined, the negative regulatory circuits that limit these pathways are poorly understood. Here we show that the E3 ubiquitin ligase adaptor Ndfip1 directs one such negative regulatory circuit. T cells lacking Ndfip1 produce IL-2, upregulate IL-2 receptor alpha (IL-2Rα), and proliferate, in the absence of CD28 co-stimulation. Furthermore, T cells in mice lacking both Ndfip1 and CD28 become activated, produce IL-4, and drive inflammation at barrier surfaces. Ndfip1 constrains T cell activation by limiting the duration of IL-2 mRNA expression after TCR stimulation. Ndfip1 and IL-2 have a similar expression pattern and, following TCR stimulation, expression of both Ndfip1 and IL-2 require the activity of NFAT and Erk. Taken together, these data support a negative regulatory circuit in which factors that induce IL-2 expression downstream of TCR engagement also induce the expression of Ndfip1 to limit the extent of IL-2 production and, thus, dampen T cell activation.

Itch is a HECT type E3 ubiquitin ligase that is required to prevent the development of autoimmune disease in both mice and humans. Itch is expressed in most mammalian cell types, and, based on published data, it regulates many cellular pathways ranging from T cell differentiation to liver tumorigenesis. Since 1998, when Itch was first discovered, hundreds of publications have described mechanisms through which Itch controls various biologic activities in both immune and non-immune cells. Other studies have provided insight into how Itch catalytic activity is regulated. However, while autoimmunity is the primary clinical feature that occurs in both mice and humans lacking Itch, and Itch control of immune cell function has been well-studied, it remains unclear how Itch prevents the emergence of autoimmune disease. In this review, we explore recent discoveries that advance our understanding of how Itch regulates immune cell biology, and the extent to which these clarify how Itch prevents autoimmune disease. Additionally, we discuss how molecular regulators of Itch impact its ability to control these processes, as this may provide clues on how to therapeutically target Itch to treat patients with autoimmune disease.