Multiple functions of the E3 ubiquitin ligase CHIP in immunity

The carboxyl terminal of Hsp70-interacting protein (CHIP) is an E3 ubiquitin ligase that plays a pivotal role in the protein quality control system by shifting the balance of the folding-refolding machinery toward the degradative pathway. However, the precise mechanisms by which nonnative proteins are selected for degradation by CHIP either directly or indirectly via chaperone Hsp70 or Hsp90 are still not clear. In this review, we aim to provide a comprehensive model of the mechanism by which CHIP degrades its substrate in a chaperone-dependent or direct manner. In addition, through tight regulation of the protein level of its substrates, CHIP plays important roles in many physiological and pathological conditions, including cancers, neurological disorders, cardiac diseases, bone metabolism, immunity, and so on. Nonetheless, the precise mechanisms underlying the regulation of the immune system by CHIP are still poorly understood despite accumulating developments in our understanding of the regulatory roles of CHIP in both innate and adaptive immune responses. In this review, we also aim to provide a view of CHIP-mediated regulation of immune responses and the signaling pathways involved in the model described. Finally, we discuss the roles of CHIP in immune-related diseases.     

The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development

Polycomb-repressive complex 1 (PRC1) and PRC2 maintain repression at many developmental genes in mouse embryonic stem cells and are required for early development. However, it is still unclear how they are targeted and how they function. We show that the ability of RING1B, a core component of PRC1, to ubiquitinate histone H2A is dispensable for early mouse embryonic development and much of the gene repression activity of PRC1. Our data support a model in which PRC1 and PRC2 reinforce each other''s binding but suggest that the key functions of PRC1 lie beyond the enzymatic capabilities of RING1B.     

The E3 ubiquitin ligase Itch deficiency promotes antigen-driven B-cell responses in mice

Deficiency of Itch, an E3 ubiquitin ligase, usually induced severe systemic and progressive autoimmune disease. The Itch function is well studied in T cells but not in B cells. We hypothesize that B-cell-specific Itch deficiency promoted antigen-induced B-cell activation and antibody-expressing plasma cell (PC) production. We found that unlike Itch KO, Itch cKO (CD19^creItch^f/f) mice did not demonstrated a significant increase in the sizes of spleens and LNs, antibody level, and base mutation of antibody gene. However, in line with the fact that Itch expression decreased in GC B cells, PCs, and plasmablast (PB)-like SP 2/0 cells, Itch deficiency promoted B-cell activation and antibody production induced by antigens including lipopolysaccharide (LPS) and sheep red blood cells (SRBCs). Mechanistically, we found that Itch deficiency promotes antigen-induced cytokine production because Itch controls the proteins (e.g., eIF3a, eIF3c, eIF3h) with translation initiation factor activity. Altogether, our data suggest that Itch deficiency promotes antigen-driven B-cell response. This may provide hints for Itch-targeted treatment of patients with autoimmune disease.     

E3 ubiquitin ligase E6AP-mediated TSC2 turnover in the presence and absence of HPV16 E6

We previously found that HPV16 E6 causes the degradation of the tumor suppressor protein TSC2, resulting in the phosphorylation of S6 kinase and S6 even in the absence of insulin. In the present study, we investigated the role of E6-associated protein (E6AP) in HPV16 E6-induced TSC2 degradation. Our results demonstrated that TSC2 was targeted for degradation in the presence or absence of HPV16 E6. Over-expression of E6AP enhanced the degradation of TSC2 by HPV16 E6, while expression of a dominant negative E6AP (C833A) inhibited the E6-induced degradation. Additionally, by using shRNAs to block E6AP expression in HPV16 positive and negative cells, we found a significantly prolonged TSC2 half-life. An in vivo ubiquitination assay was done to reveal that E6AP promoted the ubiquitination of TSC2 independent of HPV16 E6. We further found that TSC2 bound E6AP in the presence as well as in the absence of HPV16 E6. The binding regions on E6AP and TSC2 have been identified as amino acid (aa) 260–316, aa 428–500 and aa 1–175, aa 1251–1807, respectively. Taken together, degradation of TSC2 is mediated by E6AP ubiquitin ligase.     

The adenovirus E4orf6 E3 ubiquitin ligase complex assembles in a novel fashion

The human adenovirus E4orf6 and E1B55K proteins are part of an E3 ubiquitin ligase complex that degrades p53, Mre11 and probably other cellular polypeptides. Our group has demonstrated previously that this complex contains Cul5, Rbx1 and Elongin B and C and is formed through interactions of these cellular proteins with E4orf6. Although this E4orf6 complex is similar in many ways to the cellular SCF and VBC E3 ligase complexes, our previous work indicated that unlike all known Cullin-containing complexes, E4orf6 contains two functional BC-box motifs that permit interactions with Elongin B and C. Here we show that a third BC-box exists that also appears to be fully functional. In addition, we attempted to identify a region in E4orf6 responsible for the specific selection of Cul5, which we show herein by knocking down Cul5 protein levels, is essential for p53 degradation. One sequence within E4orf6 shares limited homology with the 'Cul5 box motif', a recently identified sequence found to be responsible for selection of Cul5 in some cellular Cullin-containing E3 ligase complexes; however, genetic analysis indicated that this motif is not involved in Cullin binding or p53 degradation. Thus E4orf6 appears to utilize a different mechanism for Cul5 selection, and, both in terms of interactions with Elongin B and C and with Cul5, assembles the E3 ligase complex in a highly novel fashion.     

The multifaceted role of the E3 ubiquitin ligase HOIL-1: beyond linear ubiquitination

Ubiquitination controls and fine-tunes many signaling processes driving immunity, inflammation, and cancer. The E3 ubiquitin ligase HOIL-1 (heme-oxidized IRP2 ubiquitin ligase-1) is increasingly implicated in different signaling pathways and plays a vital role in immune regulation. HOIL-1 co operates with the E3 ubiquitin ligase HOIP (HOIL-1 interacting protein) to modify specific nuclear factor-κB (NF-κB) signaling proteins with linear M1-linked polyubiquitin chains. In addition, through its ability to also add K48-linked polyubiquitin chains to specific substrates, HOIL-1 has been linked with antiviral signaling, iron and xenobiotic metabolism, cell death, and cancer. HOIL-1 deficiency in humans leads to myopathy, amylopectinosis, auto-inflammation, and immunodeficiency associated with an increased frequency of bacterial infections. HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium, pathogen-specific immunodeficiency, but minimal signs of hyper-inflammation. This review summarizes current knowledge on the mechanism of action of HOIL-1 and highlights recent advances regarding its role in health and disease.     

The E3 ubiquitin ligase Itch in T cell activation, differentiation, and tolerance

Tagging a small molecule ubiquitin to a protein substrate, or protein ubiquitination, plays an important role in the immune responses. This process is catalyzed by a cascade of enzymatic reactions, with the E3 ubiquitin ligases being the critical enzymes that determine the specificity of substrate recognition. The E3 ligase Itch was identified from a mutant mouse which displays skin scratching and abnormal immune disorders. In the past few years, much progress has been made in our understanding of Itch-promoted protein ubiquitination, modulation of its ligase activity by upstream kinases, and the kinase-ligase interaction in T cell differentiation and tolerance induction.     

Essential role of the E3 ubiquitin ligase Cbl-b in T cell anergy induction

Antigen-specific immunotolerance limits the expansion of self-reactive T cells involved in autoimmune diseases. Here, we show that the E3 ubiquitin ligase Cbl-b is upregulated in T cells after tolerizing signals. Loss of Cbl-b in mice results in impaired induction of T cell tolerance both in vitro and in vivo. Importantly, rechallenge of Cbl-b mutant mice with the tolerizing antigen results in massive lethality. Moreover, ablation of Cbl-b resulted in exacerbated autoimmunity. Mechanistically, loss of Cbl-b rescues reduced calcium mobilization of anergic T cells, which was attributed to Cbl-b-mediated regulation of PLCgamma-1 phosphorylation. Our results show a critical role for Cbl-b in the regulation of peripheral tolerance and anergy of T cells.     

Expression of the Rho-GEF Pbl/ECT2 is regulated by the UBE3A E3 ubiquitin ligase

We applied genetic tools available in Drosophila to identify candidate substrates of the UBE3A ubiquitin ligase, the gene responsible for Angelman syndrome (AS). Human UBE3A was expressed in Drosophila heads to identify proteins differentially regulated in UBE3A-expressing versus wild-type extracts. Using two-dimensional gel and MALDI-TOF analysis, we detected 20 proteins that were differentially regulated by over-expression of human UBE3A in Drosophila heads. One protein responsive to UBE3A was the Rho-GEF pebble (pbl). Here, we present three lines of evidence suggesting that UBE3A regulates Pbl. First, we show genetic evidence that UBE3A and the Drosophila de-ubiquitinase fat facets (faf) exert opposing effects on Pbl function. Secondly, we find that both Pbl and ECT2, the mammalian orthologue of Pbl called epithelial cell transforming sequence 2 oncogene, physically interact with their respective ubiquitin E3 ligases. Finally, we show that Ect2 expression is regulated by Ube3a in mouse neurons as the pattern of Ect2 expression is dramatically altered in the hippocampus and cerebellum of Ube3a null mice. These results suggest that an orthologous UBE3A post-translational regulatory pathway regulates neuronal outgrowth in the mammalian brain and that dysregulation of this pathway may result in neurological phenotypes including AS and possibly other autism spectrum disorders.     

The E3 ubiquitin ligase c-Cbl restricts development and functions of hematopoietic stem cells

Hematopoietic stem cells (HSCs) are multipotent progenitors that give rise to all types of blood cells. In the present study, we document that HSC development and functions are negatively regulated by the E3 ubiquitin ligase c-Cbl (casitas B-cell lymphoma). HSCs of c-Cbl(-/-) mice exhibit augmented pool size, hyperproliferation, greater competence, and enhanced long-term repopulating capacity. Our mechanistic studies identified that c-Cbl(-/-) HSCs are hyperresponsive to thrombopoietin (TPO) and display elevated levels of STAT5 phosphorylation, thus leading to increased c-Myc expression. In essence, our data unequivocally identify c-Cbl as a novel negative regulator of developmental and functional properties of HSCs.     

Regulation of peripheral T cell tolerance by the E3 ubiquitin ligase Cbl-b

The family of the Casitas B-lineage Lymphoma (Cbl) proteins, c-Cbl, Cbl-b, and Cbl-3, function as E3 ubiquitin ligases and molecular adaptors. In particular, Cbl-b acts as a gatekeeper in T cell activation that controls activation thresholds and the requirement for co-stimulation. Loss of Cbl-b expression renders animals susceptible to antigen-triggered autoimmunity suggesting that Cbl-b is a key autoimmunity gene. In addition, Cbl-b plays a critical role in T cell anergy and escape from regulatory T cells (Treg) suppression. Modulation of Cbl-b might provide us with a unique opportunity for future immune treatment of human disorders such as autoimmunity, immunodeficiency, or cancer.     

Itch相关免疫调控的研究进展

Itch, one of the main E3 ligases, plays an important role in immune responses as a negative regulator in the ubiquitination of PLC-γ, PKC-θ, JunB,and notch. Itch deficiency leads to sever inflammatory disorders and immunological abnormality. Many efforts have been made in disclosing the Itch-promoted protein ubiquitination, modulation of its ligase, transdution of downstream signals in T cell anergy and differentiation in the past years.     

The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

Fission yeast strains auxotrophic for leucine are unable to proliferate in normally supplemented minimal media adjusted to pH 6.4 or above. High-pH sensitivity can be suppressed by the loss of Pub1, an E3 ubiquitin ligase, or by the replacement of NH₄ ⁺ with a non-repressing source of nitrogen such as L-proline. In this report we show pub1 to be required for the rapid down-regulation of leucine uptake observed in response to the addition of NH₄ ⁺ to the growth media. Furthermore, we corroborate earlier results demonstrating the transport of leucine to be negatively influenced by high extracellular pH. pub1 is homologous to the budding yeast nitrogen permease inactivator, NPI1/RSP5, which mediates the ubiquitination and subsequent destruction of NH₄ ⁺-sensitive permeases. The high-pH sensitivity of cells auxotrophic for leucine thus seems to reflect an inability of NH₄ ⁺-insensitive permeases to transport sufficient leucine under conditions where the proton gradient driving nutrient transport is low, and NH₄ ⁺-sensitive permeases have been destroyed. Intriguingly, the partial suppression of both high pH sensitivity, and the inactivating effect of NH₄ ⁺ on leucine transport, seen in pub1-1 point mutants, becomes as complete as seen in pub1Δ backgrounds when cells have concomitantly lost the function of the spc1 stress-activated MAPK. Received: 10 November 1998 / 15 March 1999     

SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage

Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displayed increased sensitivity to DNA-damaging agents. Recruitment of RNF4 to double-strand breaks required its RING and SUMO interaction motif (SIM) domains and DNA damage factors such as NBS1, mediator of DNA damage checkpoint 1 (MDC1), RNF8, 53BP1, and BRCA1. In the absence of RNF4, these factors were still recruited to sites of DNA damage, but 53BP1, RNF8, and RNF168 displayed delayed clearance from such foci. SILAC-based proteomics of SUMO substrates revealed that MDC1 was SUMO-modified in response to ionizing radiation. As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. Failure to recruit RNF4 resulted in defective loading of replication protein A (RPA) and Rad51 onto ssDNA. This appeared to be a consequence of reduced recruitment of the CtIP nuclease, resulting in inefficient end resection. Thus, RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress.     

RhoBTB2 is a substrate of the mammalian Cul3 ubiquitin ligase complex

Rhobtb2 is a candidate tumor suppressor located on human chromosome 8p21, a region commonly deleted in cancer. Rhobtb2 is homozygously deleted in 3.5% of primary breast cancers, and gene expression is ablated in approximately 50% of breast and lung cancer cell lines. RhoBTB2 is an 83-kD, atypical Rho GTPase of unknown function, comprising an N-terminal Rho GTPase domain and two tandem BTB domains. In this report, we demonstrate that RhoBTB2 binds to the ubiquitin ligase scaffold, Cul3, via its first BTB domain and show in vitro and in vivo that RhoBTB2 is a substrate for a Cul3-based ubiquitin ligase complex. Moreover, we show that a RhoBTB2 missense mutant identified in a lung cancer cell line is neither able to bind Cul3 nor is it regulated by the ubiquitin/proteasome system, resulting in increased RhoBTB2 protein levels in vivo. We suggest a model in which RhoBTB2 functions as a tumor suppressor by recruiting proteins to a Cul3 ubiquitin ligase complex for degradation.