Yongge Zhao

Optineurin negatively regulates TNFα-induced NF-κB activation by competing with NEMO for ubiquitinated RIP

NF-kappaB essential modulator (NEMO), the regulatory subunit of the IkappaB kinase (IKK) that activates NF-kappaB, is essential for NF-kappaB activation. NEMO was recently found to contain a region that preferentially binds Lys (K)63-linked but not K48-linked polyubiquitin (polyUb) chains, and the ability of NEMO to bind to K63-linked polyUb RIP (receptor-interacting protein) is necessary for efficient tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. Optineurin is a homolog of NEMO, and mutations in the optineurin gene are found in a subset of patients with glaucoma, a neurodegenerative disease involving the loss of retinal ganglion cells. Although optineurin shares considerable homology with NEMO, in resting cells, it is not present in the high-molecular-weight complex containing IKKalpha and IKKbeta, and optineurin cannot substitute for NEMO in lipopolysaccharide (LPS)-induced NF-kappaB activation. On the other hand, the overexpression of optineurin blocks the protective effect of E3-14.7K on cell death caused by the overexpression of TNFalpha receptor 1 (TNFR1). Here we show that optineurin has a K63-linked polyUb-binding region similar to that of NEMO, and like NEMO, it bound K63- but not K48-linked polyUb. Optineurin competitively antagonized NEMOs binding to polyUb RIP, and its overexpression inhibited TNFalpha-induced NF-kappaB activation. This competition occurs at physiologic protein levels because microRNA silencing of optineurin resulted in markedly enhanced TNFalpha-induced NF-kappaB activity. These results reveal a physiologic role for optineurin in dampening TNFalpha signaling, and this role might provide an explanation for its association with glaucoma.

Tumor Necrosis Factor Receptor 2 Signaling Induces Selective c-IAP1-dependent ASK1 Ubiquitination and Terminates Mitogen-activated Protein Kinase Signaling

TRAF2 and ASK1 play essential roles in tumor necrosis factor α (TNF-α)-induced mitogen-activated protein kinase signaling. Stimulation through TNF receptor 2 (TNFR2) leads to TRAF2 ubiquitination and subsequent proteasomal degradation. Here we show that TNFR2 signaling also leads to selective ASK1 ubiquitination and degradation in proteasomes. c-IAP1 was identified as the ubiquitin protein ligase for ASK1 ubiquitination, and studies with primary B cells from c-IAP1 knock-out animals revealed that c-IAP1 is required for TNFR2-induced TRAF2 and ASK1 degradation. Moreover, in the absence of c-IAP1 TNFR2-mediated p38 and JNK activation was prolonged. Thus, the ubiquitin protein ligase activity of c-IAP1 is responsible for regulating the duration of TNF signaling in primary cells expressing TNFR2.

Aurora B Interacts with NIR-p53, Leading to p53 Phosphorylation in Its DNA-binding Domain and Subsequent Functional Suppression

NIR (novel INHAT repressor) is a transcriptional co-repressor with inhibitor of histone acetyltransferase (INHAT) activity and has previously been shown to physically interact with and suppress p53 transcriptional activity and function. However, the mechanism by which NIR suppresses p53 is not completely understood. Using a proteomic approach, we have identified the Aurora kinase B as a novel binding partner of NIR. We show that Aurora B, NIR and p53 exist in a protein complex in which Aurora B binds to NIR, thus also indirectly associates with p53. Functionally, overexpression of Aurora B or NIR suppresses p53 transcriptional activity, and depletion of Aurora B or NIR causes p53-dependent apoptosis and cell growth arrest, due to the up-regulation of p21 and Bax. We then demonstrate that Aurora B phosphorylates multiple sites in the p53 DNA-binding domain in vitro, and this phosphorylation probably also occurs in cells. Importantly, the Aurora B-mediated phosphorylation on Ser269 or Thr284 significantly compromises p53 transcriptional activity. Taken together, these results provide novel insight into NIR-mediated p53 suppression and also suggest an additional way for p53 regulation.

Non-Canonical NF-κB Activation and Abnormal B Cell Accumulation in Mice Expressing Ubiquitin Protein Ligase-Inactive c-IAP2

Loss of c-IAP2 ubiquitin ligase activity, which occurs in the lymphoma-causing c-IAP2/MALT1 fusion protein, activates non-canonical NF-κB signaling and results in B cell abnormalities characteristic of MALT lymphoma.

The Deubiquitinase CYLD Targets Smad7 Protein to Regulate Transforming Growth Factor β (TGF-β) Signaling and the Development of Regulatory T Cells

Background: CYLD is a deubiquitinating enzyme (DUB) that hydrolyzes Lys-63-linked polyubiquitin chains that are attached covalently to cellular proteins. Results: CYLD knock-out mice have increased numbers of regulatory T cells (Tregs) in peripheral lymphoid organs but not in the thymus. Conclusion: CYLD regulates lysine 63-linked ubiquitination of Smad7 to control the development of peripheral Tregs. Significance: TGF-β signaling in T cells is regulated by lysine 63-Linked ubiquitination. CYLD is a lysine 63-deubiquitinating enzyme that inhibits NF-κB and JNK signaling. Here, we show that CYLD knock-out mice have markedly increased numbers of regulatory T cells (Tregs) in peripheral lymphoid organs but not in the thymus. In vitro stimulation of CYLD-deficient naive T cells with anti-CD3/28 in the presence of TGF-β led to a marked increase in the number of Foxp3-expressing T cells when compared with stimulated naive control CD4+ cells. Under endogenous conditions, CYLD formed a complex with Smad7 that facilitated CYLD deubiquitination of Smad7 at lysine 360 and 374 residues. Moreover, this site-specific ubiquitination of Smad7 was required for activation of TAK1 and p38 kinases. Finally, knockdown of Smad7 or inhibition of p38 activity in primary T cells impaired Treg differentiation. Together, our results show that CYLD regulates TGF-β signaling function in T cells and the development of Tregs through deubiquitination of Smad7.

Antibody deficiency associated with an inherited autosomal dominant mutation in TWEAK

Mutations in the TNF family of proteins have been associated with inherited forms of immune deficiency. Using an array-based sequencing assay, we identified an autosomal-dominant deficiency in TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) in a kindred with recurrent infection and impaired antibody responses to protein and polysaccharide vaccines. This mutation occurs in the sixth exon of TWEAK and results in the amino acid substitution R145C within the conserved TNF-homology domain of the full-length protein. TWEAK mutant protein formed high molecular weight aggregates under nonreducing conditions, suggesting an increased propensity for intermolecular interactions. As a result, mutant TWEAK associated with B-cell–activating factor (BAFF) protein and down-regulated the BAFF-mediated activation of the noncanonical NF-κB pathway through inhibition of p100 processing to p52, resulting in inhibition of BAFF-dependent B-cell survival and proliferation. As BAFF mediates T-cell–independent isotype switching and B-cell survival, our data implicate TWEAK as a disease-susceptibility gene for a humoral immunodeficiency.

The deubiquitinating enzyme CYLD controls apical docking of basal bodies in ciliated epithelial cells

CYLD is a tumour suppressor gene mutated in familial cylindromatosis, a genetic disorder leading to the development of skin appendage tumours. It encodes a deubiquitinating enzyme that removes Lys63- or linear-linked ubiquitin chains. CYLD was shown to regulate cell proliferation, cell survival and inflammatory responses, through various signalling pathways. Here we show that CYLD localizes at centrosomes and basal bodies via interaction with the centrosomal protein CAP350 and demonstrate that CYLD must be both at the centrosome and catalytically active to promote ciliogenesis independently of NF-κB. In transgenic mice engineered to mimic the smallest truncation found in cylindromatosis patients, CYLD interaction with CAP350 is lost disrupting CYLD centrosome localization, which results in cilia formation defects due to impairment of basal body migration and docking. These results point to an undiscovered regulation of ciliogenesis by Lys63 ubiquitination and provide new perspectives regarding CYLD function that should be considered in the context of cylindromatosis.

CYLD and the NEMO Zinc Finger Regulate Tumor Necrosis Factor Signaling and Early Embryogenesis

Background: Nuclear factor-κB (NF-κB) regulates expression of genes responsible for cell survival and immunity. Results: Cylindromatosis (CYLD) protein activated NF-κB through interaction with other pathway proteins. Conclusion: The non-catalytic function of CYLD is important for TNF-induced NF-κB signaling during embryogenesis. Significance: Our findings may explain some of the developmental and immunologic findings in patients with immune deficiency disorders. NF-κB essential modulator (NEMO) and cylindromatosis protein (CYLD) are intracellular proteins that regulate the NF-κB signaling pathway. Although mice with either CYLD deficiency or an alteration in the zinc finger domain of NEMO (K392R) are born healthy, we found that the combination of these two gene defects in double mutant (DM) mice is early embryonic lethal but can be rescued by the absence of TNF receptor 1 (TNFR1). Notably, NEMO was not recruited into the TNFR1 complex of DM cells, and consequently NF-κB induction by TNF was severely impaired and DM cells were sensitized to TNF-induced cell death. Interestingly, the TNF signaling defects can be fully rescued by reconstitution of DM cells with CYLD lacking ubiquitin hydrolase activity but not with CYLD mutated in TNF receptor-associated factor 2 (TRAF2) or NEMO binding sites. Therefore, our data demonstrate an unexpected non-catalytic function for CYLD as an adapter protein between TRAF2 and the NEMO zinc finger that is important for TNF-induced NF-κB signaling during embryogenesis.

Dendritic cells from humans with hypomorphic mutations in IKBKG/NEMO have impaired mitogen-activated protein kinase activity.

The covalent attachment of lysine 63‐linked polyubiquitin to the zinc‐finger domain of IKBKG/NEMO (also known as IKKγ) is necessary for full activation of NF‐κB. Impairments of this biochemical mechanism explain the deleterious effects of hypomorphic NEMO mutations on NF‐κB signaling function in humans suffering from X‐linked ectodermal dysplasia and immunodeficiency. Nevertheless, the biological function of the NEMO zinc‐finger domain in the regulation of mitogen‐activated protein kinase (MAPK) activity is poorly understood. Here we show that dendritic cells from patients with EDI caused by a C‐terminal E391X deletion of the zinc finger of NEMO exhibit impaired MAPK activation in response to lipopolysaccharide (LPS) stimulation. Interestingly, DCs from patients with a C417R missense mutation within the zinc finger domain of NEMO in which ubiquitination of NEMO is preserved are also defective in JNK and ERK activity following LPS stimulation. Our findings indicate that the structural integrity of the NEMO ZF domain is more important than its polyubiquitination for full activation of the MAPK. Furthermore, phosphorylation and polyubiquitination of upstream TAK1 were significantly reduced in the E391X zinc‐finger deleted patients, indicating that the NEMO zinc finger may play an important role in assembling the proximal signaling complex for MAPK activation. Hum Mutat 32:318–324, 2011. Published 2011 by Wiley‐Liss, Inc.

Novel INHAT Repressor (NIR) is required for early lymphocyte development

Significance Novel inhibitor of histone acetyltransferase repressor (NIR) is a transcriptional corepressor that can bind to p53 at promoters and suppress p53-transcriptional activity by inhibiting histone acetylation. We found that lymphoid-restricted deletion of NIR resulted in the absence of mature B and T lymphocytes, which is partially, but not completely, rescued by the combined deletion of p53 with NIR. Thus, NIR cooperates with p53 to impose a checkpoint for the generation of mature B and T lymphocytes in vivo. Further delineation of additional protein interactions with NIR may lead to the better understanding of the mechanisms that regulate cell-cycle regulation, apoptosis, and lymphocyte differentiation. Novel inhibitor of histone acetyltransferase repressor (NIR) is a transcriptional corepressor with inhibitor of histone acetyltransferase activity and is a potent suppressor of p53. Although NIR deficiency in mice leads to early embryonic lethality, lymphoid-restricted deletion resulted in the absence of double-positive CD4+CD8+ thymocytes, whereas bone-marrow-derived B cells were arrested at the B220+CD19− pro–B-cell stage. V(D)J recombination was preserved in NIR-deficient DN3 double-negative thymocytes, suggesting that NIR does not affect p53 function in response to physiologic DNA breaks. Nevertheless, the combined deficiency of NIR and p53 provided rescue of DN3L double-negative thymocytes and their further differentiation to double- and single-positive thymocytes, whereas B cells in the marrow further developed to the B220+CD19+ pro–B-cell stage. Our results show that NIR cooperate with p53 to impose checkpoint for the generation of mature B and T lymphocytes.