Employing the candidate genetic approach, we previously identified mutations in the gene encoding NEMO (NF- kB essential modulator), an intracellular signaling constituent of the NF-kB pathway, results in ectodermal dysplasia with an immune deficiency. Mutations in the zinc finger domain of NEMO block CD40 mediated activation of NF- kB and prevent B cells from undergoing class switch recombination (CSR) and APCs from synthesizing NF -kB regulated cytokines such as IL -12 or TNF-a when stimulated with CD40 ligand. The zinc finger domain of NEMO is a site for the covalent attachment of ubiquitin and this step is necessary for the full activation of NF-kappa B. The majority of patients with ectodermal dysplasia with immune deficiency (EDI) have mutations in the zinc finger domain and we have found that such mutations impair the post- translational modification of NEMO by ubiquitin. In subsequent studies we demonstrated that the NEMO zinc finger domain in the regulation of mitogen-activated protein kinase (MAPK) activity. We found 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. More recently we have studied patients with ectodermal dysplasia with immune deficiency who have normal NEMO coding sequence but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of antigen presenting cells (APCs) from these patients induced normal cytoplasmic activation and nuclear translocation of NF-kappa B. However, cells deficient in-full length NEMO were defective in expression of NF-kappa B regulated cytokines such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-kappa B transcription factors. TLR4-stimulated APCs from these patients were defective in IKK alpha-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-kappa B heterodimers Rel A and cRel to the promoter. Expression of a super active form of IKK alpha E, E restored IL-12 production in NEMO knockdown THP-1 cells following LPS treatment. Our findings indicate that failure to express full-length NEMO is associated with an immune deficiency phenotype, and offer new insights into diminished NF-kappa B signaling in patients with EDI. We found that EDI patient B cells can be characterized by an absence of Ig somatic hypermutation (SHM) and defective class switch recombination (CSR) despite normal induction of activation-induced cytidine deaminase (AID) and Iepsilon-Cepsilon transcripts. We used microarray analysis compared the gene expression profiles of activated B cells from patients with EDI to reference controls to identify candidate factors that may be necessary for CSR and SHM in B cells. 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. NIR expression was induced at 6 hours and peaked at 24 hours following CD40 stimulation of control B cells. However, such regulation was absent in patient B cells. We therefore subjected NIR to detailed analysis. Using a proteomic approach, we havve 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 Ser(269) or Thr(284) 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. We have recently completed a conditional knockout mouse for NIR and plan to study its function in B cell development. We have also undertaken efforts to identity new disease susceptibility genes for Common Variable Immunodeficiency (CVI). These are heterogeneous disorders characterized by a predisposition to serious infection and impaired or absent neutralizing antibody responses. Although a number of single gene defects have been associated with these immune deficiency disorders, the genetic basis of many cases is not known. To facilitate mutation screening in patients with these syndromes, we have developed a custom 300-kb resequencing array, the Hyper-IgM/CVID chip, which interrogates 1,576 coding exons and intron-exon junction regions from 148 genes implicated in B-cell development and immunoglobulin isotype switching. Genomic DNAs extracted from patients were hybridized to the array using a high-throughput protocol for target sequence amplification, pooling, and hybridization. A Web-based application, SNP Explorer, was developed to directly analyze and visualize the single nucleotide polymorphism (SNP) annotation and for quality filtering. Several mutations in known disease-susceptibility genes such as CD40LG, TNFRSF13B, IKBKG, AICDA, as well as rare nucleotide changes in other genes such as TRAF3IP2, were identified in patient DNA samples and validated by direct sequencing. We conclude that the Hyper-IgM/CVID chip combined with SNP Explorer may provide a cost-effective tool for high-throughput discovery of novel mutations among hundreds of disease-relevant genes in patients with inherited antibody deficiency. We are now subjecting candidate disease susceptibility genes to detailed analysis. Related efforts in the laboratory include making genetically altered mice with targeted mutations in signaling molecules, which regulate NF-kappaB. CYLD is a deubiquitinating enzyme that targets signaling constituents of the NF-kB signaling pathway, including NEMO. Alteration in CYLD have been described in patients with familial cylindromatosis, a condition characterized by numerous benign adnexal tumors. In mice deficient deficient in CYLD, we found that regulatory T cells (Tregs) are markedly increased numbers in the peripheral lymphoid organs, but not in the thymus. In vitro stimulation of CYLD-deficient nave T cells with anti-CD3/28 in the presence of TGF-βled to a marked increase in the number of Foxp3-expressing T cells compared with stimulated naive control CD4+ cells. Under endogenous conditions, CYLD formed a complex with Smad7 in which CYLD regulated Smad7 through deubiquitination of K360 and K374 residues. Moreover, this site-specific ubiquitination of Smad7 was required for activation of TAK1 and p38 kinases. Our results show that CYLD plays a role in regulating TGF-βsignaling in T cells and the development of Treg cells through deubiquitination of Smad7.

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