Regulation of many immune response genes depend on a 10 bp DNA sequence termed kappaB. This sequence is bound by a family of protein factors related to the Rel oncogene. The prototype transcription complex binding to the sequence, termed NF-kappaB, has been conventionally defined as a heterodimer between a P50 DNA binding protein and a P65 (RelA) activation protein that is typically sequestered in the cytoplasm by a protein called I-kappaB. Following certain types of stimulation to the cell, a specific protein kinase complex called I-kappaB kinase causes the phosphorylation of I-kappaB followed by its ubiquitination and degradation. Among the stimuli that can release NF-kappaB is the triggering of the T cell receptor (TCR) or B cell receptor (BCR) by antigen during an immune response. While studying a rare clinical condition of immunodeficiency, we discovered that NF-kappaB has a more complex subunit composition than previously suspected. Specifically, we found that genes such as IL-2 and I-kB harbor kappaB sites of a particular sequence that bind a trimeric complex containing p50, p65, and ribosomal protein small subunit 3 (RPS3). RPS3 is a K-homology (KH) domain-containing protein that binds single-stranded nucleic acids and dramatically enhances the affinity of the p50 and p65 Rel-homology proteins to these select kappaB sites. A subset of all NF-kappaB genes are dependent on RPS3 including crucial physiological functions such as expression of the immunoglobulin kappa light chain gene in B cells and the interleukin-2 gene in T cells. Rapid transcription of specific NF-B target genes is vital for an effective immune response. We recently discovered that in response to stimuli that induce NF-kappaB, RPS3 also independently translocates to the nucleus in parallel to p65, where it facilitates high affinity binding of NF-kappaB to certain kB sites. We went on to show that specific activating signals cause the stimulation-induced phosphorylation of RPS3 which is critical for its nuclear translocation. We found that the Inhibitor of kB (IkB) kinase beta (IKKbeta), when activated, specifically phosphorylates RPS3 at serine 209 (S209). Serine phosphorylation of RPS3, which also depends on contemporaneous IKKbeta-mediated phosphorylation and degradation of IkBalpha, augments its association with importin-alpha in the classic cytoplasmic karyopherin pathway which governs subsequent nuclear translocation. Moreover, the Escherichia coli O157:H7 type III secretion system effector protein NleH1 specifically inhibits RPS3 S209 phosphorylation to block subsequent RPS3 nuclear translocation. By selectively attenuating transcription of certain RPS3-dependent NF-kappaB target genes, E. coli O157:H7 fine-tunes the NF-kappaB response to suppress host inflammation in the gut, which may improve bacterial dissemination. In summary, our results indicate that the IKKbeta-dependent modification of a single amino acid in RPS3 promotes the specific recognition of certain kappaB sites by NF-kappaB, unveiling a novel mechanism for RPS3 that underpins its regulatory role in transcription versus translation. This mechanism also illuminates novel therapeutic targets for the control of foodborne pathogens, especially during early E. coli O157:H7 infection . We have further explored this new paradigm for NF-kappaB gene regulation to potentially explains the selective activation of genes harboring distinct versions of NF-kappaB binding sites. This has led us to search for other subunits that may also participate in the binding complex. We have determined that there is a distinct KH domain protein called wan23 is a critical subunit of the NF-kappaB complex that binds to the particular kappaB binding site found in the promoter of the CD25 (the IL-2 receptor alpha chain) gene. DNA-binding analyses reveal that wan23 dramatically enhances the binding of the p50 and p65 Rel subunits to the CD25 promoter site. Moreover, the transcriptional activity of this site mediated by NF-kappaB is profoundly suppressed if wan23 is depleted by siRNA. In functional analyses, we have determined that the participation of wan23 in NF-kappaB-mediated gene regulation in human leukemia cell lines governs the survival of these cells in response to interleukin-2 in cell culture. If we reduce the expression of wan23, various human leukemia cell lines such as Hut-102 or MT-2 have markedly reduced viability. Therefore, wan23 is a critical physiological component of particular NF-kappaB regulatory complexes. This has verified our hypothesis that there are a group of nuclear NF-kappaB complexes that have distinct subunit compositions that have previously been viewed as a single NF-kappaB complex. These discoveries elucidate a fundamental mechanism by which NF-kappaB mediates specific gene regulatory effects. Further work is being directed to understanding how the RPS3 and wan23 KH domain proteins are regulated to become incorporated into the NF-kappaB complexes at specific genes. Preliminary analyses suggest that specific phosphorylation events play a key role in this process. We are also attempting to identify inhibitors of this component, since inhibition of NF-kappaB is a prime therapeutic target for number of inflammatory and degenerative conditions. Inhibition of NF-kappaB may also be useful for various infectious diseases involving pathogens, such as HIV, that utilize this factor for their life cycle or pathogenic effects. In particular, we are studying the subunit composition of the NF-kappaB complex that binds to the kappaB sites in the HIV long terminal repeat transcriptional promoter. Experiments are underway to biophysically characterize the complexes of the Rel subunits of NF-kappaB with the additional subunits to understand how they mediate high affinity DNA interactions. We have also been studying the activation circuitry that induces NF-kappaB after triggering the antigen receptor on B cells or T cells. We have found a new kinase that plays a direct role in physically linking the membrane-associated protein complex containing the Carma 1, MALT 1, and the Bcl-10 (CBM) proteins to the I-kappaB kinase complex. We have demonstrated a vital role for the cellular kinase in the induction pathway. This novel kinase has both a positive and negative regulatory role is transducing the signals from antigen receptors at the surface of lymphocytes to the gene induction apparatus. This kinase has been shown to be involved in developmental and circadian rhythm pathways and now appears to play a key role in immune function. We will be studying the biochemical features of its regulation to understand how it might be modulated in various diseases of the immune system. Mass spectrometry analysis has also revealed other proteins in the signal transduction pathway involving the CBM complex and experimentation will be directed to elucidating the functional role of these proteins.
| Comrie, William A; Faruqi, Aiman J; Price, Susan et al. (2018) RELA haploinsufficiency in CD4 lymphoproliferative disease with autoimmune cytopenias. J Allergy Clin Immunol 141:1507-1510.e8 |
| Grönholm, Juha; Pagni, Philippe P; Pham, Minh N et al. (2017) Metabolically inactive insulin analogue does not prevent autoimmune diabetes in NOD mice. Diabetologia 60:1475-1482 |
| Ozen, Ahmet; Comrie, William A; Ardy, Rico C et al. (2017) CD55 Deficiency, Early-Onset Protein-Losing Enteropathy, and Thrombosis. N Engl J Med 377:52-61 |
| Lenardo, Michael; Lo, Bernice; Lucas, Carrie L (2016) Genomics of Immune Diseases and New Therapies. Annu Rev Immunol 34:121-49 |
| Lucas, Carrie L; Lenardo, Michael J (2015) Identifying genetic determinants of autoimmunity and immune dysregulation. Curr Opin Immunol 37:28-33 |
| Kanellopoulou, Chryssa; Gilpatrick, Timothy; Kilaru, Gokhul et al. (2015) Reprogramming of Polycomb-Mediated Gene Silencing in Embryonic Stem Cells by the miR-290 Family and the Methyltransferase Ash1l. Stem Cell Reports 5:971-978 |
| Chaigne-Delalande, Benjamin; Li, Feng-Yen; O'Connor, Geraldine M et al. (2013) Mg2+ regulates cytotoxic functions of NK and CD8 T cells in chronic EBV infection through NKG2D. Science 341:186-91 |
| Lo, Bernice; Ramaswamy, Madhu; Davis, Joie et al. (2013) A rapid ex vivo clinical diagnostic assay for fas receptor-induced T lymphocyte apoptosis. J Clin Immunol 33:479-88 |
| Pandiyan, Pushpa; Lenardo, Michael (2012) Comment on ""Cutting edge: regulatory T cells do not mediate suppression via programmed cell death pathways"". J Immunol 188:5203-4; author reply 5204-5 |
| Snow, Andrew L; Xiao, Wenming; Stinson, Jeffrey R et al. (2012) Congenital B cell lymphocytosis explained by novel germline CARD11 mutations. J Exp Med 209:2247-61 |
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