Cysteine 38 Holds the Key to NF-κB Activation

The importance of parallel signaling pathways controlling NF-κB subunit posttranslational modifications is demonstrated by Sen et al., 2012Sen N. Paul B.D. Gadalla M.M. Mustafa A.K. Sen T. Xu R. Kim S. Snyder S.H. Mol. Cell. 2012; 45 (this issue): 13-24Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, who reveal that RelA (p65) sulfhydration, at its highly conserved cysteine 38 residue, regulates association with the coactivator RPS3, DNA binding, and antiapoptotic gene expression. The importance of parallel signaling pathways controlling NF-κB subunit posttranslational modifications is demonstrated by Sen et al., 2012Sen N. Paul B.D. Gadalla M.M. Mustafa A.K. Sen T. Xu R. Kim S. Snyder S.H. Mol. Cell. 2012; 45 (this issue): 13-24Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, who reveal that RelA (p65) sulfhydration, at its highly conserved cysteine 38 residue, regulates association with the coactivator RPS3, DNA binding, and antiapoptotic gene expression. The nuclear factor κB (NF-κB) family of transcription factors, comprising homo- and heterodimers formed from RelA (p65), c-Rel, RelB, p50/p105 (NF-κB1), and p52/p100 (NF-κB2), are critical regulators of the cellular response to stress and infection (Hayden and Ghosh, 2008Hayden M.S. Ghosh S. Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3151) Google Scholar). The majority of NF-κB-inducing stimuli, such as inflammatory cytokines, bacterial and viral components, and cell stresses such as DNA damage, result in the activation of the inhibitor of NF-κB (IκB) kinase (IKK) complex, leading to the phosphorylation and ubiquitin-mediated degradation of a member of the IκB family of proteins (Hayden and Ghosh, 2008Hayden M.S. Ghosh S. Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3151) Google Scholar). This "classical" pathway of NF-κB activation typically results in the rapid nuclear localization of a p50/RelA heterodimer. The cellular consequences of NF-κB activation include resistance to apoptosis induced by tumor necrosis factor (TNF)-α and other stimuli, which is also an important component of the ability of aberrantly active NF-κB to drive the progression of inflammatory diseases and promote the survival of cancer cells (Fan et al., 2008Fan Y. Dutta J. Gupta N. Fan G. Gélinas C. Adv. Exp. Med. Biol. 2008; 615: 223-250Crossref PubMed Scopus (108) Google Scholar). In addition to release from IκB, "activation" of NF-κB requires a wide variety of subunit posttranslational modifications (PTMs), including phosphorylation, acetylation, ubiquitylation, and methylation, which can control nuclear translocation, target gene specificity, transcriptional activity, and subunit degradation (Huang et al., 2010Huang B. Yang X.D. Lamb A. Chen L.F. Cell. Signal. 2010; 22: 1282-1290Crossref PubMed Scopus (206) Google Scholar). In this edition of Molecular Cell, a manuscript from the Snyder laboratory reveals a new and critical RelA modification (referred to as p65 by Sen et al.) required for its ability to bind DNA and induce antiapoptotic target gene expression (Sen et al., 2012Sen N. Paul B.D. Gadalla M.M. Mustafa A.K. Sen T. Xu R. Kim S. Snyder S.H. Mol. Cell. 2012; 45 (this issue): 13-24Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Almost all NF-κB subunits, with the exceptions being some isoforms from nonvertebrate species, contain a highly conserved cysteine residue in the N-terminal region of the Rel homology domain (RHD) (Cys-38 in human RelA; Figure 1). The importance of this residue has been appreciated for many years: it interacts with the phosphate backbone of NF-κB binding sites (Chen et al., 1998Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (323) Google Scholar) (Figure 2) while its oxidation or nitrosylation are known to inhibit DNA binding (Kelleher et al., 2007Kelleher Z.T. Matsumoto A. Stamler J.S. Marshall H.E. J. Biol. Chem. 2007; 282: 30667-30672Crossref PubMed Scopus (165) Google Scholar). Moreover, it is the target of many naturally occurring NF-κB inhibitors such as the sesquiterpene lactones (Gilmore and Herscovitch, 2006Gilmore T.D. Herscovitch M. Oncogene. 2006; 25: 6887-6899Crossref PubMed Scopus (425) Google Scholar). Here, Sen et al. demonstrate that RelA Cys-38 is also subject to hydrogen sulfide-linked sulfhydration (Sen et al., 2012Sen N. Paul B.D. Gadalla M.M. Mustafa A.K. Sen T. Xu R. Kim S. Snyder S.H. Mol. Cell. 2012; 45 (this issue): 13-24Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). This modification, which creates a hydropersulfide (−SSH) by attaching an additional sulfur to the thiol (−SH) group, is performed by cystathionine γ-lyase (CSE), the expression of which is induced in an NF-κB- and IKK-independent manner following TNF stimulation (Sen et al., 2012Sen N. Paul B.D. Gadalla M.M. Mustafa A.K. Sen T. Xu R. Kim S. Snyder S.H. Mol. Cell. 2012; 45 (this issue): 13-24Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). Sen et al. convincingly demonstrate that impairment of this modification inhibits RelA DNA binding and compromises its ability to induce antiapoptotic gene expression (Sen et al., 2012Sen N. Paul B.D. Gadalla M.M. Mustafa A.K. Sen T. Xu R. Kim S. Snyder S.H. Mol. Cell. 2012; 45 (this issue): 13-24Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). These effects are at least partly explained by sulfhydration of Cys-38 being required for RelA interaction with ribosomal protein S3 (RPS3), which can function as a NF-κB coregulator (Wan et al., 2007Wan F. Anderson D.E. Barnitz R.A. Snow A. Bidere N. Zheng L. Hegde V. Lam L.T. Staudt L.M. Levens D. et al.Cell. 2007; 131: 927-939Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar).Figure 2Structure of the RelA/p50 Dimer Bound to the IgH/HIV κB SiteShow full caption(A) The positions of RelA Cys-38 (red) and Cys-95 (purple), together with p50 Cys-59 (blue) and Cys-119 (green), are shown. The DNA κB site is shown in gold.(B) Space-filling structures of the RelA/p50/DNA complex from an "underside" (top image) and "side on" (bottom image) perspective, showing RelA Cys-38 (red) and p50 Cys-59 (blue). The likely binding site for RPS3 suggested by the work of Sen et al. is shown. Images were created from PDB file 1VKW (Chen et al., 1998Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (323) Google Scholar) using the Jmol interface of DNADynamo software.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) The positions of RelA Cys-38 (red) and Cys-95 (purple), together with p50 Cys-59 (blue) and Cys-119 (green), are shown. The DNA κB site is shown in gold. (B) Space-filling structures of the RelA/p50/DNA complex from an "underside" (top image) and "side on" (bottom image) perspective, showing RelA Cys-38 (red) and p50 Cys-59 (blue). The likely binding site for RPS3 suggested by the work of Sen et al. is shown. Images were created from PDB file 1VKW (Chen et al., 1998Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (323) Google Scholar) using the Jmol interface of DNADynamo software. The evolutionary conservation of Cys-38 suggests that this is a highly conserved mechanism, and it will be interesting to see if this pathway also functions across species. However, it is not currently clear if cysteine sulfhydration will regulate the other NF-κB subunits. Sen et al. did examine the p50 subunit and found that CSE did not modify the Cys-38 equivalent, Cys-59, unless Cys-119 was mutated to alanine (Sen et al., 2012Sen N. Paul B.D. Gadalla M.M. Mustafa A.K. Sen T. Xu R. Kim S. Snyder S.H. Mol. Cell. 2012; 45 (this issue): 13-24Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar) (note: human numberings are Cys-62 and -122). One possibility, noted by the authors, is that this might result from disulfide bond formation, although analysis of the structure of p50/RelA bound to DNA suggests this is unlikely to occur in that conformation of the protein (Figure 2). Moreover, Cys-119 is also conserved in RelA, albeit with differing flanking residues (Figure 1). An alternative possibility is that a CSE docking motif on RelA or an associated protein is required. Some specificity determinant must exist, as Cys-38 is the only one of eight cysteines in RelA to be modified by CSE. Analysis of the p50/RelA structure also reveals that while Cys-38 is closely associated with the DNA, it is also partially exposed and accessible for protein binding to RPS3 (Figure 2). This suggests that the docking site for RPS3 will be in the "cleft" at the underside of the NF-κB/DNA complex, with the potential to make contact with the exposed DNA surface, thus stabilizing DNA binding (Figure 2). Although Sen et al. demonstrated that LPS stimulation also induces RelA Cys-38 sulfhydration, it is not yet clear how universally applicable this mechanism of regulation will be to other inducers of NF-κB and other biological contexts. Indeed, deletion or loss of CSE does not abolish NF-κB DNA binding but instead reduces it (Sen et al., 2012Sen N. Paul B.D. Gadalla M.M. Mustafa A.K. Sen T. Xu R. Kim S. Snyder S.H. Mol. Cell. 2012; 45 (this issue): 13-24Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar), while CSE knockout mice are viable (Yang et al., 2008Yang G. Wu L. Jiang B. Yang W. Qi J. Cao K. Meng Q. Mustafa A.K. Mu W. Zhang S. et al.Science. 2008; 322: 587-590Crossref PubMed Scopus (1733) Google Scholar) in contrast to RelA null mice, which die in utero as a consequence of TNF-induced liver apoptosis (Fan et al., 2008Fan Y. Dutta J. Gupta N. Fan G. Gélinas C. Adv. Exp. Med. Biol. 2008; 615: 223-250Crossref PubMed Scopus (108) Google Scholar). Therefore, in contexts where Cys-38 sulfhydration does not occur, NF-κB promoter targeting may be directed toward variant κB sites where RPS3 is not required or where other proteins, such as HMGA1, can fulfill the role of a DNA binding coregulator. The order, location, and timing of these regulatory events are not yet established. For example, RPS3 undergoes IKKβ-dependent phosphorylation and nuclear translocation (Wan et al., 2011Wan F. Weaver A. Gao X. Bern M. Hardwidge P.R. Lenardo M.J. Nat. Immunol. 2011; 12: 335-343Crossref PubMed Scopus (98) Google Scholar), raising the possibility that cytoplasmic RelA Cys-38 sulfhydration results in the formation of the RelA/RPS3 complex, which then moves to the nucleus. Alternatively, RPS3 might associate with RelA in the nucleus after induction or even bind and stabilize a prebound RelA/DNA complex. TNF activation of NF-κB can also be very rapid, occurring within minutes and before induction of the CSE gene will have had time to affect CSE enzymatic activity. Sen et al. do not look at time points prior to 60 min, and so the status of RelA modification at these earlier time points is not known. It is possible that "early" activated RelA is not modified at this residue and that CSE induction has the effect of prolonging and establishing an NF-κB response. Alternatively, pre-existing CSE may be sufficient to ensure this "early" RelA is appropriately modified. It will also be interesting to see if Cys-38 sulfhydration is linked to other RelA PTMs. For example, Ser-276 phosphorylation is a critical activator of RelA (Chen et al., 1998Chen F.E. Huang D.B. Chen Y.Q. Ghosh G. Nature. 1998; 391: 410-413Crossref PubMed Scopus (323) Google Scholar, Hayden and Ghosh, 2008Hayden M.S. Ghosh S. Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3151) Google Scholar) and may provide a conformational trigger to allow CSE access to the Cys-38 residue. Sen et al. also show that RelA Cys-38 nitrosylation replaces sulfhydration and that this is associated with switching off the NF-κB response. Sulfhydrated RelA can be nitrosylated, indicating that active repression of RelA activity by this second modification may occur (Sen et al., 2012Sen N. Paul B.D. Gadalla M.M. Mustafa A.K. Sen T. Xu R. Kim S. Snyder S.H. Mol. Cell. 2012; 45 (this issue): 13-24Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). However, it cannot be ruled out that, in vivo, Cys-38 sulfhydrated "active" RelA will be degraded, with de novo synthesized RelA being subject to Cys-38 nitrosylation. This study demonstrates that there is still much to be learned about NF-κB subunit function and regulation. Moreover, this could lead to new drugs and therapies. Many natural products covalently modify Cys-38 as a means of inhibiting or modulating NF-κB activity, implying that inhibiting CSE or the pathway leading to its inducible expression may provide a way to indirectly target NF-κB as a treatment for inflammatory diseases and cancer. That the CSE knockout mice are viable suggests that such drugs will be tolerated (Yang et al., 2008Yang G. Wu L. Jiang B. Yang W. Qi J. Cao K. Meng Q. Mustafa A.K. Mu W. Zhang S. et al.Science. 2008; 322: 587-590Crossref PubMed Scopus (1733) Google Scholar). Given the likely problems of targeting IKKβ, due to its widespread effects and the toxicity associated with its inhibition, this should warrant further investigation. Hydrogen Sulfide-Linked Sulfhydration of NF-κB Mediates Its Antiapoptotic ActionsSen et al.Molecular CellJanuary 13, 2012In BriefNuclear factor κB (NF-κB) is an antiapoptotic transcription factor. We show that the antiapoptotic actions of NF-κB are mediated by hydrogen sulfide (H2S) synthesized by cystathionine gamma-lyase (CSE). TNF-α treatment triples H2S generation by stimulating binding of SP1 to the CSE promoter. H2S generated by CSE stimulates DNA binding and gene activation of NF-κB, processes that are abolished in CSE-deleted mice. As CSE deletion leads to decreased glutathione levels, resultant oxidative stress may contribute to alterations in CSE mutant mice. Full-Text PDF Open Archive