In the present study we have identified a signaling cascade that mediates the anti-apoptotic actions of NFκB in response to TNFα activation (Figure S9I
). TNF-α stimulates transcription of the H2
S generating enzyme CSE, whose promoter region has a recognition motif for SP1, a transcription factor activated by TNFα. The synthesized H2
S sulfhydrates the p65 subunit of NF-κB at cysteine-38 which augments its ability to bind its co-activator RPS3. The activator/co-activator complex then stimulates transcription of anti-apoptotic genes.
We have measured sulfhydration of p65 both by the modified biotin switch assay and the maleimide assay. Maleimides do not react with methionine, histidine or tyrosine, instead specifically targeting thiol groups of cysteine residues. In proteins with multiple cysteine residues, the reactivity of an individual cysteine is dependent both on its local environment and the hydrophobicity of the reactive dye (Brustad et al., 2008
; Kim et al., 2008
; Tyagarajan et al., 2003
). Moreover, the polarity of Alexa Fluor conjugated maleimides permits the sensitive detection of exposed or accessible thiols (Gelderman et al., 2006
; Sahaf et al., 2003
). This may explain the fact that in our assay system Alexa conjugated C2 or C5 maleimide interacts selectively with cysteine-38 of p65. The similarity of results with the maleimide and modified biotin switch methods supports the reliability of the two techniques as assays for sulfhydration. The exact molecular mechanism, whereby the modified biotin switch assay differentiates sulfhydrated and unmodified cysteine is not altogether clear. We presume that MMTS can react with both but that biotin selectively links to the more chemically reactive sulfhydryl moiety.
Lenardo and associates (Wan et al., 2007
) established that RPS3 mediates interactions of p65 with its gene promoter targets. Heretofore, specific cellular functions of RPS3 have not been delineated. Our findings establish an important role for RPS3 in mediating the anti-apoptotic influences of p65. Sulfhydration of p65 enables it to bind to RPS3 whose co-activator effects are required for transcriptional activation. Earlier studies have shown that oxidizing and reducing agents, such as DTT, respectively decrease and increase p65 binding to DNA (Gius et al., 1999
). In our study, DTT (up to around 2 mM) treatment of intact cells also enhances p65 binding to DNA due to increases in p65 sulfhydration and binding to RPS3, findings that may account for the earlier observations. Higher concentrations of DTT (2.5 mM and above), abolish sulfhydration of p65, which in turn decreases its binding to RPS3 and its DNA binding to promoters of anti-apoptotic genes leading to cell death. Interestingly, C38 of p65 lies in the DNA binding domain, and its sulfhydration enhances binding to RPS3 leading to increased transcriptional activation by p65. Exactly how p65, RPS3 and the target DNA promoters interface is unclear and presumably awaits structural studies.
Sulfhydration and nitrosylation both appear to regulate p65 and do so reciprocally. Following TNF-α treatment, sulfhydration of p65 occurs first, leading to an early activation of its promoter targets. Subsequently, NO generated by iNOS nitrosylates p65, which reverses the sulfhydration-elicited activation. Conceivably, reciprocal influences of sulfhydration and nitrosylation represent a mode for determining NF-κB physiology and, perhaps, actions of other signaling proteins.
NF-κB mediates inflammatory effects of TNF-α with ramifications for diseases such as arthritis, diabetes, sepsis and osteoporosis (Ghosh and Karin, 2002
). NF-κB physiologically suppresses the pro-apoptotic effects of TNF-α, leading to diminished apoptosis. Our findings indicate that the anti-apoptotic actions of NF-κB are dependent on the generation of H2
S which sulfhydrates p65. Decreasing p65 sulfhydration and thereby augmenting apoptosis may be beneficial in the therapy of diseases that involve excessive cell growth and division, such as cancer.