Spatial Redox Regulation of a Critical Cysteine Residue of NF-κB in Vivo

Abstract

Reduction-oxidation (redox) regulation has been implicated in the activation of the transcription factor NF-B. However, the significance and mechanism of the redox regulation remain elusive, mainly due to the technical limitations caused by rapid proton transfer in redox reactions and by the presence of many redox molecules within cells. Here we establish versatile methods for measuring redox states of proteins and their individual cysteine residues in vitro and in vivo, involving thiolmodifying reagents and LC-MS analysis. Using these methods, we demonstrate that the redox state of NF-B is spatially regulated by its subcellular localization. While the p65 subunit and most cysteine residues of the p50 subunit are reduced similarly in the cytoplasm and in the nucleus, Cys-62 of p50 is highly oxidized in the cytoplasm and strongly reduced in the nucleus. The reduced form of Cys-62 is essential for the DNA binding activity of NF-B. Several lines of evidence suggest that the redox factor Ref-1 is involved in Cys-62 reduction in the nucleus. We propose that the Ref-1-dependent reduction of p50 in the nucleus is a necessary step for NF-B activation. This study also provides the first example of a drug that inhibits the redox reaction between two specific proteins.The redox states of cysteine residues, which can change reversibly within cells, often greatly influence the various properties of proteins, such as protein stability, chaperone activity, enzymatic activity, and protein structure (1-5). It has also been suggested that several transcription factors bind to their cognate sites in a redox-regulated manner. Well characterized cases include the prokaryotic transcription factors SoxR and OxyR, which function as oxidative stress sensors, their DNA binding activated through oxidation of critical cysteine residues (6 -7). In most cases, however, the roles and mechanisms of redox regulation are not fully defined because it is difficult to monitor the alteration of redox states of proteins mainly due to the rapid proton transfer in redox reactions. A few have directly quantified the redox state of cysteine clustered with iron or amounts of oxidized cysteines using physicochemical or biochemical techniques (3, 8 -9), but these methods cannot describe the whole picture of redox states of a protein and are not widely applicable to other proteins. Therefore, most researchers have chosen an indirect way of using cysteine-substitution mutant proteins (3-5, 7).NF-B 1 is a eukaryotic transcription factor that regulates a wide variety of genes involved in immune function and development (10). NF-B is composed of two subunits, p50 and p65, both of which are members of the Rel family of transcription factors. NF-B normally exists in the cytoplasm, forming an inactive ternary complex with the inhibitor protein IB␣. Following the application of appropriate stimuli, NF-B is released from IB␣ and translocates into the nucleus, where it binds DNA and activates transcription of target genes. Mechanisms of NF-B activation have been exten...

The Requirement of Reversible Cysteine Sulfenic Acid Formation for T Cell Activation and Function

“…NF-B is also subject to redox regulation on cysteine 62 on the p50 subunit (49). The DNA binding activity of the transcription factor is inhibited by oxidation (23). Although cysteine oxidation inhibits the function of some proteins, in others it promotes activation.…”

“…Phosphatases, such as phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and Src homology 2 domain-containing phosphatase (SHP)-2, as well as transcription factors, such as NF-B and AP-1, use reversible cysteine oxidation to modulate protein function (21)(22)(23)(24). In the presence of ROI, cysteine thiolates can be transiently oxidized to cysteine sulfenic acid (-SOH).…”

Acrolein: Sources, metabolism, and biomolecular interactions relevant to human health and disease

“…The in vitro experiment also revealed acrolein adduction to Arg-307, forming six-membered heterocycle. From previous mutation studies in combination with thiol labeling approaches, it was known that Cys-61 is critical for the DNA-binding activity of p50 [103,104]. Analysis of the crystal structure of the NF-κB (p50/p65 heterodimer) bound to DNA supports a role of Arg-307 in mediating binding of p50 to DNA [105].…”

APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing

“…While a number of studies have investigated genes regulated by APE1, and specifically its redox signaling function (Cardoso et al ., 2012; Fishel et al ., 2015; Gaiddon et al ., 1999; Jiang et al ., 2010; Kelley et al ., 2012; Lando et al ., 2000; Logsdon et al ., 2016; Nishi et al ., 2002; Xanthoudakis et al ., 1992), it has been difficult to compile a comprehensive list of genes regulated by APE1 as it is essential for cell viability. APE1‐knockout in mice results in embryonic lethality, postimplantation, between days E5‐E9 (Ludwig et al ., 1998; Xanthoudakis et al ., 1996).…”