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Covalent 1. by a Key Metabolic Cofactor Coenzyme A Under Oxidative and Metabolic Stress

Submitted:

25 April 2026

Posted:

28 April 2026

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Abstract

Kelch-like ECH-associated protein 1 (Keap1) acts as a repressor of nuclear factor-erythroid 2-related factor 2 (Nrf2), a major transcription factor regulating cellular antioxidant response. Keap1 is the substrate adaptor subunit of the cullin 3-RING E3 ubiquitin ligase complex that specifically facilitates Nrf2 ubiquitination and its proteasomal degradation. Keap1 is rich in cysteine residues and several of them undergo various modifications, such as sulfhydration, nitrosylation and glutathionylation under cellular stress conditions. Some of these modifications alter the conformation of Keap1, preventing Nrf2 from ubiquitination and subsequent proteasome-mediated degradation. As a result, newly synthesised Nrf2 translocates to the nucleus to induce the expression of diverse genes involved in protecting cells against oxidative stress. Protein CoAlation is a reversible redox-dependent post-translational modification (PTM) in which coenzyme A (CoA) forms disulphide bonds with oxidised cysteine residues under oxidative or metabolic stress. In this study, we demonstrate for the first time that disulphide Keap1 dimer undergoes CoAlation in cellular response to oxidative stress induced by various oxidising compounds. Furthermore, glucose deprivation also induces CoAlation of disulphide Keap1 dimer in HEK293/Pank1β cells. We also demonstrate that Keap111 Cys-less mutant is not CoAlated in response to diamide treatment or glucose deprivation. In summary, this study uncovers a novel PTM of Keap1 by the key metabolic integrator CoA, which provides new insights into the regulation of the Keap1-Nrf2 antioxidant pathway under oxidative and metabolic stress.

Keywords: 
Keap1/Nrf2
;  
coenzyme A
;  
post-translational modification
;  
CoAlation
;  
antioxidant response
Subject: 
Biology and Life Sciences  -   Biochemistry and Molecular Biology

1. Introduction

Reduction-oxidation (redox) homeostasis is pivotal for maintaining the biological functions and health of all living cells. Reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and superoxide anion radical, are generated during aerobic metabolic processes. ROS can also function as critical signalling molecules that modulate diverse biological processes under physiological conditions. However, high levels of ROS deviate redox balance towards oxidative stress that damages biological macromolecules, including proteins, nucleic acids, lipids and carbohydrates. Excessive oxidative stress could lead to ageing and various pathologies, such as cancer, metabolic disorders, neurodegeneration and cardiovascular diseases [1,2].
Intricate cellular defence systems are implicated to counteract oxidative stress, including low-molecular-weight (LMW) thiols and antioxidant enzymes such as catalases, thioredoxins, peroxiredoxins, and redox-sensitive transcription factors [1,2]. LMW thiols, such as glutathione (GSH), bacillithiol (BSH), mycothiol (MSH) and CoA, are a class of compounds characterised by a highly nucleophilic thiol group that can react with ROS for detoxification or protects cellular macromolecules from oxidative or metabolic stress [3,4].
CoA is an indispensable metabolic cofactor generated from cysteine, pantothenic acid and ATP through a highly conserved five-step enzymatic pathway [5]. CoA comprises an ADP moiety linked to a flexible pantetheine tail that ends with a thiol group. Under physiological conditions, CoA forms energy-rich CoA thioesters, which take part in diverse biological processes, such as the tricarboxylic acid (TCA) cycle, the metabolism of amino acids and lipids, signal transduction and regulation of gene expression [5]. Recent findings revealed that in addition to these cellular regulatory processes, CoA acts as an important cellular antioxidant by forming a reversible mixed disulphide bond with protein cysteines (termed protein CoAlation) under oxidative or metabolic stress. Protein CoAlation has been shown to take place in both eukaryotes and prokaryotes exposed to cellular stress. A mass spectrometry-based methodology for the identification of CoAlated proteins has been recently established [5]. More than 2,300 proteins have been identified to be targeted by CoAlation [6,7]. CoAlation not only protects protein thiol groups from irreversible oxidation, but can also modulate the conformation, subcellular localisation and enzymatic activities of CoAlated proteins [5,8,9,10,11,12,13].
Nrf2 is a major transcription factor coordinating cellular antioxidant events, which upregulates various cytoprotective and antioxidant genes under cellular stress. When translocated to the nucleus, the Neh1 domain of Nrf2 heterodimerises with small musculoaponeurotic fibrosarcoma (sMAF) proteins and then binds to the Cap’n’collar (CNC)-sMaf binding elements (CsMBEs) in the promoters of target genes [14,15,16]. Under physiological conditions, the Neh2 domain of Nrf2 undergoes Keap1-mediated ubiquitination and degradation in the cytoplasm.
Keap1 is the substrate adaptor subunit of the cullin 3 (Cul3)–RING E3 ubiquitin ligase complex that specifically targets Nrf2 for ubiquitination and subsequent proteasomal degradation [17,18,19,20]. Keap1 is a cysteine-rich redox sensor. Several highly reactive cysteine residues in both human and mouse Keap1 were shown to be directly modified by electrophiles and oxidants, including Cys151, Cys273, Cys288, Cys226, Cys613, Cys622 and Cys624 [21,22,23,24,25,26,27,28]. Under stressed conditions, PTMs of Keap1 oxidised cysteine residues impair Nrf2 ubiquitination and subsequent degradation. Consequently, newly synthesised Nrf2 translocates to the nucleus, where it binds to CsMBEs to enhance cellular defence mechanisms against redox imbalance [15].
Keap1 is composed of five domains: the N-terminal domain (NTD), the Broad complex-Tramtrack-Bric-a-brac (BTB) domain, the intervening region (IVR), the Kelch domain and the C-terminal domain (CTD) [15,29]. Keap1 forms a non-covalent homodimer via its BTB domain. Under physiological conditions, Keap1 binds Nrf2 via its Kelch domain. Through its BTB domain, Keap1 then binds to Cul3 which is pre-associated with RING-box protein 1 (RBX1). This assembly forms the active Cul3–RING E3 ubiquitin ligase complex that targets Nrf2 for ubiquitination and proteasome-mediated degradation. [18,29]. Oxidative stress induces modifications of Keap1 cysteine residues in some of these domains, specifically C151 in the BTB domain and C273 and C288 in the IVR domain, reduce Keap1-Cul3 E3 ubiquitin ligase activity, while modifications of other cysteine residues could interfere with Keap1-Nrf2 association and ubiquitination [15].
Studies have shown various PTMs on Keap1 cysteine residues, including alkylation, sulfhydration, nitrosylation, lactoylation, succinylation, and the formation of methylimidazole (a crosslink between cysteine and arginine). Some of these modifications lead to the activation of the Nrfrf2 pathway [30,31,32,33,34,35,36,37]. Notably, several studies reported Keap1 glutathionylation under oxidative stress, promoting Nrf2 nuclear translocation and upregulation of Nrf2 downstream genes [30,38,39,40,41,42]. Published studies on Keap1 glutathionylation indicate that it is not restricted to monomeric Keap1, as oxidative stress can also modify the disulphide Keap1 dimer in human lung epithelial cells and mouse lung tissues [42]. These findings emphasise the impact of oxidative stress-induced cysteine modifications on Keap1 and subsequent Nrf2 activation, indicating that Keap1 CoAlation, may also modulate the antioxidant events mediated by the Keap1-Nrf2 pathway.
Initially recombinant Keap1 was shown to be CoAlated in vitro, and therefore, we hypothesised that Keap1 may undergo CoAlation in cellular response to oxidative or metabolic stress. We were surprised to find that disulphide Keap1 dimer is CoAlated in cells exposed to diamide-induced oxidative stress or glucose deprivation. Furthermore, we showed that the Keap111 Cys-less mutant is not CoAlated in response to tested stress conditions. In summary, we demonstrate for the first time a novel PTM of Keap1 by CoA in cells under oxidative and metabolic stress. These findings initiate research on the regulation of the Keap1-Nrf2 antioxidant pathway by CoA, which is known as an essential cofactor of cellular metabolism.

2. Materials and Methods

2.1. Reagents and Chemicals

H2O2, diamide, rotenone, menadione, tert-Butyl hydroperoxide (TBH), N-ethylmaleimide (NEM), β-glycerolphosphate, sodium dodecyl sulfate (SDS), dithiothreitol (DTT) and 1M Tris hydrochloride (Tris-HCl) were obtained from Sigma-Aldrich. The cOmplete™ Protease Inhibitor Cocktail (PIC) EDTA-free was obtained from Roche (#11836170001), and the Precision Plus Protein Dual Colour Standards protein marker was obtained from Bio-Rad (#1610374). The mouse anti-CoA monoclonal antibody (mAb) 1F10 was produced and characterised as reported by Malanchuk et al. [43]. The rabbit anti-HA recombinant antibody (81290-1-RR) polyclonal antibodies were obtained from Proteintech. Secondary antibodies Alexa Fluor 680 goat anti-mouse IgG H&L (#A21057) and IRdye 800 CW goat anti-rabbit IgG H&L (#A32735) were obtained from Invitrogen.

2.2. Mammalian Cell Culture and Transient Transfection

Human embryonic kidney 293 (HEK293) cell line as well as HEK293 cells stably overexpressing pantothenate kinase 1β (HEK293/Pank1β) were used in cell-based experiments. The construction of HEK293/Pank1β cells was reported in a previous study [5]. HEK293 and HEK293/Pank1β cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza) containing 10% fetal bovine serum (FBS) (HyClone), 50 units/mL penicillin and 50 µg/mL streptomycin (Lonza) and incubated at 37 °C and 5% CO2.
pEF-HA-mKeap1WT and pEF-HA-mKeap111Cys-less plasmids were kindly provided by Prof. M. Yamamoto. Escherichia coli XL-10 Gold competent cells (Agilent Technologies) were used for plasmid amplification. Plasmids were purified using the Monarch Spin Plasmid Miniprep Kit (New England Biolabs), following the manufacturer’s protocols.
6x105 million of HEK293 and HEK293/Pank1β cells were seeded onto 10-cm tissue culture dishes and transfected at 65% confluence with pEF-HA-mKeap1WT or pEF-HA-mKeap111Cys-less using the TurboFect transfection reagent (ThermoScientific Dionex), following the manufacturer’s protocols.

2.3. Cell Treatment with Oxidising Agents and Metabolic Stress

Twenty-four hours after transfection, the complete DMEM medium was replaced with pyruvate-free DMEM containing 5 mM glucose and 10% FBS prior to treatment with oxidising agents. Cells were incubated in this media for a further 24 hours to prime them for oxidative stress and were then treated with diamide (20 μM, 100 μM, 250 μM, or 500 μM), H2O2 (500 μM), menadione (500 μM), rotenone (50 μM), or TBH (1 mM) for 30 minutes at 37 °C. To induce metabolic stress, the medium was changed 24 hours after transfection to pyruvate-free DMEM containing either 5 mM or 0 mM glucose and 10% FBS. Cells subjected to oxidative or metabolic stress were incubated for further 24 hours and then harvested by gentle pressure washing and centrifuged at 2630 x g for 5 minutes at room temperature (RT). The cell pellets were stored at -80 °C.

2.4. Cell Lysis and Immunoprecipitation

Cell pellets were resuspended in lysis buffer and incubated on ice for 20 min. The lysis buffer contains 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 5 mM Na4P2O7 and 1% Triton X-100, with freshly supplemented 100 mM NEM to alkylate unmodified thiol groups, 20 mM β-glycerophosphate and 1x PIC before usage. Total cell lysates (TCLs) were centrifuged at 20817 x g for 20 minutes at 4 °C and the supernatant was collected for subsequent immunoprecipitation and Western blot (WB) analysis. Protein concentrations were determined by the Pierce Bradford Protein Assay Kit (#23200, Thermo Scientific).
The immunoprecipitation of transiently expressed HA-tagged mouse Keap1 wild-type (HA-mKeap1WT) protein or a mutant containing 11 substituted cysteine residues (HA-mKeap111Cys-less) from TCLs was performed using 20 μL 100% Protein G Sepharose (Generon) and 2 μg rabbit anti-HA recombinant antibody (81290-1-RR, Proteintech) per sample. The protein-bound beads were then mixed with an equal volume of 2× SDS loading buffer, boiled and analysed by WB.

2.5. Western Blot Analysis

For cell lysates, samples were prepared under non-reducing conditions by mixing with 5× SDS loading buffer without DTT, boiled for 5 minutes and centrifuged at 16100 x g for 1 minute.
Immunoprecipitated protein samples were resolved by non-reducing SDS polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% Precast Gel (Merck Life Science UK Limited) and TCLs were resolved by non-reducing SDS-PAGE using a 4-20% Precast Gel (Merck Life Science UK Limited). SDS-PAGE was performed at 180 V for 60 minutes. Separated proteins were then transferred to a PVDF membrane (Millipore) using 5x Trans-Blot Turbo Transfer Buffer (#10026938, Bio-Rad) and a Trans-Blot Turbo transfer system (Bio-Rad). The membrane was then blocked with Bio-Rad EveryBlot blocking buffer (#12010020) for 10 minutes at RT. The membrane was incubated with primary antibodies overnight at 4 °C on a shaker. The membrane was then washed with Tris-buffer saline containing 0.1% Tween 20 (TBSt) and then incubated with the secondary antibodies for 30 minutes at RT. After incubation, the membrane was washed extensively using TBSt followed by TBS for imaging.
Immunoprecipitated protein samples and TCLs were analysed by WB using anti-CoA (1:6000) and anti-HA (1:5000) antibodies. Primary antibodies were diluted in Bio-Rad EveryBlot blocking buffer. Secondary antibodies were diluted (1:10000) in the same blocking buffer supplemented 0.02% SDS.
Fluorescent immunoreactivities were imaged using Odyssey Scanner CLx and Image Studio Lite software version 5.0 (LI-COR Biosciences).

3. Results

3.1. Oxidative Stress Induces CoAlation of HA-mKeap1 in HEK293/Pank1β Cells

We were prompted to investigate Keap1 CoAlation in cells by strong in vitro CoAlation of GST-Keap1 (data not shown). To validate this finding in cells, we used HEK293 cells with stable expression of Pank1β (HEK293/Pank1β), which produce a comparable CoA level to that in rat liver, kidney and primary cardiomyocytes, but much higher in comparison with parental HEK293 cell or other established cell lines [5]. The level of CoA in cells/tissues determines the extent of protein CoAlation, and therefore, HEK293/Pank1β cells have been used widely for the investigation of protein CoAlation under oxidative and metabolic stress [5,8,9,10,11,12,13].
Initially, we examined whether transiently overexpressed HA-mKeap1WT is CoAlated in HEK293/Pank1β cells exposed to diamide in a concentration-dependent manner. In this study, HEK293/Pank1β cells were transfected with HA-mKeap1WT and treated with increasing concentrations of diamide or left untreated as the negative control. The lysates from HA-mKeap1WT transfected or mock-transfected cells were then immunoprecipitated using the anti-HA antibody. Immunoprecipitated proteins and TCLs were resolved by non-reducing SDS-PAGE and analysed by WB with the anti-CoA and anti-HA antibodies. Anti-HA immunoprecipitated samples from mock-transfected cells were used as negative controls. The efficiency of expression and immunoprecipitation of HA-mKeap1WT was confirmed by anti-HA WB of TCL and immunoprecipitated samples (Figure 1). mKeap1 is composed of 624 amino acids with a molecular weight of around 70 kDa [21]. It forms a homodimer (~150 kDa) by non-covalent interactions via the BTB domain in solution [44]. Under non-reducing SDS-PAGE, transiently overexpressed HA-mKeap1WT in untreated cells is predominantly separated as a monomeric form and a less intense covalent dimeric form mediated by disulphide bond formation. Separation of prepared samples under reducing conditions (+DTT) revealed only the monomeric form of HA-Keap1WT in cells, regardless of exposure to diamide or glucose deprivation (Figure S1). The disulphide Keap1 dimer corresponds to two bands at around 150 kDa in TCLs (Figure 1 lanes 6-10). The difference in migration could be attributed to different intramolecular disulphide bond formation within a Keap1 dimer [21]. The intensity of Keap1 monomer and disulphide dimer bands on the anti-HA blot stayed nearly the same in cells treated with 20 μM diamide compared with untreated cells (~2 : 1 ratio), but the shift from monomer to disulphide dimer was noticeable when cells were treated for 30min with 100 μM or higher concentrations of diamide (Figure 1 lanes 8-10). Immunoprecipitated HA-mKeap1WT disulphide dimer was found to be strongly CoAlated in a dose-dependent manner, but not the monomer. The intensity of CoAlation increased with higher doses of diamide, corresponding to the decreased mobility of CoAlated HA-mKeap1WT disulphide dimer. The anti-HA IP and WB of mock-transfected cells showed strong immunoreactivity, corresponding to IgG bands (Figure 1).
Diamide induces oxidative stress by perturbing the balance between oxidised and reduced thiols, whereas other oxidising agents function through different mechanisms. H2O2 and TBH are oxidants while menadione and rotenone increase the production of ROS via redox cycling or disrupting electron transport via complex I, respectively [45,46,47]. After treatment of HEK293/Pank1β cells overexpressing HA-mKeap1WT with different oxidising agents, we observed that diamide and TBH caused the most significant modifications of HA-KEAP1 by CoA and showed a noticeable shift from its monomer to disulphide dimer form on the anti-HA immunoblot (Figure 2 lanes 9 and 12). Cells treated by menadione and H2O2 showed a significant reduction of HA-mKeap1 monomer in TCLs, while rotenone treatment had a much weaker effect. When the immunoprecipitated samples were immunoblotted with anti-CoA antibody, diamide and TBH treatments showed the strongest CoAlation of the HA-mKeap1 disulphide dimer (Figure 2 lanes 9 and 12). A relatively weak CoAlation of HA-mKeap1 disulphide dimer was observed in rotenone-treated cells (Figure2 lane 10), but not in cellular response to H2O2 and menadione when compared to untreated cells (Figure 2 lanes 8 and 11). A weak band of CoAlated HA-mKeap1 in control cells was most likely caused by priming cells (cultured in the pyruvate-free DMEM containing 5 mM glucose overnight, which caused mild metabolic stress) to enhance protein CoAlation under oxidative stress.

3.2. Metabolic Stress Induces CoAlation of HA-mKeap1WT in HEK293/Pank1β Cells

In addition to oxidative stress, metabolic stress caused by glucose/pyruvate depletion has been demonstrated to induce protein CoAlation in both mammalian cells and bacteria [5,8]. Therefore, we investigated whether Keap1 is CoAlated in HEK293/Pank1β cells transiently overexpressing HA-mKeap1WT under metabolic stress. The anti-HA immunoblotting of TCLs and anti-HA immunoprecipitated samples under the non-reducing condition revealed that metabolic stress shifted the monomer:disulphide dimer ratio of HA-mKeap1WT towards the dimeric form (Figure 3 lanes 5-6). Probing the same samples with anti-CoA antibody showed that CoAlation of immunoprecipitated HA-mKeap1WT is strongly induced under complete glucose/pyruvate deprivation (Figure 3 lane 6).

3.3. Oxidative Stress Does Not Induce CoAlation of the HA-mKeap111Cys-less Mutant

To further investigate Keap1 CoAlation, HEK293/Pank1β cells were transiently transfected with pEF-HA-mKeap1WT or pEF-HA-mKeap111Cys-less plasmids, respectively, and then treated with 500 μM diamide. The Keap111Cys-less mutant lacks 11 cysteine residues that have been implicated in the regulation of the Keap1 function. These include C151, C226, C257, C273, C288, C319, C434, C489, C613, C622 and C624 [28]. The HA-mKeap111Cys-less was detected as a monomer in untreated cells (Figure 4 lane 5), while diamide treatment caused the appearance of a weak disulphide dimer band on the anti-HA immunoblot (Figure 4 lane 6). The immunoprecipitated HA-mKeap1WT disulphide dimer was strongly CoAlated in response to diamide treatment (Figure 4). However, HA-mKeap111Cys-less CoAlation was absent after diamide treatment (Figure 4 lane 6), indicating that CoAlation occurs on one or more of these 11 cysteine residues.

4. Discussion

In this study, we report for the first time CoAlation as a novel PTM of Keap1 under oxidative and metabolic stress. Covalent modification of Keap1 by CoA expands regulatory mechanisms of the Keap1-Nrf2 pathway and sheds light on the antioxidant role of CoA by modulating a major signalling pathway of oxidative stress response.
It is well known that two Keap1 molecules form a homodimer via non-covalent interactions mediated by the BTB domain under physiological conditions [44,48]. The existence of the Keap1 disulphide dimer has been also reported in cells exposed to oxidative stress. A study using HeLa cells overexpressing mKeap1 demonstrated that the H2O2-induced Keap1 dimer formation was mediated by a disulphide bond formation between C151 residues of two Keap1 monomers [21]. The formation of covalently linked Keap1 disulphide dimer was also detected in the mouse liver under stressed conditions [49].
A central discovery of this study is the CoAlation of the HA-mKeap1WT disulphide dimer in HEK293/Pank1β cells under oxidative stress induced by a range of oxidising agents, including diamide and TBH. Readily detectable CoAlation of HA-Keap1 disulphide dimer was also observed under metabolic stress, induced by glucose deprivation. Notably, CoAlation was not observed in a Keap1 mutant lacking 11 key cysteine residues, which encompass C151 in the BTB domain, C226, C257, C273 and C288 in the IVR domain, C319, C434 and C489 in the Kelch domain, C613, C622 and C624 in the CTR domain (Figure 5) [28]. We speculate that oxidative and metabolic stress induces the formation of Keap1 disulfide dimers, which promote Keap1 conformational changes, leading to Keap1 CoAlation.
Previous site-directed mutagenesis studies have suggested that C273, C288 and C434 are residues for glutathionylation [41,42]. Mass spectrometry and molecular docking studies demonstrated that C434 can be glutathionylated, which impacts Nrf2 ubiquitination and therefore activates the Nrf2 pathway [42]. An in vitro study has reported that C319 of Keap1 has a high reactivity to form a mixed disulphide bond with GSH [50]. It has been reported that Keap1 C319 is one of the most reactive cysteine residues, which has been reported to be modified by several electrophiles by alkylation to upregulate Nrf2 downstream genes [31,51,52,53,54]. Moreover, it has been shown that C319 can be succinylated by fumarate, contributing to Nrf2 overactivation in fumarate hydratase-related cysts and tumours [33]. Therefore, these four residues could be promising CoAlation sites. Our preliminary data (not shown) revealed that Keap1 C151S, C273W, C288E, C434S single mutants were still CoAlated under oxidative stress, indicating that different cysteine residue(s) may be CoAlated.
Under physiological conditions, CoA is mainly involved in intermediary metabolism via the formation of metabolically active thioesters. However, CoA switches to function as a major cellular antioxidant under oxidative or metabolic stress and may mediate stress-induced redox signalling [6,55]. Previous studies have demonstrated that Keap1 is redox-sensitive and can be glutathionylated under oxidative stress in lymphocytes, brain, pancreatic β cells, lung epithelial cells and several cancer cell lines. Keap1 glutathionylation activates Nrf2-regulated antioxidant and cytoprotective events [30,38,39,40,41,42]. Keap1 CoAlation may cooperate with glutathionylation in facilitating Nrf2-mediated downstream antioxidant responses when cells respond to oxidative or metabolic stress. It has been reported that Nrf2 is activated in skeletal muscles under the fasting condition, which could be related to the Keap1 CoAlation [56].
Several endogenous metabolites have been identified to modify Keap1 and therefore activate Nrf2, including methylglyoxal and glyceraldehyde 3-phosphate that are produced during glycolysis as well as fumarate and itaconate that are derived from the TCA cycle [32,33,34,35]. These findings uncovered the crosstalk between cellular metabolism and stress-response signalling mediated by the Keap1-Nrf2 pathway. Therefore, the discovery of Keap1 CoAlation may highlight a new regulatory mechanism of Keap1 and uncover how a vital metabolic integrator, CoA, functions as a redox effector that connects cellular metabolic states and Nrf2-regulated stress responses. As Keap1 CoAlation is a potential mechanism to protect key cysteines residues from irreversible oxidative damage, Keap1 de-CoAlation to restore its regulatory activity is also worth further investigation [5,57].
The dysregulation of the Keap1-Nrf2 pathway has been implicated in numerous pathologies, including cancer, neurodegenerative disorders and chronic inflammatory diseases [58,59,60]. Our previous study has illustrated extensive anti-CoA immunoreactivity in Alzheimer’s disease post-mortem brain samples as well as tau CoAlation in vitro and in vivo [61]. Thus, understanding how Keap1 CoAlation regulates the Keap1-Nrf2 signalling pathway may facilitate the identification of potential therapeutic targets. Modulating CoA levels could represent an innovative strategy for treating diseases associated with aberrant Keap1-Nrf2 signalling pathway.
In conclusion, this study adds a novel layer to the complex PTMs of Keap1, demonstrating that CoAlation modifies Keap1 in response to oxidative and metabolic stress. By acting as an antioxidant, CoA transcends its conventional metabolic role and may participate in regulating cellular stress responses through the Keap1-Nrf2 pathway. The Keap1 CoAlation sites will be investigated by mass spectrometry and site-specific mutagenesis. Further studies will evaluate the expression levels of Nrf2 and its downstream genes, such as heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1, as well as analysis of Nrf2 nuclear translocation. This work provides a foundation for future studies aiming to delineate the mechanism and functional consequences of Keap1 CoAlation and may offer novel directions for the development of therapeutic interventions that modulate the Keap1-Nrf2 pathway.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org.

Author Contributions

Conceptualization, Ivan Gout and Jerome Gouge; methodology, Oksana Malanchuk and Xuezhe Zhou; validation, Xuezhe Zhou and Oksana Malanchuk; formal analysis, Xuezhe Zhou and Oksana Malanchuk; investigation, Xuezhe Zhou and Oksana Malanchuk.; resources, Takafumi Suzuki, Masayuki Yamamoto, Valeriy Filonenko and Ivan Gout; data curation, Xuezhe Zhou and Oksana Malanchuk.; writing—original draft preparation, Xuezhe Zhou; writing—review and editing, Oksana Malanchuk, Dejun Zhang, Alexander Zhyvoloup, Maria-Armineh Tossounian, Jerome Gouge, Ivan Gout; supervision, Oksana Malanchuk, Jerome Gouge and Ivan Gout; project administration, Ivan Gout; funding acquisition, Ivan Gout. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Biotechnology and Biological Sciences Research Council under Grant BB/L010410/1 and BB/S009027/1 to I.G, and The British Academy of Science Fellowship to O.M. (RaR 100514).

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
Keap1 Kelch-like ECH-associated protein 1
Nrf2 Nuclear factor-erythroid 2-related factor 2
PTM Post-translational modification
CoA Coenzyme A
Redox Reduction-oxidation
ROS Reactive oxygen species
H2O2 Hydrogen peroxide
LMW Low-molecular-weight
GSH Glutathione
BSH Bacillithiol
MSH Mycothiol
TCA Tricarboxylic acid
sMAF Small musculoaponeurotic fibrosarcoma
CNC Cap’n’collar
CsMBE CNC-sMaf binding element
Cul3
RBX1		 Cullin3
RING-box protein 1
NTD N-terminal domain
BTB Broad complex-Tramtrack-Bric-a-brac
IVR Intervening region
CTD C-terminal domain
TBH tert-Butyl hydroperoxide
NEM N-ethylmaleimide
SDS Sodium dodecyl sulfate
DTT Dithiothreitol
Tris-HCl Tris hydrochloride
PIC Protease Inhibitor Cocktail
mAb Monoclonal antibody
HEK293 Human embryonic kidney 293
Pank1β Pantothenate kinase 1β
DMEM Dulbecco’s Modified Eagle Medium
FBS Fetal bovine serum
RT Room temperature
TCL Total cell lysate
WB Western blot
HA-mKeap1WT HA-tagged wild-type mouse Keap1
HA-mKeap111Cys-less HA-tagged mouse Keap1 mutant containing 11 substituted cysteine residues
SDS-PAGE SDS polyacrylamide gel electrophoresis
TBSt Tris-buffer saline containing 0.1% Tween 20

References

  1. Sies, H.; Berndt, C.; Jones, D.P. Oxidative Stress. Annu Rev. Biochem. 2017, 86, 715–48. [Google Scholar] [CrossRef] [PubMed]
  2. Sies, H.; Belousov, V.V.; Chandel, N.S.; Davies, M.J.; Jones, D.P.; Mann, G.E.; et al. Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. Nat. Rev. Mol. Cell. Biol. 2022, 23(7), 499–515. [Google Scholar] [CrossRef]
  3. Tossounian, M.-A.; Zhao, Y.; Yu, B.Y.K.; Markey, S.A.; Malanchuk, O.; Zhu, Y.; et al. Low-molecular-weight thiol transferases in redox regulation and antioxidant defence. Redox Biol. 2024, 71, 103094. [Google Scholar] [CrossRef]
  4. Van Laer, K.; Hamilton, C.J.; Messens, J. Low-molecular-weight thiols in thiol-disulfide exchange. Antioxid. Redox Signal. 2013, 18(13), 1642–53. [Google Scholar] [CrossRef]
  5. Tsuchiya, Y.; Peak-Chew, S.Y.; Newell, C.; Miller-Aidoo, S.; Mangal, S.; Zhyvoloup, A.; et al. Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells. Biochem J. 2017, 474(14), 2489–508. [Google Scholar] [CrossRef]
  6. Brett, C.; Gout, I. The two faces of coenzyme A in cellular biology. Free Radic. Biol. Med. 2025, 233, 162–73. [Google Scholar] [CrossRef]
  7. Tossounian, M.A.; Baczynska, M.; Dalton, W.; Newell, C.; Ma, Y.; Das, S.; et al. Profiling the Site of Protein CoAlation and Coenzyme A Stabilization Interactions. Antioxidants 2022, 11(7). [Google Scholar] [CrossRef] [PubMed]
  8. Tsuchiya, Y.; Zhyvoloup, A.; Baković, J.; Thomas, N.; Yu, B.Y.K.; Das, S.; et al. Protein CoAlation and antioxidant function of coenzyme A in prokaryotic cells. Biochem J. 2018, 475(11), 1909–37. [Google Scholar] [CrossRef] [PubMed]
  9. Baković, J.; Yu, B.Y.K.; Silva, D.; Chew, S.P.; Kim, S.; Ahn, S.H.; et al. A key metabolic integrator, coenzyme A, modulates the activity of peroxiredoxin 5 via covalent modification. Mol. Cell. Biochem. 2019, 461(1-2), 91–102. [Google Scholar] [CrossRef] [PubMed]
  10. Lin, C.C.; Lin, Y.T.; Chen, S.Y.; Setayeshpour, Y.; Chen, Y.; Dunn, D.E.; et al. Coenzyme A protects against ferroptosis via CoAlation of mitochondrial thioredoxin reductase. J. Clin. Invest. 2025, 135(19). [Google Scholar] [CrossRef] [PubMed]
  11. Malanchuk, O.; Bdzhola, A.; Palchevskyi, S.; Bdzhola, V.; Chai, P.; Pardo, O.E.; et al. Investigating the Regulation of Ribosomal Protein S6 Kinase 1 by CoAlation. Int. J. Mol. Sci. 2024, 25(16). [Google Scholar] [CrossRef]
  12. Tsuchiya, Y.; Byrne, D.P.; Burgess, S.G.; Bormann, J.; Baković, J.; Huang, Y.; et al. Covalent Aurora A regulation by the metabolic integrator coenzyme A. Redox Biol. 2020, 28, 101318. [Google Scholar] [CrossRef]
  13. Yu, B.Y.K.; Tossounian, M.A.; Hristov, S.D.; Lawrence, R.; Arora, P.; Tsuchiya, Y.; et al. Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A. Redox Biol. 2021, 44, 101978. [Google Scholar] [CrossRef]
  14. Suzuki, T.; Motohashi, H.; Yamamoto, M. Toward clinical application of the Keap1-Nrf2 pathway. Trends Pharmacol. Sci. 2013, 34(6), 340–6. [Google Scholar] [CrossRef] [PubMed]
  15. Yamamoto, M.; Kensler, T.W.; Motohashi, H. The KEAP1-NRF2 System: a Thiol-Based Sensor-Effector Apparatus for Maintaining Redox Homeostasis. Physiol. Rev. 2018, 98(3), 1169–203. [Google Scholar] [CrossRef] [PubMed]
  16. Tossounian, M.-A.; Zhyvoloup, A.; Chatterjee, R.; Gouge, J. Dissecting the Interplay Between NRF2 and BACH1 at CsMBEs. Antioxidants 2025, 14, 1203. [Google Scholar] [CrossRef]
  17. Zhang, D.D.; Lo, S.C.; Cross, J.V.; Templeton, D.J.; Hannink, M. Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Mol. Cell. Biol. 2004, 24(24), 10941–53. [Google Scholar]
  18. Kobayashi, A.; Kang, M.I.; Okawa, H.; Ohtsuji, M.; Zenke, Y.; Chiba, T.; et al. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol. Cell. Biol. 2004, 24(16), 7130–9. [Google Scholar] [CrossRef]
  19. Furukawa, M.; Xiong, Y. BTB protein Keap1 targets antioxidant transcription factor Nrf2 for ubiquitination by the Cullin 3-Roc1 ligase. Mol. Cell. Biol. 2005, 25(1), 162–71. [Google Scholar] [CrossRef] [PubMed]
  20. Cullinan, S.B.; Gordan, J.D.; Jin, J.; Harper, J.W.; Diehl, J.A. The Keap1-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase: oxidative stress sensing by a Cul3-Keap1 ligase. Mol. Cell. Biol. 2004, 24(19), 8477–86. [Google Scholar] [PubMed]
  21. Fourquet, S.; Guerois, R.; Biard, D.; Toledano, M.B. Activation of NRF2 by nitrosative agents and H2O2 involves KEAP1 disulfide formation. J. Biol. Chem. 2010, 285(11), 8463–71. [Google Scholar] [CrossRef]
  22. Yamamoto, T.; Suzuki, T.; Kobayashi, A.; Wakabayashi, J.; Maher, J.; Motohashi, H.; et al. Physiological significance of reactive cysteine residues of Keap1 in determining Nrf2 activity. Mol. Cell. Biol. 2008, 28(8), 2758–70. [Google Scholar] [CrossRef]
  23. Saito, R.; Suzuki, T.; Hiramoto, K.; Asami, S.; Naganuma, E.; Suda, H.; et al. Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response. Mol. Cell. Biol. 2016, 36(2), 271–84. [Google Scholar] [CrossRef] [PubMed]
  24. Zhang, D.D.; Hannink, M. Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress. Mol. Cell. Biol. 2003, 23(22), 8137–51. [Google Scholar] [CrossRef]
  25. Takaya, K.; Suzuki, T.; Motohashi, H.; Onodera, K.; Satomi, S.; Kensler, T.W.; et al. Validation of the multiple sensor mechanism of the Keap1-Nrf2 system. Free Radic. Biol. Med. 2012, 53(4), 817–27. [Google Scholar] [PubMed]
  26. McMahon, M.; Lamont, D.J.; Beattie, K.A.; Hayes, J.D. Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals. Proc. Natl. Acad. Sci. U S A 2010, 107(44), 18838–43. [Google Scholar] [CrossRef]
  27. Kobayashi, M.; Li, L.; Iwamoto, N.; Nakajima-Takagi, Y.; Kaneko, H.; Nakayama, Y.; et al. The antioxidant defense system Keap1-Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds. Mol. Cell. Biol. 2009, 29(2), 493–502. [Google Scholar] [CrossRef] [PubMed]
  28. Suzuki, T.; Muramatsu, A.; Saito, R.; Iso, T.; Shibata, T.; Kuwata, K.; et al. Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1. Cell. Rep. 2019, 28(3), 746–58.e4. [Google Scholar] [CrossRef]
  29. Ogura, T.; Tong, K.I.; Mio, K.; Maruyama, Y.; Kurokawa, H.; Sato, C.; et al. Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains. Proc. Natl. Acad. Sci. U S A. 2010, 107(7), 2842–7. [Google Scholar] [CrossRef]
  30. Carvalho, A.N.; Marques, C.; Guedes, R.C.; Castro-Caldas, M.; Rodrigues, E.; van Horssen, J.; et al. S-Glutathionylation of Keap1: a new role for glutathione S-transferase pi in neuronal protection. FEBS Lett. 2016, 590(10), 1455–66. [Google Scholar] [CrossRef]
  31. Luo, Y.; Eggler, A.L.; Liu, D.; Liu, G.; Mesecar, A.D.; van Breemen, R.B. Sites of alkylation of human Keap1 by natural chemoprevention agents. J. Am. Soc. Mass. Spectrom. 2007, 18(12), 2226–32. [Google Scholar] [PubMed]
  32. Mills, E.L.; Ryan, D.G.; Prag, H.A.; Dikovskaya, D.; Menon, D.; Zaslona, Z.; et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature. 2018, 556(7699), 113–7. [Google Scholar] [PubMed]
  33. Adam, J.; Hatipoglu, E.; O’Flaherty, L.; Ternette, N.; Sahgal, N.; Lockstone, H.; et al. Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling. Cancer Cell. 2011, 20(4), 524–37. [Google Scholar]
  34. Bollong, M.J.; Lee, G.; Coukos, J.S.; Yun, H.; Zambaldo, C.; Chang, J.W.; et al. A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling. Nature 2018, 562(7728), 600–4. [Google Scholar]
  35. Ko, Y.; Hong, M.; Lee, S.; Kumar, M.; Ibrahim, L.; Nutsch, K.; et al. S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling. Proc. Natl. Acad. Sci. U S A. 2023, 120(20), e2300763120. [Google Scholar]
  36. Um, H.C.; Jang, J.H.; Kim, D.H.; Lee, C.; Surh, Y.J. Nitric oxide activates Nrf2 through S-nitrosylation of Keap1 in PC12 cells. Nitric Oxide. 2011, 25(2), 161–8. [Google Scholar]
  37. Xie, L.; Gu, Y.; Wen, M.; Zhao, S.; Wang, W.; Ma, Y.; et al. Hydrogen Sulfide Induces Keap1 S-sulfhydration and Suppresses Diabetes-Accelerated Atherosclerosis via Nrf2 Activation. Diabetes 2016, 65(10), 3171–84. [Google Scholar] [CrossRef] [PubMed]
  38. Li, N.; Wang, J.; Zang, X.; Wang, Z.; Zhang, T.; Zhao, B.; et al. H(2)S probe CPC inhibits autophagy and promotes apoptosis by inhibiting glutathionylation of Keap1 at Cys434. Apoptosis. 2021, 26(1-2), 111–31. [Google Scholar] [CrossRef]
  39. Gambhir, L.; Checker, R.; Thoh, M.; Patwardhan, R.S.; Sharma, D.; Kumar, M.; et al. 1,4-Naphthoquinone, a pro-oxidant, suppresses immune responses via KEAP-1 glutathionylation. Biochem Pharmacol. 2014, 88(1), 95–105. [Google Scholar]
  40. Wang, L.; Qu, G.; Gao, Y.; Su, L.; Ye, Q.; Jiang, F.; et al. A small molecule targeting glutathione activates Nrf2 and inhibits cancer cell growth through promoting Keap-1 S-glutathionylation and inducing apoptosis. RSC Adv. 2018, 8(2), 792–804. [Google Scholar]
  41. Chen, X.; Zhou, Q.; Chen, H.; Bai, J.; An, R.; Zhang, K.; et al. Glutathione Induces Keap1 S-Glutathionylation and Mitigates Oscillating Glucose-Induced β-Cell Dysfunction by Activating Nrf2. Antioxidants . 2024, 13(4). [Google Scholar] [CrossRef]
  42. Sun, X.; Guo, C.; Huang, C.; Lv, N.; Chen, H.; Huang, H.; et al. GSTP alleviates acute lung injury by S-glutathionylation of KEAP1 and subsequent activation of NRF2 pathway. Redox Biol. 2024, 71, 103116. [Google Scholar] [CrossRef]
  43. Malanchuk, O.; Panasyuk, G.; Serbin, N.; Gout, I.; Filonenko, V. Generation and characterization of monoclonal antibodies specific to Coenzyme A. Biopolym. Cell. 2015, 31, 187–92. [Google Scholar] [CrossRef]
  44. Wakabayashi, N.; Dinkova-Kostova, A.T.; Holtzclaw, W.D.; Kang, M.I.; Kobayashi, A.; Yamamoto, M.; et al. Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers. Proc. Natl. Acad. Sci. U S A 2004, 101(7), 2040–5. [Google Scholar] [CrossRef]
  45. Criddle, D.N.; Gillies, S.; Baumgartner-Wilson, H.K.; Jaffar, M.; Chinje, E.C.; Passmore, S.; et al. Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells. J. Biol. Chem. 2006, 281(52), 40485–92. [Google Scholar] [CrossRef]
  46. Li, N.; Ragheb, K.; Lawler, G.; Sturgis, J.; Rajwa, B.; Melendez, J.A.; et al. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J. Biol. Chem. 2003, 278(10), 8516–25. [Google Scholar] [CrossRef]
  47. Kosower, N.S.; Kosower, E.M. Diamide: an oxidant probe for thiols. Methods Enzymol. 1995, 251, 123–33. [Google Scholar]
  48. Zipper, L.M.; Mulcahy, R.T. The Keap1 BTB/POZ dimerization function is required to sequester Nrf2 in cytoplasm. J. Biol. Chem. 2002, 277(39), 36544–52. [Google Scholar] [CrossRef]
  49. Sato, M.; Yaguchi, N.; Iijima, T.; Muramatsu, A.; Baird, L.; Suzuki, T.; et al. Sensor systems of KEAP1 uniquely detecting oxidative and electrophilic stresses separately In vivo. Redox Biol. 2024, 77, 103355. [Google Scholar] [CrossRef]
  50. Holland, R.; Hawkins, A.E.; Eggler, A.L.; Mesecar, A.D.; Fabris, D.; Fishbein, J.C. Prospective type 1 and type 2 disulfides of Keap1 protein. Chem. Res. Toxicol. 2008, 21(10), 2051–60. [Google Scholar] [CrossRef]
  51. Dunlap, T.; Piyankarage, S.C.; Wijewickrama, G.T.; Abdul-Hay, S.; Vanni, M.; Litosh, V.; et al. Quinone-induced activation of Keap1/Nrf2 signaling by aspirin prodrugs masquerading as nitric oxide. Chem. Res. Toxicol. 2012, 25(12), 2725–36. [Google Scholar] [CrossRef]
  52. Hong, F.; Sekhar, K.R.; Freeman, M.L.; Liebler, D.C. Specific patterns of electrophile adduction trigger Keap1 ubiquitination and Nrf2 activation. J. Biol. Chem. 2005, 280(36), 31768–75. [Google Scholar] [CrossRef]
  53. Eggler, A.L.; Liu, G.; Pezzuto, J.M.; van Breemen, R.B.; Mesecar, A.D. Modifying specific cysteines of the electrophile-sensing human Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2. Proc. Natl. Acad. Sci. U S A. 2005, 102(29), 10070–5. [Google Scholar] [CrossRef]
  54. Dietz, B.M.; Liu, D.; Hagos, G.K.; Yao, P.; Schinkovitz, A.; Pro, S.M.; et al. Angelica sinensis and its alkylphthalides induce the detoxification enzyme NAD(P)H: quinone oxidoreductase 1 by alkylating Keap1. Chem. Res. Toxicol. 2008, 21(10), 1939–48. [Google Scholar] [CrossRef]
  55. Desideri, E.; Filomeni, G.; Ciriolo, M.R. Glutathione participates in the modulation of starvation-induced autophagy in carcinoma cells. Autophagy. 2012, 8(12), 1769–81. [Google Scholar] [CrossRef]
  56. Lettieri-Barbato, D.; Minopoli, G.; Caggiano, R.; Izzo, R.; Santillo, M.; Aquilano, K.; et al. Fasting Drives Nrf2-Related Antioxidant Response in Skeletal Muscle. Int. J. Mol. Sci. 2020, 21(20). [Google Scholar] [CrossRef]
  57. Tossounian, M.A.; Baczynska, M.; Dalton, W.; Peak-Chew, S.Y.; Undzenas, K.; et al. Bacillus subtilis YtpP and Thioredoxin A Are New Players in the Coenzyme-A-Mediated Defense Mechanism against Cellular Stress. Antioxidants 2023, 12(4), 938. [Google Scholar] [CrossRef]
  58. Ahmed, S.M.; Luo, L.; Namani, A.; Wang, X.J.; Tang, X. Nrf2 signaling pathway: Pivotal roles in inflammation. Biochim Biophys. Acta Mol. Basis Dis. 2017, 1863(2), 585–97. [Google Scholar] [CrossRef]
  59. Adinolfi, S.; Patinen, T.; Deen, J.A.; Pitkänen, S.; Härkönen, J.; Kansanen, E.; et al. The KEAP1-NRF2 pathway: Targets for therapy and role in cancer. Redox Biol. 2023, 63, 102726. [Google Scholar] [CrossRef]
  60. Yamazaki, H.; Tanji, K.; Wakabayashi, K.; Matsuura, S.; Itoh, K. Role of the Keap1/Nrf2 pathway in neurodegenerative diseases. Pathol. Int. 2015, 65(5), 210–9. [Google Scholar] [CrossRef]
  61. Lashley, T.; Tossounian, M.-A.; Heaven, C.N.; Wallworth, S.; Peak-Chew, S.; Bradshaw, A.; et al. Extensive Anti-CoA Immunostaining in Alzheimer’s Disease and Covalent Modification of Tau by a Key Cellular Metabolite Coenzyme A. Front Cell. Neurosci. 2021, 15, 739425. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. HA-mKeap1WT is CoAlated in HEK293/Pank1β cells treated with diamide. HEK293/Pank1β cells were transiently transfected with or without the pEF-HA-mKeap1WT plasmid. 24 hours later, the medium was replaced with fresh pyruvate-free DMEM medium containing 5 mM glucose to prime cells for oxidative stress. After culturing for additional 24 hours, cells were treated with or without diamide. TCLs and anti-HA immunoprecipitated samples were separated by SDS-PAGE under the non-reducing condition and analysed by Western blotting with anti-CoA and anti-HA antibodies. The positions of Keap1 monomer and disulphide dimer, and anti-HA antibody heavy and light chains) are indicated. N=3.
Figure 1. HA-mKeap1WT is CoAlated in HEK293/Pank1β cells treated with diamide. HEK293/Pank1β cells were transiently transfected with or without the pEF-HA-mKeap1WT plasmid. 24 hours later, the medium was replaced with fresh pyruvate-free DMEM medium containing 5 mM glucose to prime cells for oxidative stress. After culturing for additional 24 hours, cells were treated with or without diamide. TCLs and anti-HA immunoprecipitated samples were separated by SDS-PAGE under the non-reducing condition and analysed by Western blotting with anti-CoA and anti-HA antibodies. The positions of Keap1 monomer and disulphide dimer, and anti-HA antibody heavy and light chains) are indicated. N=3.
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Figure 2. Induction of Keap1 CoAlation in response to oxidising agents in HEK293/Pank1β cells transiently overexpressing HA-mKeap1WT. HEK293/Pank1β cells were transiently transfected with or without the pEF-HA-mKeap1WT plasmid. 24 hours later, the medium was replaced with fresh pyruvate-free DMEM medium containing 5 mM glucose to prime cells for oxidative stress. After culturing for additional 24 hours, cells were treated with or without a panel of oxidising agents. TCLs and anti-HA immunoprecipitated samples were separated by SDS-PAGE under the non-reducing condition and analysed by Western blotting with anti-CoA and anti-HA antibodies. The positions of Keap1 monomer and disulphide dimer, and anti-HA antibody (heavy and light chains) are indicated. N=3.
Figure 2. Induction of Keap1 CoAlation in response to oxidising agents in HEK293/Pank1β cells transiently overexpressing HA-mKeap1WT. HEK293/Pank1β cells were transiently transfected with or without the pEF-HA-mKeap1WT plasmid. 24 hours later, the medium was replaced with fresh pyruvate-free DMEM medium containing 5 mM glucose to prime cells for oxidative stress. After culturing for additional 24 hours, cells were treated with or without a panel of oxidising agents. TCLs and anti-HA immunoprecipitated samples were separated by SDS-PAGE under the non-reducing condition and analysed by Western blotting with anti-CoA and anti-HA antibodies. The positions of Keap1 monomer and disulphide dimer, and anti-HA antibody (heavy and light chains) are indicated. N=3.
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Figure 3. Metabolic stress induces CoAlation of HA-mKeap1WT in HEK293/Pank1β cells. HEK293/Pank1β cells were transiently transfected with or without pEF-HA-mKeap1WT plasmid. 24 hours later, the medium was replaced with fresh pyruvate-free DMEM medium containing 5 mM glucose or fresh DMEM lacking glucose and pyruvate to induce metabolic stress for 24 hours. TCLs and anti-HA immunoprecipitated samples were separated by SDS-PAGE under the non-reducing condition and analysed by Western blotting with anti-CoA and anti-HA antibodies. The positions of Keap1 monomer and disulphide dimer, and anti-HA antibody (heavy and light chains) are indicated. N=3.
Figure 3. Metabolic stress induces CoAlation of HA-mKeap1WT in HEK293/Pank1β cells. HEK293/Pank1β cells were transiently transfected with or without pEF-HA-mKeap1WT plasmid. 24 hours later, the medium was replaced with fresh pyruvate-free DMEM medium containing 5 mM glucose or fresh DMEM lacking glucose and pyruvate to induce metabolic stress for 24 hours. TCLs and anti-HA immunoprecipitated samples were separated by SDS-PAGE under the non-reducing condition and analysed by Western blotting with anti-CoA and anti-HA antibodies. The positions of Keap1 monomer and disulphide dimer, and anti-HA antibody (heavy and light chains) are indicated. N=3.
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Figure 4. CoAlation of the HA-mKeap111Cys-less mutant is not induced by diamide. HEK293/Pank1β cells were transiently transfected with pEF-HA-mKeap1WT or pEF-HA-mKeap111Cys-less mutant plasmids. 24 hours later, the medium was replaced with fresh pyruvate-free DMEM medium containing 5 mM glucose to prime cells for oxidative stress. After culturing for additional 24 hours, cells were treated with or without diamide. TCLs and anti-HA immunoprecipitated samples were separated by SDS-PAGE under the non-reducing condition and analysed by Western blotting with anti-CoA and anti-HA antibodies. The positions of Keap1 monomer and disulphide dimer, and anti-HA antibody heavy and light chains) are indicated. N=3.
Figure 4. CoAlation of the HA-mKeap111Cys-less mutant is not induced by diamide. HEK293/Pank1β cells were transiently transfected with pEF-HA-mKeap1WT or pEF-HA-mKeap111Cys-less mutant plasmids. 24 hours later, the medium was replaced with fresh pyruvate-free DMEM medium containing 5 mM glucose to prime cells for oxidative stress. After culturing for additional 24 hours, cells were treated with or without diamide. TCLs and anti-HA immunoprecipitated samples were separated by SDS-PAGE under the non-reducing condition and analysed by Western blotting with anti-CoA and anti-HA antibodies. The positions of Keap1 monomer and disulphide dimer, and anti-HA antibody heavy and light chains) are indicated. N=3.
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Figure 5. Schematic demonstration of domain organisation of cysteine-rich wild-type mouse Keap1 (A) and an 11 Cys-less mutant (B).
Figure 5. Schematic demonstration of domain organisation of cysteine-rich wild-type mouse Keap1 (A) and an 11 Cys-less mutant (B).
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