4-Hydroxynonenal and 4-Oxononenal Differentially Bind to the Redox

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4‑Hydroxynonenal and 4‑Oxononenal Differentially Bind to the Redox Sensor MitoNEET Dayna Arnett,† Alexandria Quillin,† Werner J. Geldenhuys,‡ Michael A. Menze,§ and Mary Konkle*,† †

Department of Chemistry, Ball State University, Muncie, Indiana 47304, United States, School of Pharmacy, West Virginia University, Morgantown, West Virginia 26506, United States § Department of Biology, University of Louisville, Louisville, Kentucky 40292, United States ‡

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S Supporting Information *

ABSTRACT: MitoNEET is a CDGSH iron−sulfur protein that has been a target for drug development for diseases such as type-2 diabetes, cancer, and Parkinson’s disease. Functions proposed for mitoNEET are as a redox sensor and regulator of free iron in the mitochondria. We have investigated the reactivity of mitoNEET toward the reactive electrophiles 4-hydroxynonenal (HNE) and 4-oxononenal (ONE) that are produced from the oxidation of polyunsaturated fatty acid during oxidative stress. Proteomic, electrophoretic, and spectroscopic analysis has shown that HNE and ONE react in a sequence selective manner that was unexpected considering the structure similarity of these two reactive electrophiles.

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considers the three ligating cysteine residues (Cys72, Cys74, and Cys83), the ligating histidine residues (His87), and a lysine (Lys55′) from the opposite protomer (Figure S1.). The reactivity of these three residues for reactions toward electrophile HNE and ONE is considered, absent additional factors, to be Cys > His > Lys.1,2 Interestingly, the ligating histidine of mitoNEET, His87, is in a hydrogen bonding network with Lys55′ of the other protomer in the dimer (mediated by a structural water), which would activate Lys55′ (Figure S1) for Michael addition or imine formation.18,19 Previous studies have discovered that the ligating cysteine residues of mitoNEET are reactive to forming disulfide bonds in a manner that is dependent on sequence of the cysteine-supplying polypeptide chains. Cys83 of mitoNEET will form a disulfide bond with its noncovalent dimer partner at either Cys74 or Cys72.20 In comparison, mitoNEET will form a mixed disulfide linkage with Cys74 and Cys319 of glutamate dehydrogenase 1 (GDH1).20 These results indicate that the ligating cysteine residues of mitoNEET are activated to react with atoms other than the canonical iron.

roducts of cellular oxidized phospholipids include the reactive electrophiles 4-hydroxynonenal (HNE) and 4oxononenal (ONE). The α,β-unsaturated aldehyde moiety in both compounds is known to covalently modify both DNA and proteins. Specifically, HNE and ONE readily form Michael adducts with the nucleophilic side chains of lysine, histidine, and cysteine.1,2 Additionally, HNE and ONE can bind to lysine residues to form an imine. HNE is found in higher concentrations in vivo, but ONE is considered more toxic in vitro. However, the specific cellular detoxification processes ultimately impact the toxicity profile of HNE and ONE.3,4 MitoNEET is a [2Fe-2S] cluster-containing protein of whose physiological function(s) is still unclear.5−7 However, several of the proposed functions, such as iron−sulfur cluster transfer, electron transport, or redox sensing, are focused around the metal cluster. The crystal structures of wild-type (2QD0, 3REE, 2R13, 3EW0, and 2QH7) and mutant mitoNEET (3LPQ, 4F1E, 4F28, 4EZF, and 4F2C) in the Protein Data Bank are homodimers and are not bound to any organic ligands, despite a wide variety of ligands that were identified through other techniques.8−17 The area within 7 A of the metal cluster in mitoNEET is rich with residues that may be modified by HNE or ONE through Michael addition or imine formation when one © XXXX American Chemical Society

Received: April 16, 2019

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DOI: 10.1021/acs.chemrestox.9b00166 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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mitoNEET monomers (Figure 1B). This covalent dimer formed after treatment with ONE could not be reduced to the monomer by the addition of ΒΜΕ post modification (Figure 1B). This is in stark contrast to the previously observed higher order oligomers of mitoNEET linked by a disulfide bond.20 Treatment of mitoNEET with ONE also caused a shift from the bands being majority monomer (in the control samples) to majority covalent dimer (in the treated samples) (Figure 1D). Cross-linking has been previously noted with both HNE and ONE treatment of tubulins and pyruvate kinase isoform M2 but was not selective in regard to the reactive electrophile.4,22 Other reports of crosslinking that are ONE specific either require a participating cysteine residue, Michael adducts, or pyrrole formation.22−24 Considering this, a possible model for the cross-linking of mitoNEET monomers is an initial Michael reaction with His87 with a subsequent imine formation with Lys55′ of the other monomer. The sites of modification on mitoNEET by HNE were determined by proteomic analysis. MitoNEET contains 10 lysine residues in its soluble portion. Similar to a previous report, His90 was transformed into the Michael adduct by HNE. However, the ligating His87 was also modified under the in vitro conditions. Four lysine residues, Lys55, Lys68, Lys89, and Lys104, were modified by HNE (Figure 2). The fact that the Cys residues (all of which ligate the [2Fe-2S] cluster) remained unmodified indicates that the propensity to form the Michael adduct is less than the stability of the Fe−S ligating bond. No evidence of an imine formation (+138 m/z) was observed. Samples of mitoNEET treated with either ONE or ONE and the reductant sodium borohydride were also analyzed by proteomic techniques. A mass shift of +136 m/z for the imine and +138 m/z for the amine in the reduced samples was observed for Lys55, Lys68, Lys104, and Lys105 (Figure 3A) and was consistent with other reports of ONE protein adducts. However, the ONE-imine adduct was selective for Lys55 in that 50% of that fragments containing Lys55 were modified. This is compared to less than 10% for any other modified Lys (Figure 3B). There is a possibility that the modified peptides containing ONE-modified Lys55 ionize exceptionally well. However, given the consistent percentage modified versus total peptides for all other modification sites (for both ONE and HNE treatment), this is unlikely. Additionally, the reduction of the imine to the amine by sodium borohydride did not increase the yield. The samples were also analyzed for the expected Michael adducts of +158 m/z. Only Lys104 and Lys105 were available for Michael chemistry with ONE and at a level comparable to that of HNE adduction (Figure 3C). None of the His or Cys residues was modified. Of particular note, the ligating His87 was not modified. The modification of His87 upon treatment of mitoNEET by HNE was confirmed by monitoring the spectroscopy of the ligand to metal charge transfer (LMCT) bands. Covalent modification of the ligating histidine residue(s) of the [2Fe-2S] cluster containing Rieske protein cause a weakening and shape change of the LMCT bands.25,26 In stark contrast, the modification of the lone ligating histidine residue of mitoNEET by HNE caused an increase of the LMCT bands over time (Figure S2A,C). One explanation of this observation is that the accumulation of the adduct promotes a steric restriction that makes the transition of the LMCT bands more allowed. In stark contrast, the LMCT bands of mitoNEET are not increased upon treatment with ONE (Figure S2B). This is consistent with the

Previous work identified mitoNEET as modified by HNE (at His90) in a mouse model of alcoholic liver disease by two distinct methods of proteomic analysis.21 While these authors classified mitoNEET as having a low reactivity with HNE in the quantitative portion of the study, it is worth noting that the correlation value in the dose-dependent treatment was high (0.982) despite a low signal.21 However, this analysis was of a sample with a complicated matrix of proteins, only looked for HNE adducts, and gave no mechanistic insight on the modification(s). To bridge this knowledge gap, we have undertaken in vitro studies using purified mitoNEET and treated with HNE and ONE. HNE was added to either mitoNEET or a mixture of mitoNEET and GDH1 and analyzed by SDS-PAGE gel electrophoresis to determine if HNE would interrupt the formation of the covalent mitoNEET-mitoNEET or mitoNEET−GDH1 complexes. HNE did not interrupt the formation of the mixed GDH1−mitoNEET complex (Figure 1A). In contrast, the addition of HNE did interrupt the

Figure 1. Gel analysis of the modification of mitoNEET by reactive electrophiles. (A) SDS-PAGE gel of mitoNEET (Lane 1), GDH1 (Lane 2), mitoNEET + GDH (Lane 3), mitoNEET + HNE (Lane 4), mitoNEET + HNE + GDH1 (Lane 5), and GDH1 + HNE (Lane 6). (B) SDS-PAGE gel of mitoNEET (Lane 1) and mitoNEET + ONE (Lane 2). The addition of BME to the sample buffer is denoted with a + in panels A and B. (C) Area for the mitoNEET monomer band (13 kDa) and covalent dimer band (26 kDa) in control (black bars) and HNE treated samples (dotted bars). (D) Area for the mitoNEET monomer band (13 kDa) and covalent dimer band (26 kDa) for the control (black bars), mitoNEET + ONE (dotted bars), and mitoNEET + ONE + BME (checkered boxes). All errors shown for panels C and D are SEM, n = 3.

formation of the covalent dimer of mitoNEET formed through a disulfide bond between Cys83 and either Cys72/Cys74. This was demonstrated by the 26 kDa band disappearing upon treatment with β-mercaptoethanol (BME) after the modification reaction.20 On Coomassie-blue stained SDS-PAGE gels without HNE treatment, the monomer band is 1.6-times more intense than the dimer band, whereas that same ratio shifts to 3.9-times more intensity when treated with HNE (Figure 1C). In a similar fashion, mitoNEET was treated with ONE. ONE also did not disrupt the formation of the mixed GDH1− mitoNEET product (data not shown). Strikingly, the addition of ONE caused the formation of a covalently linked dimer of B

DOI: 10.1021/acs.chemrestox.9b00166 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Figure 3. Proteomic analysis of mitoNEET reacted with ONE. (A) Sites of modification on mitoNEET to form Michael adducts (in salmon) or imine adducts (in dark purple). (B) Percentage of modification to form the (a) imine or (b) amine after treatment with NaBH4 both dehydrated in source to give a +136 or +138 m/z shift, respectively. (C) Modification to form the Michael adduct (under both nonreducing and reducing conditions) as a percentage of the total counts of identified peptide. (D) Scheme for ONE addition to Lys55 of mitoNEET.

adducts. The reaction of mitoNEET with ONE, however, unveiled a particularly reactive Lys55 residue. Additionally, the amine of Lys55 is prone to form an unusually stable imine with ONE. The imine formation with HNE or ONE has certainly been reported previously, but the dogma is that the imine formation is only preferred for buried lysine residues in a hydrophobic environment.2 All of the crystal structures show Lys55 in a solvent exposed environment so either the crystal structures are not entirely representative of the mitoNEET structure in solution or the local environment of this residue significantly increases the Lys55 reactivity. MitoNEET has been identified as a redox mediator in the cell.27 Cells from knockout animals show altered oxidative phosphorylation and significantly increased amount of reactive oxygen species.8 Additionally, mitoNEET appears to have a similar role in cancer cells through the (dys)regulation of ligating free iron in the cell.28 A higher level of free iron in the cell promotes the production of HNE and ONE through Fenton chemistry. Taken together, this mechanistic in vitro work places the protein mitoNEET in the context of reactive electrophiles.

Figure 2. Proteomic analysis of mitoNEET by HNE. (A) Location of Michael adducts on mitoNEET (+156 m/z). All modified residues are shown in salmon, and the modified locations on one side of the dimer are labeled. (B) Modification as a percentage of the total counts of identified peptide. (C) Scheme for Michael addition to histidine and lysine residues.

proteomic results of the selective modification of predominantly Lys55 residue but none of the ligating residues. The selectivity of binding and subsequent modification of HNE and ONE with mitoNEET was unexpected. HNE modified over the whole structure of mitoNEET at both histidine and lysine residues and forming the canonical Michael C

DOI: 10.1021/acs.chemrestox.9b00166 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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linked specifically by a thiazolidinedione photoprobe. Am. J. Phys.Endo. Met. 286 (2), E252−60. (8) Geldenhuys, W. J., Benkovic, S. A., Lin, L., Yonutas, H. M., Crish, S. D., Sullivan, P. G., Darvesh, A. S., Brown, C. M., and Richardson, J. R. (2017) MitoNEET (CISD1) Knockout Mice Show Signs of Striatal Mitochondrial Dysfunction and a Parkinson’s Disease Phenotype. ACS Chem. Neurosci. 8 (12), 2759−2765. (9) Vernay, A., Marchetti, A., Sabra, A., Jauslin, T. N., Rosselin, M., Scherer, P. E., Demaurex, N., Orci, L., and Cosson, P. (2017) MitoNEET-dependent formation of intermitochondrial junctions. Proc. Natl. Acad. Sci. U. S. A. 114 (31), 8277−8282. (10) Patel, S. P., Cox, D. H., Gollihue, J. L., Bailey, W. M., Geldenhuys, W. J., Gensel, J. C., Sullivan, P. G., and Rabchevsky, A. G. (2017) Pioglitazone treatment following spinal cord injury maintains acute mitochondrial integrity and increases chronic tissue sparing and functional recovery. Exp. Neurol. 293, 74−82. (11) Landry, A. P., Wang, Y., Cheng, Z., Crochet, R. B., Lee, Y. H., and Ding, H. (2017) Flavin nucleotides act as electron shuttles mediating reduction of the [2Fe-2S] clusters in mitochondrial outer membrane protein mitoNEET. Free Radical Biol. Med. 102, 240−247. (12) Geldenhuys, W. J., Yonutas, H. M., Morris, D. L., Sullivan, P. G., Darvesh, A. S., and Leeper, T. C. (2016) Identification of small molecules that bind to the mitochondrial protein mitoNEET. Bioorg. Med. Chem. Lett. 26 (21), 5350−5353. (13) Kusminski, C. M., Chen, S., Ye, R., Sun, K., Wang, Q. A., Spurgin, S. B., Sanders, P. E., Brozinick, J. T., Geldenhuys, W. J., Li, W. H., Unger, R. H., and Scherer, P. E. (2016) MitoNEET-Parkin Effects in Pancreatic alpha- and beta-Cells, Cellular Survival, and Intrainsular Cross Talk. Diabetes 65 (6), 1534−55. (14) Tan, G., Liu, D., Pan, F., Zhao, J., Li, T., Ma, Y., Shen, B., and Lyu, J. (2016) His-87 ligand in mitoNEET is crucial for the transfer of iron sulfur clusters from mitochondria to cytosolic aconitase. Biochem. Biophys. Res. Commun. 470 (1), 226−232. (15) Bai, F., Morcos, F., Sohn, Y. S., Darash-Yahana, M., Rezende, C. O., Lipper, C. H., Paddock, M. L., Song, L., Luo, Y., Holt, S. H., Tamir, S., Theodorakis, E. A., Jennings, P. A., Onuchic, J. N., Mittler, R., and Nechushtai, R. (2015) The Fe-S cluster-containing NEET proteins mitoNEET and NAF-1 as chemotherapeutic targets in breast cancer. Proc. Natl. Acad. Sci. U. S. A. 112 (12), 3698−703. (16) Takahashi, T., Yamamoto, M., Amikura, K., Kato, K., Serizawa, T., Serizawa, K., Akazawa, D., Aoki, T., Kawai, K., Ogasawara, E., Hayashi, J., Nakada, K., and Kainoh, M. (2015) A novel MitoNEET ligand, TT01001, improves diabetes and ameliorates mitochondrial function in db/db mice. J. Pharmacol. Exp. Ther. 352 (2), 338−45. (17) Kusminski, C. M., Park, J., and Scherer, P. E. (2014) MitoNEETmediated effects on browning of white adipose tissue. Nat. Commun. 5, 3962. (18) Bak, D. W., and Elliott, S. J. (2013) Conserved hydrogen bonding networks of MitoNEET tune Fe-S cluster binding and structural stability. Biochemistry 52 (27), 4687−96. (19) Bak, D. W., Zuris, J. A., Paddock, M. L., Jennings, P. A., and Elliott, S. J. (2009) Redox characterization of the FeS protein MitoNEET and impact of thiazolidinedione drug binding. Biochemistry 48 (43), 10193−5. (20) Roberts, M. E., Crail, J. P., Laffoon, M. M., Fernandez, W. G., Menze, M. A., and Konkle, M. E. (2013) Identification of disulfide bond formation between MitoNEET and glutamate dehydrogenase 1. Biochemistry 52 (50), 8969−71. (21) Tzeng, S. C., and Maier, C. S. (2016) Label-Free Proteomics Assisted by Affinity Enrichment for Elucidating the Chemical Reactivity of the Liver Mitochondrial Proteome toward Adduction by the Lipid Electrophile 4-hydroxy-2-nonenal (HNE). Front. Chem. 4, 2. (22) Stewart, B. J., Doorn, J. A., and Petersen, D. R. (2007) Residuespecific adduction of tubulin by 4-hydroxynonenal and 4-oxononenal causes cross-linking and inhibits polymerization. Chem. Res. Toxicol. 20 (8), 1111−9. (23) Aluise, C. D., Camarillo, J. M., Shimozu, Y., Galligan, J. J., Rose, K. L., Tallman, K. A., and Marnett, L. J. (2015) Site-specific,

MitoNEET itself may, in fact, have cellular roles in regulating while simultaneously uncovering the selective reactivity of Lys55 toward imine formation and stability.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.9b00166. Experimental section; molecular model of mitoNEET from PDB file EW0; spectroscopy of mitoNEET modification; mass spectra of modified peptides (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (765)285-2195. ORCID

Werner J. Geldenhuys: 0000-0002-2405-376X Michael A. Menze: 0000-0003-1072-5462 Mary Konkle: 0000-0003-0959-3714 Funding

We thank the National Science Foundation (RUI-#1806266, M.K. and M.M.) and the National Institutes of Health (R41NS110070 and GM109098, W.G.) for the funding. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Dr. Phil Albiniak for helpful discussions. ABBREVIATIONS BME, β-mercaptoethanol; CISD, CDGSH iron−sulfur domain; HNE, 4-hydroxynonenal; ONE, 4-oxononenal; GDH1, glutamate dehydrogenase 1

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REFERENCES

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intramolecular cross-linking of Pin1 active site residues by the lipid electrophile 4-oxo-2-nonenal. Chem. Res. Toxicol. 28 (4), 817−27. (24) Zhu, X., and Sayre, L. M. (2007) Mass spectrometric evidence for long-lived protein adducts of 4-oxo-2-nonenal. Redox Rep. 12 (1), 45− 9. (25) Karagas, N. E., Jones, C. N., Osborn, D. J., Dzierlenga, A. L., Oyala, P., Konkle, M. E., Whitney, E. M., David Britt, R., and HunsickerWang, L. M. (2014) The reduction rates of DEPC-modified mutant Thermus thermophilus Rieske proteins differ when there is a negative charge proximal to the cluster. JBIC, J. Biol. Inorg. Chem. 19 (7), 1121− 35. (26) Konkle, M. E., Elsenheimer, K. N., Hakala, K., Robicheaux, J. C., Weintraub, S. T., and Hunsicker-Wang, L. M. (2010) Chemical modification of the Rieske protein from Thermus thermophilus using diethyl pyrocarbonate modifies ligating histidine 154 and reduces the [2FE-2S] cluster. Biochemistry 49 (34), 7272−81. (27) Landry, A. P., Cheng, Z., and Ding, H. (2015) Reduction of mitochondrial protein mitoNEET [2Fe-2S] clusters by human glutathione reductase. Free Radical Biol. Med. 81, 119. (28) Mittler, R., Darash-Yahana, M., Sohn, Y. S., Bai, F., Song, L., Cabantchik, I. Z., Jennings, P. A., Onuchic, J. N., and Nechushtai, R. (2019) NEET Proteins: A New Link Between Iron Metabolism, Reactive Oxygen Species, and Cancer. Antioxid. Redox Signaling 30 (8), 1083−1095.

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DOI: 10.1021/acs.chemrestox.9b00166 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX