Browsing by Author "Gross, Michael L."
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Item Characterization of the redox activity and disulfide bond formation in Apurinic/apyrimidinic endonuclease(2012-01) Luo, Meihua; Zhang, Jun; He, Hongzhen; Su, Dian; Chen, Qiujia; Gross, Michael L.; Kelley, Mark R.; Georgiadis, Millie M.Apurinic/apyrimidinic endonuclease (APE1) is an unusual nuclear redox factor in which the redox-active cysteines identified to date, C65 and C93, are surface inaccessible residues whose activities may be influenced by partial unfolding of APE1. To assess the role of the five remaining cysteines in APE1’s redox activity, double-cysteine mutants were analyzed, excluding C65A, which is redox-inactive as a single mutant. C93A/C99A APE1 was found to be redox-inactive, whereas other double-cysteine mutants retained the same redox activity as that observed for C93A APE1. To determine whether these three cysteines, C65, C93, and C99, were sufficient for redox activity, all other cysteines were substituted with alanine, and this protein was shown to be fully redox-active. Mutants with impaired redox activity failed to stimulate cell proliferation, establishing an important role for APE1’s redox activity in cell growth. Disulfide bond formation upon oxidation of APE1 was analyzed by proteolysis of the protein followed by mass spectrometry analysis. Within 5 min of exposure to hydrogen peroxide, a single disulfide bond formed between C65 and C138 followed by the formation of three additional disulfide bonds within 15 min; 10 total disulfide bonds formed within 1 h. A single mixed-disulfide bond involving C99 of APE1 was observed for the reaction of oxidized APE1 with thioredoxin (TRX). Disulfide-bonded APE1 or APE1–TRX species were further characterized by size exclusion chromatography and found to form large complexes. Taken together, our data suggest that APE1 is a unique redox factor with properties distinct from those of other redox factors.Item Functional Analysis of Novel Analogues of E3330 That Block the Redox Signaling Activity of the Multifunctional AP Endonuclease/Redox Signaling Enzyme APE1/Ref-1(2011-03) Kelley, Mark R.; Luo, Meihua; Reed, April; Su, Dian; Delaplane, Sarah; Borch, Richard F.; Nyland II, Rodney L.; Gross, Michael L.; Georgiadis, Millie M.APE1 is a multifunctional protein possessing DNA repair and redox activation of transcription factors. Blocking these functions leads to apoptosis, antiangiogenesis, cell-growth inhibition, and other effects, depending on which function is blocked. Because a selective inhibitor of the APE redox function has potential as a novel anticancer therapeutic, new analogues of E3330 were synthesized. Mass spectrometry was used to characterize the interactions of the analogues (RN8-51, 10-52, and 7-60) with APE1. RN10-52 and RN7-60 were found to react rapidly with APE1, forming covalent adducts, whereas RN8-51 reacted reversibly. Median inhibitory concentration (IC50 values of all three compounds were significantly lower than that of E3330. EMSA, transactivation assays, and endothelial tube growth-inhibition analysis demonstrated the specificity of E3330 and its analogues in blocking the APE1 redox function and demonstrated that the analogues had up to a sixfold greater effect than did E3330. Studies using cancer cell lines demonstrated that E3330 and one analogue, RN8-51, decreased the cell line growth with little apoptosis, whereas the third, RN7-60, caused a dramatic effect. RN8-51 shows particular promise for further anticancer therapeutic development. This progress in synthesizing and isolating biologically active novel E3330 analogues that effectively inhibit the APE1 redox function validates the utility of further translational anticancer therapeutic development.Item Inhibition of Apurinic/apyrimidinic endonuclease I’s redox activity revisited(2013-04) Zhang, Jun; Luo, Meihua; Marasco, Daniela; Logsdon, Derek; LaFavers, Kaice A.; Chen, Qiujia; Reed, April; Kelley, Mark R.; Gross, Michael L.; Georgiadis, Millie M.The essential base excision repair protein, apurinic/apyrimidinic endonuclease 1 (APE1), plays an important role in redox regulation in cells and is currently targeted for the development of cancer therapeutics. One compound that binds APE1 directly is (E)-3-[2-(5,6-dimethoxy-3-methyl-1,4-benzoquinonyl)]-2-nonylpropenoic acid (E3330). Here, we revisit the mechanism by which this negatively charged compound interacts with APE1 and inhibits its redox activity. At high concentrations (millimolar), E3330 interacts with two regions in the endonuclease active site of APE1, as mapped by hydrogen–deuterium exchange mass spectrometry. However, this interaction lowers the melting temperature of APE1, which is consistent with a loss of structure in APE1, as measured by both differential scanning fluorimetry and circular dichroism. These results are consistent with other findings that E3330 concentrations of >100 μM are required to inhibit APE1’s endonuclease activity. To determine the role of E3330’s negatively charged carboxylate in redox inhibition, we converted the carboxylate to an amide by synthesizing (E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methylene]-N-methoxy-undecanamide (E3330-amide), a novel uncharged derivative. E3330-amide has no effect on the melting temperature of APE1, suggesting that it does not interact with the fully folded protein. However, E3330-amide inhibits APE1’s redox activity in in vitro electrophoretic mobility shift redox and cell-based transactivation assays, producing IC50 values (8.5 and 7 μM) lower than those produced with E3330 (20 and 55 μM, respectively). Thus, E3330’s negatively charged carboxylate is not required for redox inhibition. Collectively, our results provide additional support for a mechanism of redox inhibition involving interaction of E3330 or E3330-amide with partially unfolded APE1.Item Interactions of APE1 with a redox inhibitor: Evidence for an alternate conformation of the enzyme(2011-01) Su, Dian; Delaplane, Sarah; Luo, Meihua; Rempel, Don L.; Vu, Bich; Kelley, Mark R.; Gross, Michael L.; Georgiadis, Millie M.Apurinic/apyrimidinic endonuclease (APE1) is an essential base excision repair protein that also functions as a reduction and oxidation (redox) factor in mammals. Through a thiol-based mechanism, APE1 reduces a number of important transcription factors, including AP-1, p53, NF-κB, and HIF-1α. What is known about the mechanism to date is that the buried residues Cys 65 and Cys 93 are critical for APE1’s redox activity. To further detail the redox mechanism, we developed a chemical footprinting−mass spectrometric assay using N-ethylmaleimide (NEM), an irreversible Cys modifier, to characterize the interaction of the redox inhibitor, E3330, with APE1. When APE1 was incubated with E3330, two NEM-modified products were observed, one with two and a second with seven added NEMs; this latter product corresponds to a fully modified APE1. In a similar control reaction without E3330, only the +2NEM product was observed in which the two solvent-accessible Cys residues, C99 and C138, were modified by NEM. Through hydrogen−deuterium amide exchange with analysis by mass spectrometry, we found that the +7NEM-modified species incorporates approximately 40 more deuterium atoms than the native protein, which exchanges nearly identically as the +2NEM product, suggesting that APE1 can be trapped in a partially unfolded state. E3330 was also found to increase the extent of disulfide bond formation involving redox critical Cys residues in APE1 as assessed by liquid chromatography and tandem mass spectrometry, suggesting a basis for its inhibitory effects on APE1’s redox activity. Collectively, our results suggest that APE1 adopts a partially unfolded state, which we propose is the redox active form of the enzyme.Item Kinetic Isotope Effects and Hydrogen/Deuterium Exchange Reveal Large Conformational Changes During the Catalysis of the Clostridium acetobutylicum Spore Photoproduct Lyas(Wiley, 2017-01) Yang, Linlin; Adhikari, Jagat; Gross, Michael L.; Li, Lei; Chemistry and Chemical Biology, School of ScienceItem Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting(PNAS, 2014-06-24) Poor, Taylor A.; Jones, Lisa M.; Sood, Amika; Leser, George P.; Plasencia, Manolo D.; Rempel, Don L.; Jardetzky, Theodore S.; Woods, Robert J.; Gross, Michael L.; Lamb, Robert A.; Department of Chemistry & Chemical Biology, School of ScienceTo infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event.