F. Schopfer - Academia.edu (original) (raw)

Papers by F. Schopfer

Research paper thumbnail of Detection of nitrated fatty acids in olive oils: endogenous formation versus acidic nitration

Research paper thumbnail of P2-OLIVES AND OLIVE OIL AS A SOURCE OF ELECTROPHILIC LIPID SIGNALING MEDIATORS

Research paper thumbnail of HPLC separation of human serum albumin isoforms based on their isoelectric points

Journal of Chromatography B, 2014

Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residu... more Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg + ), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO 2 − ) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA 3-585 ) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA

Research paper thumbnail of Reversible Post-translational Modification of Proteins by Nitrated Fatty Acids in Vivo

Journal of Biological Chemistry, 2006

Nitric oxide (˙NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene... more Nitric oxide (˙NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon β to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9-and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC 50 of ∼3 μM for both nitroalkenes, an IC 50 equivalent to the potent thiol oxidant peroxynitrite (ONOO − ) and an IC 50 30-fold less than H 2 O 2 , indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiolreversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this

Research paper thumbnail of Detection of Nitrated Fatty Acids in Olives and Olive Oils

Free Radical Biology and Medicine, 2012

Research paper thumbnail of Reactivity of Sulfenic Acid in Human Serum Albumin †

Biochemistry, 2008

Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modul... more Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modulation of an increasing number of proteins. Methods for quantifying or even detecting sulfenic acid are scarce. Herein, the reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA-SOH) because of lack of specificity. Thionitrobenzoate (TNB) reacted with HSA exposed to hydrogen peroxide, but not control or thiol-blocked HSA. The reaction was biphasic. The first phase was ∼20-fold faster than the second phase and first order in HSA-SOH and TNB (105 ( 11 M -1 s -1 , 25°C, pH 7.4), allowing quantitative data on HSA-SOH formation and reactivity to be obtained. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37°C, 4 min) yielded 0.18 ( 0.02 mol of HSA-SOH per mol of HSA. HSA-SH reacted with hydrogen peroxide at 2.7 ( 0.7 M -1 s -1 (37°C, pH 7.4), while HSA-SOH reacted at 0.4 ( 0.2 M -1 s -1 , yielding sulfinic acid (HSA-SO 2 H), as detected by mass spectrometry. The rate constants of HSA-SOH with targets of analytical interest such as dimedone and sodium arsenite were determined. HSA-SOH did not react appreciably with the plasma reductants ascorbate or urate, nor with free basic amino acids. In contrast, HSA-SOH reacted rapidly with the plasma thiols cysteine, glutathione, homocysteine, and cysteinylglycine at 21.6 ( 0.2, 2.9 ( 0.5, 9.3 ( 0.9, and 55 ( 3 M -1 s -1 (25°C, pH 7.4), respectively, supporting a role for HSA-SOH in the formation of mixed disulfides.

Research paper thumbnail of Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe–S center

Free Radical Biology and Medicine, 2013

PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic pro... more PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N-and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9-and 10-nitro-octadeca-9-cis-enoic acid; OA-NO 2 ) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein functioCn and subcellular distribution in a reversible manner. Here, we show that OA-NO 2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO 2 -mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO 2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.

Research paper thumbnail of Detection of nitrated fatty acids in olive oils: endogenous formation versus acidic nitration

Research paper thumbnail of P2-OLIVES AND OLIVE OIL AS A SOURCE OF ELECTROPHILIC LIPID SIGNALING MEDIATORS

Research paper thumbnail of HPLC separation of human serum albumin isoforms based on their isoelectric points

Journal of Chromatography B, 2014

Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residu... more Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg + ), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO 2 − ) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA 3-585 ) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA

Research paper thumbnail of Reversible Post-translational Modification of Proteins by Nitrated Fatty Acids in Vivo

Journal of Biological Chemistry, 2006

Nitric oxide (˙NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene... more Nitric oxide (˙NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon β to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9-and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC 50 of ∼3 μM for both nitroalkenes, an IC 50 equivalent to the potent thiol oxidant peroxynitrite (ONOO − ) and an IC 50 30-fold less than H 2 O 2 , indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiolreversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this

Research paper thumbnail of Detection of Nitrated Fatty Acids in Olives and Olive Oils

Free Radical Biology and Medicine, 2012

Research paper thumbnail of Reactivity of Sulfenic Acid in Human Serum Albumin †

Biochemistry, 2008

Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modul... more Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modulation of an increasing number of proteins. Methods for quantifying or even detecting sulfenic acid are scarce. Herein, the reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA-SOH) because of lack of specificity. Thionitrobenzoate (TNB) reacted with HSA exposed to hydrogen peroxide, but not control or thiol-blocked HSA. The reaction was biphasic. The first phase was ∼20-fold faster than the second phase and first order in HSA-SOH and TNB (105 ( 11 M -1 s -1 , 25°C, pH 7.4), allowing quantitative data on HSA-SOH formation and reactivity to be obtained. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37°C, 4 min) yielded 0.18 ( 0.02 mol of HSA-SOH per mol of HSA. HSA-SH reacted with hydrogen peroxide at 2.7 ( 0.7 M -1 s -1 (37°C, pH 7.4), while HSA-SOH reacted at 0.4 ( 0.2 M -1 s -1 , yielding sulfinic acid (HSA-SO 2 H), as detected by mass spectrometry. The rate constants of HSA-SOH with targets of analytical interest such as dimedone and sodium arsenite were determined. HSA-SOH did not react appreciably with the plasma reductants ascorbate or urate, nor with free basic amino acids. In contrast, HSA-SOH reacted rapidly with the plasma thiols cysteine, glutathione, homocysteine, and cysteinylglycine at 21.6 ( 0.2, 2.9 ( 0.5, 9.3 ( 0.9, and 55 ( 3 M -1 s -1 (25°C, pH 7.4), respectively, supporting a role for HSA-SOH in the formation of mixed disulfides.

Research paper thumbnail of Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe–S center

Free Radical Biology and Medicine, 2013

PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic pro... more PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N-and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9-and 10-nitro-octadeca-9-cis-enoic acid; OA-NO 2 ) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein functioCn and subcellular distribution in a reversible manner. Here, we show that OA-NO 2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO 2 -mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO 2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.