- Email: [email protected]
Oxidation of disulfide bonds: a novel pathway to protein glutathionylation
C.F. Lourenço et al. / Free Radical Biology and Medicine 120 (2018) S29–S43
OP-31
Targeting mitochondria by TrxR2 inhibition Maria Pia Rigobello 1, Valeria Scalcon 1, Federica Tonolo 1, Alessandra Folda 1, Anne Vessières 2, Michèle Salmain 2, Alberto Bindoli 3
S39
hours after initial oxidation. Disulfide oxidation can therefore give rise to long-lived oxidants on proteins that can undergo further reaction with thiols, including GSH and other proteins, to give mixed disulfides and protein dimers. These reactions may play a key role in GSH-dependent cell signaling and tissue damage.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.133
1
Dipartimento di Scienze Biomediche, Università di Padova, Padova, Italy 2 Sorbonne Université, CNRS, Institut Parisien de Chimie Moléculaire (IPCM), Paris, France 3 Istituto di Neuroscienze (CNR) Sezione di Padova, c/o Dipartimento di Scienze Biomediche, Italy
Thioredoxin reductase is often over expressed in tumor tissues and consequently, inhibitors of TrxR are actively studied. TrxR is present in different isoforms and the role of the mitochondrial isoform is still not completely elucidated. In tumors, TrxR2 inhibition can address cells to apoptosis by altering mitochondrial permeability. This enzyme, directly or through its natural substrate Trx2, is crucial in the redox regulation of many target proteins, such as Prx3 and Grx2. Another protein redox regulated by the mitochondrial thioredoxin system is Cyclophilin D, a key molecule in the permeability transition process. Many inhibitors, such as metal complexes, are able to interact with the selenol group of TrxR, but their action is often nonselective for TrxR2 isoform. In order to hamper TrxR2, determining a redox imbalance, the inhibitors have to reach the mitochondrial matrix compartment and therefore mitochondriotropic agents could be a useful tool to block TrxR2 bringing to cell death. Interesting chemical features are offered by tamoxifen-like metallocifens, that are able to reach the mitochondrial environment, driven by the negative charge of mitochondrial membrane potential, and to inhibit TrxR determining a large increase of ROS production finalized to apoptosis.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.132
OP-32
Oxidation of disulfide bonds: a novel pathway to protein glutathionylation
OP-33
A1M-035, an improved human recombinant alpha-1microglobulin, has therapeutic effects in rhabdomyolysis-induced acute kidney injury Bo Åkerström, Lena Rosenlöf, Anneli Hägerwall, Sigurbjörg Rutardottir, Jonas Ahlstedt, Maria E Johansson, Lena Erlandsson, Maria Allhorn, Magnus Gram Section for Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden
Human A1M (alpha-1-microglobulin) is a reductase and radical- and heme-binding protein with physiological antioxidant protective functions. Recombinant human A1M has been shown to have therapeutic properties in vitro and in vivo, but lacks glycosylation, and shows lower solubility and stability than A1M purified from human plasma. The aims of this work were to 1) use site-directed mutagenesis to improve the physicochemical properties of A1M, 2) demonstrate that the physicochemically improved A1M displays full in vitro cell protective effects as recombinant wild-type A1M (A1M-wt), and 3) show its therapeutic potential in vivo against acute kidney injury (AKI), a disease associated with oxidative stress, using a mouse rhabdomyolysis glycerol-injection model. A novel recombinant A1M-variant (A1M-035) with three amino acid substitutions was constructed, successfully expressed and purified. A1M-035 had improved solubility and stability compared to A1M-wt, and had intact in vitro heme-binding, reductase, antioxidation, and cell protective activities. Both A1M-035 and A1M-wt showed, for the first time, potential in vivo protective effects on acute kidney injury (AKI). It is concluded that the new A1M-035 is a better drug candidate than A1M-wt for treatment of AKI in human patients.
E-mail address: [email protected] Luke Carroll, Shuwen Jiang, Kasper Engholm-Keller, Adelina Rogowska-Wrzesinska, Michael Davies
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.134
University of Copenhagen, Denmark; University of Southern Denmark, Denmark OP-34 Disulfide bonds, formed from two cysteine (Cys) residues, play a key role in defining and stabilizing protein structures. Whilst the reactions of Cys are well characterized, little is known about oxidation of disulfides, despite their high abundance in some proteins. Recent data indicate that some disulfides are rapidly oxidized by a range of oxidants, with rate constants, k, 105–107 M-1 s-1. Here we show that these reactions yield intermediates, potentially thiosulfinates [RSS( ¼ O)R], that can undergo further reaction, including disulfide bond cleavage and reaction with another thiol. Thus, oxidation of alpha-lactalbumin, which contains no free Cys residues, by HOCl or ONOOH gives long-lived reactive thiosulfinates that can react with GSH to give a glutathionylated protein, which can be detected by both anti-GSH antibodies and mass spectrometry. The thiosulfinate has a lifetime of many hours allowing glutathionylation to occur
Amyloid-β peptide irreversibly blocks mitochondrial biogenesis and dynamics of self-renewing neural stem cells compromising neurogenesis Maria Filipe Ribeiro, Tânia Genebra, Cecilia Rodrigues, Susana Solá Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
OP-31
Targeting mitochondria by TrxR2 inhibition Maria Pia Rigobello 1, Valeria Scalcon 1, Federica Tonolo 1, Alessandra Folda 1, Anne Vessières 2, Michèle Salmain 2, Alberto Bindoli 3
S39
hours after initial oxidation. Disulfide oxidation can therefore give rise to long-lived oxidants on proteins that can undergo further reaction with thiols, including GSH and other proteins, to give mixed disulfides and protein dimers. These reactions may play a key role in GSH-dependent cell signaling and tissue damage.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.133
1
Dipartimento di Scienze Biomediche, Università di Padova, Padova, Italy 2 Sorbonne Université, CNRS, Institut Parisien de Chimie Moléculaire (IPCM), Paris, France 3 Istituto di Neuroscienze (CNR) Sezione di Padova, c/o Dipartimento di Scienze Biomediche, Italy
Thioredoxin reductase is often over expressed in tumor tissues and consequently, inhibitors of TrxR are actively studied. TrxR is present in different isoforms and the role of the mitochondrial isoform is still not completely elucidated. In tumors, TrxR2 inhibition can address cells to apoptosis by altering mitochondrial permeability. This enzyme, directly or through its natural substrate Trx2, is crucial in the redox regulation of many target proteins, such as Prx3 and Grx2. Another protein redox regulated by the mitochondrial thioredoxin system is Cyclophilin D, a key molecule in the permeability transition process. Many inhibitors, such as metal complexes, are able to interact with the selenol group of TrxR, but their action is often nonselective for TrxR2 isoform. In order to hamper TrxR2, determining a redox imbalance, the inhibitors have to reach the mitochondrial matrix compartment and therefore mitochondriotropic agents could be a useful tool to block TrxR2 bringing to cell death. Interesting chemical features are offered by tamoxifen-like metallocifens, that are able to reach the mitochondrial environment, driven by the negative charge of mitochondrial membrane potential, and to inhibit TrxR determining a large increase of ROS production finalized to apoptosis.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.132
OP-32
Oxidation of disulfide bonds: a novel pathway to protein glutathionylation
OP-33
A1M-035, an improved human recombinant alpha-1microglobulin, has therapeutic effects in rhabdomyolysis-induced acute kidney injury Bo Åkerström, Lena Rosenlöf, Anneli Hägerwall, Sigurbjörg Rutardottir, Jonas Ahlstedt, Maria E Johansson, Lena Erlandsson, Maria Allhorn, Magnus Gram Section for Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden
Human A1M (alpha-1-microglobulin) is a reductase and radical- and heme-binding protein with physiological antioxidant protective functions. Recombinant human A1M has been shown to have therapeutic properties in vitro and in vivo, but lacks glycosylation, and shows lower solubility and stability than A1M purified from human plasma. The aims of this work were to 1) use site-directed mutagenesis to improve the physicochemical properties of A1M, 2) demonstrate that the physicochemically improved A1M displays full in vitro cell protective effects as recombinant wild-type A1M (A1M-wt), and 3) show its therapeutic potential in vivo against acute kidney injury (AKI), a disease associated with oxidative stress, using a mouse rhabdomyolysis glycerol-injection model. A novel recombinant A1M-variant (A1M-035) with three amino acid substitutions was constructed, successfully expressed and purified. A1M-035 had improved solubility and stability compared to A1M-wt, and had intact in vitro heme-binding, reductase, antioxidation, and cell protective activities. Both A1M-035 and A1M-wt showed, for the first time, potential in vivo protective effects on acute kidney injury (AKI). It is concluded that the new A1M-035 is a better drug candidate than A1M-wt for treatment of AKI in human patients.
E-mail address: [email protected] Luke Carroll, Shuwen Jiang, Kasper Engholm-Keller, Adelina Rogowska-Wrzesinska, Michael Davies
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.134
University of Copenhagen, Denmark; University of Southern Denmark, Denmark OP-34 Disulfide bonds, formed from two cysteine (Cys) residues, play a key role in defining and stabilizing protein structures. Whilst the reactions of Cys are well characterized, little is known about oxidation of disulfides, despite their high abundance in some proteins. Recent data indicate that some disulfides are rapidly oxidized by a range of oxidants, with rate constants, k, 105–107 M-1 s-1. Here we show that these reactions yield intermediates, potentially thiosulfinates [RSS( ¼ O)R], that can undergo further reaction, including disulfide bond cleavage and reaction with another thiol. Thus, oxidation of alpha-lactalbumin, which contains no free Cys residues, by HOCl or ONOOH gives long-lived reactive thiosulfinates that can react with GSH to give a glutathionylated protein, which can be detected by both anti-GSH antibodies and mass spectrometry. The thiosulfinate has a lifetime of many hours allowing glutathionylation to occur
Amyloid-β peptide irreversibly blocks mitochondrial biogenesis and dynamics of self-renewing neural stem cells compromising neurogenesis Maria Filipe Ribeiro, Tânia Genebra, Cecilia Rodrigues, Susana Solá Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal