Ischemic preconditioning(IPC)is a potential intervention known to protect the heart against ischemia/reperfusion injury,but its role in the no-reflow phenomenon that follows reperfusion is unclear.Dihydrotanshinone I(...Ischemic preconditioning(IPC)is a potential intervention known to protect the heart against ischemia/reperfusion injury,but its role in the no-reflow phenomenon that follows reperfusion is unclear.Dihydrotanshinone I(DT)is a natural compound and this study illustrates its role in cardiac ischemic injury from the aspect of IPC.Pretreatment with DT induced modest ROS production and protected cardiomyocytes against oxygen and glucose deprivation(OGD),but the protection was prevented by a ROS scavenger.In addition,DT administration protected the heart against isoprenaline challenge.Mechanistically,PKM2 reacted to transient ROS via oxidization at Cys423/Cys424,leading to glutathionylation and nuclear translocation in dimer form.In the nucleus,PKM2 served as a co-factor to promote HIF-1a-dependent gene induction,contributing to adaptive responses.In mice subjected to permanent coronary ligation,cardiac-specific knockdown of Pkm2 blocked DT-mediated preconditioning protection,which was rescued by overexpression of wild-type Pkm2,rather than Cys423/424-mutated Pkm2.In conclusion,PKM2 is sensitive to oxidation,and subsequent glutathionylation promotes its nuclear translocation.Although IPC has been viewed as a protective means against reperfusion injury,our study reveals its potential role in protection of the heart from no-reflow ischemia.展开更多
Glutathione (GSH), γ-Glu-Cys-Gly, is one of the most abundant small non-protein thiol molecules in mammalian tissues, particu- larly in the liver. Although glutathione is present in thiol-reduced (GSH) and disulfide ...Glutathione (GSH), γ-Glu-Cys-Gly, is one of the most abundant small non-protein thiol molecules in mammalian tissues, particu- larly in the liver. Although glutathione is present in thiol-reduced (GSH) and disulfide oxidized (GSSG) forms, the predominant form is GSH and its content can exceed 10 mmol/L in liver cells. As an important intracellular reductant, GSH has many biological functions in cells. Its major function is as an anti-oxidant as it can protect proteins from oxidation by reversible posttranslational modification (glutathionylation) and decrease reactive oxygen species-mediated damage. However, it does have numerous other functions, including to chelate metal irons; enhance the absorption of iron, selenium and calcium; participate in lipid and insulin metabolism; regulate cellular events such as gene expression, DNA and protein synthesis, cell proliferation and apoptosis, redox-dependent signal transduction pathways, cytokine production and the immune response; and control protein glutathionylation. Therefore, GSH plays important roles in cell survival and health, and an imbalance in the GSH level can lead to many diseases. In this review, we provide an overview of the function of GSH in mammalian cells and discuss future research of GSH.展开更多
Excessive N-acetyl-p-benzoquinone imine(NAPQI)formation is a starting event that triggers oxidative stress and subsequent hepatocyte necrosis in acetaminophen(APAP)overdose caused acute liver failure(ALF).S-glutathion...Excessive N-acetyl-p-benzoquinone imine(NAPQI)formation is a starting event that triggers oxidative stress and subsequent hepatocyte necrosis in acetaminophen(APAP)overdose caused acute liver failure(ALF).S-glutathionylation is a reversible redox post-translational modification and a prospective mechanism of APAP hepatotoxicity.Glutaredoxin-1(Glrx1),a glutathione-specific thioltransferase,is a primary enzyme to catalyze deglutathionylation.The objective of this study was to explored whether and how Glrx1 is associated with the development of ALF induced by APAP.The Glrx1 knockout mice(Glrx1^(-/-))and liver-specific overexpression of Glrx1(AAV8-Glrx1)mice were produced and underwent APAPinduced ALF.Pirfenidone(PFD),a potential inducer of Glrx1,was administrated preceding APAP to assess its protective effects.Our results revealed that the hepatic total protein S-glutathionylation(PSSG)increased and the Glrx1 level reduced in mice after APAP toxicity.Glrx1^(-/-)mice were more sensitive to APAP overdose,with higher oxidative stress and more toxic metabolites of APAP.This was attributed to Glrx1 deficiency increasing the total hepatic PSSG and the S-glutathionylation of cytochrome p4503a11(Cyp3a11),which likely increased the activity of Cyp3a11.Conversely,AAV8-Glrx1 mice were defended against liver damage caused by APAP overdose by inhibiting the S-glutathionylation and activity of Cyp3a11,which reduced the toxic metabolites of APAP and oxidative stress.PFD precede administration upregulated Glrx1 expression and alleviated APAP-induced ALF by decreasing oxidative stress.We have identified the function of Glrx1 mediated PSSG in liver injury caused by APAP overdose.Increasing Glrx1 expression may be investigated for the medical treatment of APAP-caused hepatic injury.展开更多
Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox ...Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed.展开更多
The redox state of cellular thiols is widely studied because it was recently linked to many different diseases and pathologies. In this work we quantified the concentrations of protein disulfides (PSSP) and thiol-prot...The redox state of cellular thiols is widely studied because it was recently linked to many different diseases and pathologies. In this work we quantified the concentrations of protein disulfides (PSSP) and thiol-protein mixed disulfides (XSSP) in rat tissues (liver, kidney and heart) and cells (Raw 264.7) by an improved method of XSSP and PSSP determination after oxidative stress induced by diamide. Under native and denaturing conditions, a thiol block by N-ethymaleimide was introduced to avoid thiol exchange reaction activations by protein SH groups (PSH) (PSH + XSSP ←→ PSSP + XSH) and alterations of original XSSP/PSSP levels. Low molecular weight thiols (XSH) and PSH were respectively measured by HPLC on supernatants and on corresponding pellets by DTNB (Ellman’s reagent) after dithiothreitol reduction. PSSP concentrations of liver, heart and kidney were respectively 0.304, 0.605 and 0.785 μmoles/g and after diamide exposure they were significantly augmented of about 65%-70% in liver and heart, but not in the kidney. Normal XSSP, that were -20 times lower than normal PSSP were induced by diamide in liver and heart of about 40 times, but not in kidney. Thermodynamic criteria regarding the pKa values of thiols engaged as PSSP and GSSP were used to interpret dethiolation mechanisms via thiol exchange reactions.展开更多
Carbonic anhydrase isozyme CA3 protects cells against oxidative stress. Ectopic expression of murine Ca3, but not Ca2, protects proto-oncogene Evi1 expressing Rat1 fibroblast cells (ca3low) against hydrogen peroxide (...Carbonic anhydrase isozyme CA3 protects cells against oxidative stress. Ectopic expression of murine Ca3, but not Ca2, protects proto-oncogene Evi1 expressing Rat1 fibroblast cells (ca3low) against hydrogen peroxide (H2O2) induced stress. Ca3 is S-glutathionylated via glutathione adducts with cysteines 181 and 186. Substitution of both Ca3 cysteines with serine fails to protect cells from oxidative stress. Insertion of cysteine at 181 and 186 in Ca2 is insufficient for conferring efficient anti-oxidant activity. This shows for the first time that S-glutathionylation of cys181 and cys186 residues is required for Ca3 anti-oxidant activity but that additional factors are also required.展开更多
Under ultra-high cadmium(Cd)stress,large amounts of glutathione are produced in Auxenochlorella protothecoides UTEX 2341,and the lipid content increases significantly.Glutathione is the best reductant that can effecti...Under ultra-high cadmium(Cd)stress,large amounts of glutathione are produced in Auxenochlorella protothecoides UTEX 2341,and the lipid content increases significantly.Glutathione is the best reductant that can effectively remove Cd,but the relationship between lipid accumulation and the cellular response to Cd stress has not been ascertained.Integrating analyses of the transcriptomes and lipidomes,the mechanism of lipid accumulation to Cd tolerance were studied from the perspectives of metabolism,transcriptional regulation and protein glutathionylation.Under Cd stress,basic metabolic pathways,such as purine metabolism,translation and pre-m RNA splicing process,were inhibited,while the lipid accumulation pathway was significantly activated.Further analysis revealed that the transcription factors(TFs)and genes related to lipid accumulation were also activated.Analysis of the TF interaction sites showed that ABI5,MYBrel and NF-YB could further regulate the expression of diacylglycerol acyltransferase through glutathionylation/deglutathionylation,which led to increase of the triacylglycerol(TAG)content.Lipidomes analysis showed that TAG could help maintain lipid homeostasis by adjusting its saturation/unsaturation levels.This study for the first time indicated that glutathione could activate TAG synthesis in microalga A.protothecoides,leading to TAG accumulation and glutathione accumulation under Cd stress.Therefore,the accumulation of TAG and glutathione can confer resistance to high Cd stress.This study provided insights into a new operation mode of TAG accumulation under heavy metal stress.展开更多
Cytoskeletal proteins are susceptible to glutathionylation under oxidizing conditions,and oxidative damage has been implicated in several neurodegenerative diseases.End-binding protein 1(EB1)is a master regulator of m...Cytoskeletal proteins are susceptible to glutathionylation under oxidizing conditions,and oxidative damage has been implicated in several neurodegenerative diseases.End-binding protein 1(EB1)is a master regulator of microtubule plus-end tracking proteins(+TIPs)and is critically involved in the control of microtubule dynamics and cellular processes.However,the impact of glutathionylation on EB1 functions remains unknown.Here we reveal that glutathionylation is important for controlling EB1 activity and protecting EB1 from irreversible oxidation.In vitro biochemical and cellular assays reveal that EB1 is glutathionylated.Diamide,a mild oxidizing reagent,reduces EB1 comet number and length in cells,indicating the impairment of microtubule dynamics.Three cysteine residues of EB1 are glutathionylated,with mutations of these three cysteines to serines attenuating microtubule dynamics but buffering diamide-induced decrease in microtubule dynamics.In addition,glutaredoxin 1(Grx1)deglutathionylates EB1,and Grx1 depletion suppresses microtubule dynamics and leads to defects in cell division orientation and cell migration,suggesting a critical role of Grx1-mediated deglutathionylation in maintaining EB1 activity.Collectively,these data reveal that EB1 glutathionylation is an important protective mechanism for the regulation of microtubule dynamics and microtubule-based cellular activities.展开更多
Glutathionyl-haemoglobin (Hb-SSG) is a minor form of haemoglobin characterized by the presence of a disulfide bond between the β-93 cysteine residue and the thiol group of glutathione. Hb-SSG is naturally present in ...Glutathionyl-haemoglobin (Hb-SSG) is a minor form of haemoglobin characterized by the presence of a disulfide bond between the β-93 cysteine residue and the thiol group of glutathione. Hb-SSG is naturally present in the erythrocytes at levels comparable to those of glycated haemoglobin and can be measured by MALDI mass spectrometry on very small samples of erythrocytes from peripheral blood. Since Hb-SSG has been recognized as a sensitive biomarker of oxidative stress in several degenerative diseases (diabetes, hyperlipidemia, kidney disease) and in healthy workers exposed to glutathione-depleting toxic agents such as butadiene, we have measured for the first time the levels of Hb-SSG in two groups: healthy heavy cigarette smokers and overweight-obese. For both classes of subjects, the measured levels (6.4%±1.7%, n=30 for smokers;3.0%±0.8%, n=20 for overweight-obese) are in the upper 97thpercentile of those measured in the Italian general population. Levels in smokers show a small, yet statistically significant dependence on the level of smoking addiction (>20 cig./day vs. ￡20 cig./day: 7.0% ± 1.4% vs. 5.7% ± 1.1%;p < 0.05). This biomarker thus adds to those presently available to rationally assess the extent of biological damage caused by tobacco smoking.展开更多
基金supported by the National Key R&D Program of China(2019YFC1711000)the National Natural Science Foundation of China(No.81421005)the“111”Project(B16046)from the Ministry of Education of China and the State Administration of Foreign Experts Affairs of China。
文摘Ischemic preconditioning(IPC)is a potential intervention known to protect the heart against ischemia/reperfusion injury,but its role in the no-reflow phenomenon that follows reperfusion is unclear.Dihydrotanshinone I(DT)is a natural compound and this study illustrates its role in cardiac ischemic injury from the aspect of IPC.Pretreatment with DT induced modest ROS production and protected cardiomyocytes against oxygen and glucose deprivation(OGD),but the protection was prevented by a ROS scavenger.In addition,DT administration protected the heart against isoprenaline challenge.Mechanistically,PKM2 reacted to transient ROS via oxidization at Cys423/Cys424,leading to glutathionylation and nuclear translocation in dimer form.In the nucleus,PKM2 served as a co-factor to promote HIF-1a-dependent gene induction,contributing to adaptive responses.In mice subjected to permanent coronary ligation,cardiac-specific knockdown of Pkm2 blocked DT-mediated preconditioning protection,which was rescued by overexpression of wild-type Pkm2,rather than Cys423/424-mutated Pkm2.In conclusion,PKM2 is sensitive to oxidation,and subsequent glutathionylation promotes its nuclear translocation.Although IPC has been viewed as a protective means against reperfusion injury,our study reveals its potential role in protection of the heart from no-reflow ischemia.
基金the National Natural Science Foundation of China (30970630)the New Century Talents Program (NCET-08-0912)the Key Project of the Chinese Ministry of Education (210010)
文摘Glutathione (GSH), γ-Glu-Cys-Gly, is one of the most abundant small non-protein thiol molecules in mammalian tissues, particu- larly in the liver. Although glutathione is present in thiol-reduced (GSH) and disulfide oxidized (GSSG) forms, the predominant form is GSH and its content can exceed 10 mmol/L in liver cells. As an important intracellular reductant, GSH has many biological functions in cells. Its major function is as an anti-oxidant as it can protect proteins from oxidation by reversible posttranslational modification (glutathionylation) and decrease reactive oxygen species-mediated damage. However, it does have numerous other functions, including to chelate metal irons; enhance the absorption of iron, selenium and calcium; participate in lipid and insulin metabolism; regulate cellular events such as gene expression, DNA and protein synthesis, cell proliferation and apoptosis, redox-dependent signal transduction pathways, cytokine production and the immune response; and control protein glutathionylation. Therefore, GSH plays important roles in cell survival and health, and an imbalance in the GSH level can lead to many diseases. In this review, we provide an overview of the function of GSH in mammalian cells and discuss future research of GSH.
基金supported by the National Natural Science Foundation of China(Grant Nos.:82025007,81930020,and 82170874)China Postdoctoral Science Foundation(Grant No.:2022M710099).
文摘Excessive N-acetyl-p-benzoquinone imine(NAPQI)formation is a starting event that triggers oxidative stress and subsequent hepatocyte necrosis in acetaminophen(APAP)overdose caused acute liver failure(ALF).S-glutathionylation is a reversible redox post-translational modification and a prospective mechanism of APAP hepatotoxicity.Glutaredoxin-1(Glrx1),a glutathione-specific thioltransferase,is a primary enzyme to catalyze deglutathionylation.The objective of this study was to explored whether and how Glrx1 is associated with the development of ALF induced by APAP.The Glrx1 knockout mice(Glrx1^(-/-))and liver-specific overexpression of Glrx1(AAV8-Glrx1)mice were produced and underwent APAPinduced ALF.Pirfenidone(PFD),a potential inducer of Glrx1,was administrated preceding APAP to assess its protective effects.Our results revealed that the hepatic total protein S-glutathionylation(PSSG)increased and the Glrx1 level reduced in mice after APAP toxicity.Glrx1^(-/-)mice were more sensitive to APAP overdose,with higher oxidative stress and more toxic metabolites of APAP.This was attributed to Glrx1 deficiency increasing the total hepatic PSSG and the S-glutathionylation of cytochrome p4503a11(Cyp3a11),which likely increased the activity of Cyp3a11.Conversely,AAV8-Glrx1 mice were defended against liver damage caused by APAP overdose by inhibiting the S-glutathionylation and activity of Cyp3a11,which reduced the toxic metabolites of APAP and oxidative stress.PFD precede administration upregulated Glrx1 expression and alleviated APAP-induced ALF by decreasing oxidative stress.We have identified the function of Glrx1 mediated PSSG in liver injury caused by APAP overdose.Increasing Glrx1 expression may be investigated for the medical treatment of APAP-caused hepatic injury.
文摘Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed.
基金supported by National Heart, Lung, and Blood Institute Grant (No. HL-067890)National Institutes of Health Grant (No. HD060959)the American Heart Association (No. 09GRNT2010037)
文摘The redox state of cellular thiols is widely studied because it was recently linked to many different diseases and pathologies. In this work we quantified the concentrations of protein disulfides (PSSP) and thiol-protein mixed disulfides (XSSP) in rat tissues (liver, kidney and heart) and cells (Raw 264.7) by an improved method of XSSP and PSSP determination after oxidative stress induced by diamide. Under native and denaturing conditions, a thiol block by N-ethymaleimide was introduced to avoid thiol exchange reaction activations by protein SH groups (PSH) (PSH + XSSP ←→ PSSP + XSH) and alterations of original XSSP/PSSP levels. Low molecular weight thiols (XSH) and PSH were respectively measured by HPLC on supernatants and on corresponding pellets by DTNB (Ellman’s reagent) after dithiothreitol reduction. PSSP concentrations of liver, heart and kidney were respectively 0.304, 0.605 and 0.785 μmoles/g and after diamide exposure they were significantly augmented of about 65%-70% in liver and heart, but not in the kidney. Normal XSSP, that were -20 times lower than normal PSSP were induced by diamide in liver and heart of about 40 times, but not in kidney. Thermodynamic criteria regarding the pKa values of thiols engaged as PSSP and GSSP were used to interpret dethiolation mechanisms via thiol exchange reactions.
文摘Carbonic anhydrase isozyme CA3 protects cells against oxidative stress. Ectopic expression of murine Ca3, but not Ca2, protects proto-oncogene Evi1 expressing Rat1 fibroblast cells (ca3low) against hydrogen peroxide (H2O2) induced stress. Ca3 is S-glutathionylated via glutathione adducts with cysteines 181 and 186. Substitution of both Ca3 cysteines with serine fails to protect cells from oxidative stress. Insertion of cysteine at 181 and 186 in Ca2 is insufficient for conferring efficient anti-oxidant activity. This shows for the first time that S-glutathionylation of cys181 and cys186 residues is required for Ca3 anti-oxidant activity but that additional factors are also required.
基金funded by the National Natural Science Foundation of China(No.31570117)。
文摘Under ultra-high cadmium(Cd)stress,large amounts of glutathione are produced in Auxenochlorella protothecoides UTEX 2341,and the lipid content increases significantly.Glutathione is the best reductant that can effectively remove Cd,but the relationship between lipid accumulation and the cellular response to Cd stress has not been ascertained.Integrating analyses of the transcriptomes and lipidomes,the mechanism of lipid accumulation to Cd tolerance were studied from the perspectives of metabolism,transcriptional regulation and protein glutathionylation.Under Cd stress,basic metabolic pathways,such as purine metabolism,translation and pre-m RNA splicing process,were inhibited,while the lipid accumulation pathway was significantly activated.Further analysis revealed that the transcription factors(TFs)and genes related to lipid accumulation were also activated.Analysis of the TF interaction sites showed that ABI5,MYBrel and NF-YB could further regulate the expression of diacylglycerol acyltransferase through glutathionylation/deglutathionylation,which led to increase of the triacylglycerol(TAG)content.Lipidomes analysis showed that TAG could help maintain lipid homeostasis by adjusting its saturation/unsaturation levels.This study for the first time indicated that glutathione could activate TAG synthesis in microalga A.protothecoides,leading to TAG accumulation and glutathione accumulation under Cd stress.Therefore,the accumulation of TAG and glutathione can confer resistance to high Cd stress.This study provided insights into a new operation mode of TAG accumulation under heavy metal stress.
基金supported by the National Natural Science Foundation of China(31701216,31771542,31900502)the Natural Science Foundation of Shandong Province(ZR2017MC008)。
文摘Cytoskeletal proteins are susceptible to glutathionylation under oxidizing conditions,and oxidative damage has been implicated in several neurodegenerative diseases.End-binding protein 1(EB1)is a master regulator of microtubule plus-end tracking proteins(+TIPs)and is critically involved in the control of microtubule dynamics and cellular processes.However,the impact of glutathionylation on EB1 functions remains unknown.Here we reveal that glutathionylation is important for controlling EB1 activity and protecting EB1 from irreversible oxidation.In vitro biochemical and cellular assays reveal that EB1 is glutathionylated.Diamide,a mild oxidizing reagent,reduces EB1 comet number and length in cells,indicating the impairment of microtubule dynamics.Three cysteine residues of EB1 are glutathionylated,with mutations of these three cysteines to serines attenuating microtubule dynamics but buffering diamide-induced decrease in microtubule dynamics.In addition,glutaredoxin 1(Grx1)deglutathionylates EB1,and Grx1 depletion suppresses microtubule dynamics and leads to defects in cell division orientation and cell migration,suggesting a critical role of Grx1-mediated deglutathionylation in maintaining EB1 activity.Collectively,these data reveal that EB1 glutathionylation is an important protective mechanism for the regulation of microtubule dynamics and microtubule-based cellular activities.
文摘Glutathionyl-haemoglobin (Hb-SSG) is a minor form of haemoglobin characterized by the presence of a disulfide bond between the β-93 cysteine residue and the thiol group of glutathione. Hb-SSG is naturally present in the erythrocytes at levels comparable to those of glycated haemoglobin and can be measured by MALDI mass spectrometry on very small samples of erythrocytes from peripheral blood. Since Hb-SSG has been recognized as a sensitive biomarker of oxidative stress in several degenerative diseases (diabetes, hyperlipidemia, kidney disease) and in healthy workers exposed to glutathione-depleting toxic agents such as butadiene, we have measured for the first time the levels of Hb-SSG in two groups: healthy heavy cigarette smokers and overweight-obese. For both classes of subjects, the measured levels (6.4%±1.7%, n=30 for smokers;3.0%±0.8%, n=20 for overweight-obese) are in the upper 97thpercentile of those measured in the Italian general population. Levels in smokers show a small, yet statistically significant dependence on the level of smoking addiction (>20 cig./day vs. ￡20 cig./day: 7.0% ± 1.4% vs. 5.7% ± 1.1%;p < 0.05). This biomarker thus adds to those presently available to rationally assess the extent of biological damage caused by tobacco smoking.
