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
Energy stress triggers the activation of AMP-activated protein kinase(AMPK)via phosphorylation mediated by liver kinase B1(LKB1).A pivotal step during this process is the translocation of protein kinase C zeta(PKCζ)t...Energy stress triggers the activation of AMP-activated protein kinase(AMPK)via phosphorylation mediated by liver kinase B1(LKB1).A pivotal step during this process is the translocation of protein kinase C zeta(PKCζ)to the nucleus,where it facilitates the phosphorylation and subsequent nuclear export of LKB1 to the cytosol.However,the mechanism(s)by which PKCζ translocates to the nucleus remains elusive.Here we demonstrate that energy stress,including glucose starvation or metformin treatment,elevates cellular reactive oxygen species(ROS)that promotes PKCζ nuclear import to promote LKB1 cytoplasmic translocation and subsequent AMPK activation both in vitro and in vivo.Mechanistically,we show that energy stress-induced ROS promotes the S-glutathionylation of PKCζ at Cys48,and enhances the interaction of PKCζ with karyopherin subunit alpha 2(KPNA2),a key nuclear transport protein,thereby facilitating PKCζ nuclear translocation and the phosphorylation of LKB1 at Ser428,consequently leading to LKB1 cytoplasmic translocation and activation of AMPK.Importantly,the reduction of ROS significantly augments the high-fat diet-induced lipid accumulation in mouse liver and reduces the hypoglycemic efficacy of metformin in an AMPK-dependent manner.Together,these results establish a critical role of energy stress-induced PKCζ S-glutathionylation in LKB1 cytoplasmic translocation,highlighting the activation of the ROS-PKCζ-KPNA2-LKB1 axis as a vital mechanism for AMPK activation in response to energy stress.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
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
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supported by the National Natural Science Foundation of China(82073248 and 81973243)to Y.Y.and Y.C.the National Key R&D Program of China(2022YFA1103700)to Z.X.J.X.the Natural Science Foundation of Sichuan Province(2023NSFSC1859)to Y.Y.
文摘Energy stress triggers the activation of AMP-activated protein kinase(AMPK)via phosphorylation mediated by liver kinase B1(LKB1).A pivotal step during this process is the translocation of protein kinase C zeta(PKCζ)to the nucleus,where it facilitates the phosphorylation and subsequent nuclear export of LKB1 to the cytosol.However,the mechanism(s)by which PKCζ translocates to the nucleus remains elusive.Here we demonstrate that energy stress,including glucose starvation or metformin treatment,elevates cellular reactive oxygen species(ROS)that promotes PKCζ nuclear import to promote LKB1 cytoplasmic translocation and subsequent AMPK activation both in vitro and in vivo.Mechanistically,we show that energy stress-induced ROS promotes the S-glutathionylation of PKCζ at Cys48,and enhances the interaction of PKCζ with karyopherin subunit alpha 2(KPNA2),a key nuclear transport protein,thereby facilitating PKCζ nuclear translocation and the phosphorylation of LKB1 at Ser428,consequently leading to LKB1 cytoplasmic translocation and activation of AMPK.Importantly,the reduction of ROS significantly augments the high-fat diet-induced lipid accumulation in mouse liver and reduces the hypoglycemic efficacy of metformin in an AMPK-dependent manner.Together,these results establish a critical role of energy stress-induced PKCζ S-glutathionylation in LKB1 cytoplasmic translocation,highlighting the activation of the ROS-PKCζ-KPNA2-LKB1 axis as a vital mechanism for AMPK activation in response to energy stress.
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.
