Moon B. Yim

Protein glycation: creation of catalytic sites for free radical generation.

Abstract: In a glycation reaction, α‐dicarbonyl compounds such as deoxyglucosone, methylglyoxal, and glyoxal are more reactive than the parent sugars with respect to their ability to react with amino groups of proteins to form inter‐ and intramolecular cross‐links of proteins, stable end products called advanced Maillard products or advanced end products (AGEs). The AGEs, which are irreversibly formed, accumulate with aging, atherosclerosis, and diabetes mellitus, and are especially associated with long‐lived proteins such as collagens, lens crystallins, and nerve proteins. It was suggested that the formation of AGEs not only modifies protein properites but also induces biological damage in vivo. In this report, we summerize results obtained from our studies for (1) identifying the structure of the cross‐linked radical species formed in the model system—the reaction between α‐dicarbonyl methylglyoxal with amino acids, and (2) the reactivity of the radical center of the protein created by the similar reaction. These results indicate that glycation of protein generates active centers for catalyzing one‐electron oxidation‐reduction reactions. This active center, which exhibits enzyme‐like character, is suggested to be the cross‐linked Schiff‐based radical cation of the protein. It mimics the characteristics of the metal‐catalyzed oxidation system. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free redicals.

Oxidation-Reduction Properties of Methylglyoxal-modified Protein in Relation to Free Radical Generation

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation were investigated. Glycation of bovine serum albumin by methylglyoxal generated the protein-bound free radical, probably the cation radical of the cross-linked Schiff base, as observed in the reaction of methylglyoxal with l-alanine (Yim, H.-S., Kang, S.-O., Hah, Y. C., Chock, P. B., and Yim, M. B. (1995)J. Biol. Chem. 270, 28228–28233) or withN α-acetyl-l-lysine. The glycated bovine serum albumin showed increased electrophoretic mobility suggesting that the basic residues, such as lysine, were modified by methylglyoxal. The glycated protein reduced ferricytochromec to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen, whereas the protein free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like characteristic, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. The glycated bovine serum albumin cross-linked further to the cytochromec in the absence of methylglyoxal. The cross-linked cytochrome c maintains its oxidation-reduction properties. These results together indicate that glycated proteins accumulatedin vivo provide stable active sites for catalyzing the formation of free radicals.

Transcriptional Activation of the Human Manganese Superoxide Dismutase Gene Mediated by Tetradecanoylphorbol Acetate

Transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA induced by a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined to identify the responsive transcriptional regulator. The effect of various deletions and mutations within the 5′-flanking region of the human MnSOD gene promoter was evaluated using the luciferase reporter system in A549 human lung carcinoma cells. Deletion of a region between −1292 and −1202 nucleotides upstream of the transcription start site abolished TPA-responsive induction, whereas deletion of the putative binding sequence for NF-κB or AP-1 did not. The region between −1292 and −1202 contains a cAMP-responsive element-like sequence, TGACGTCT, which we identified as the manganese superoxide dismutase TPA-responsive element, MSTRE. Site-specific mutation of the MSTRE abolished the TPA-responsive induction, validating the critical role of this sequence. We detected specific MSTRE activity from nuclear extracts and demonstrated by antibody supershift assay that this activity is closely related to CREB-1/ATF-1. TPA treatment rapidly induced phosphorylation of the CREB-1/ATF-1-like factor via the protein kinase C pathway. These results led us to conclude that the human MnSOD gene having the promoter construct used in this study is induced by TPA via activation of a CREB-1/ATF-1-like factor and not via either NF-κB or AP-1. In addition, we found that this induction was blocked by inhibitors of flavoproteins and NADPH oxidases, indicating involvement of enhanced generation of superoxide radical anion as an upstream signal.

Dual Function of Protein Kinase C (PKC) in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced Manganese Superoxide Dismutase (MnSOD) Expression ACTIVATION OF CREB AND FOXO3a BY PKC-α PHOSPHORYLATION AND BY PKC-MEDIATED INACTIVATION OF Akt, RESPECTIVELY

12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA in human lung carcinoma cells, A549, mediated by a protein kinase C (PKC)-dependent activation of cAMP-responsive element-binding protein (CREB)-1/ATF-1-like factors. In this study, we showed that MnSOD protein expression was elevated in response to TPA or TNF-α, but not to hydrogen peroxide treatment. TPA-induced generation of reactive oxygen species (ROS) was blocked by pretreatment of the PKC inhibitor BIM and NADPH oxidase inhibitor DPI. Small interfering RNA (siRNA) experiments indicated that knocking down the NADPH oxidase components e.g. Rac1, p22phox, p67phox, and NOXO1 in A549 cells impaired TPA-induced MnSOD expression. To identify the PKC isozyme involved, we used a sod2 gene response reporter plasmid, pSODLUC-3340-I2E-C, capable of sensing the effect of TNF-α and TPA, to monitor the effects of PKC isozyme-specific inhibitors and siRNA-induced knockdown of specific PKC isozyme. Our data indicate that TPA-induced MnSOD expression was independent of p53 and most likely mediated by PKC-α-, and -ϵ-dependent signaling pathways. Furthermore, siRNA-induced knock-down of CREB and Forkhead box class O (FOXO) 3a led to a reduction in TPA-induced MnSOD gene expression. Together, our results revealed that TPA up-regulates, in part, two PKC-dependent transcriptional pathways to induce MnSOD expression. One pathway involves PKC-α catalyzed phosphorylation of CREB and the other involves a PKC-mediated the PP2A catalyzed dephosphorylation of Akt at Ser473 which in turn leads to FOXO3a Ser253 dephosphorylation and its activation.

Antioxidative role of selenoprotein W in oxidant-induced mouse embryonic neuronal cell death

It has been reported that selenoprotein W (SelW) mRNA is highly expressed in the developing central nerve system of rats, and its expression is maintained until the early postnatal stage. We here found that SelW protein significantly increased in mouse brains of postnatal day 8 and 20 relative to embryonic day 15. This was accompanied by increased expression of SOD1 and SOD2. When the expression of SelW in primary cultured cells derived from embryonic cerebral cortex was knocked down with small interfering RNAs (siRNAs), SelW siRNA-transfected neuronal cells were more sensitive to the oxidative stress induced by treatment of H2O2 than control cells. TUNEL assays revealed that H2O2-induced apoptotic cell death occurred at a higher frequency in the siRNA-transfected cells than in the control cells. Taken together, our findings suggest that SelW plays an important role in protection of neurons from oxidative stress during neuronal development.

Dual function of protein kinase C in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MnSOD expression: Activation of CREB and FOXO3a by PKC-α phosphorylation and by PKC-mediated Inactivation of Akt, respectively

12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA in human lung carcinoma cells, A549, mediated by a protein kinase C (PKC)-dependent activation of cAMP-responsive element-binding protein (CREB)-1/ATF-1-like factors. In this study, we showed that MnSOD protein expression was elevated in response to TPA or TNF-α, but not to hydrogen peroxide treatment. TPA-induced generation of reactive oxygen species (ROS) was blocked by pretreatment of the PKC inhibitor BIM and NADPH oxidase inhibitor DPI. Small interfering RNA (siRNA) experiments indicated that knocking down the NADPH oxidase components e.g. Rac1, p22phox, p67phox, and NOXO1 in A549 cells impaired TPA-induced MnSOD expression. To identify the PKC isozyme involved, we used a sod2 gene response reporter plasmid, pSODLUC-3340-I2E-C, capable of sensing the effect of TNF-α and TPA, to monitor the effects of PKC isozyme-specific inhibitors and siRNA-induced knockdown of specific PKC isozyme. Our data indicate that TPA-induced MnSOD expression was independent of p53 and most likely mediated by PKC-α-, and -ϵ-dependent signaling pathways. Furthermore, siRNA-induced knock-down of CREB and Forkhead box class O (FOXO) 3a led to a reduction in TPA-induced MnSOD gene expression. Together, our results revealed that TPA up-regulates, in part, two PKC-dependent transcriptional pathways to induce MnSOD expression. One pathway involves PKC-α catalyzed phosphorylation of CREB and the other involves a PKC-mediated the PP2A catalyzed dephosphorylation of Akt at Ser473 which in turn leads to FOXO3a Ser253 dephosphorylation and its activation.

Enzyme-Like Activity of Glycated Cross-Linked Proteins in Free Radical Generation

Abstract: The structure and property of cross‐linked amino acids and proteins produced by a three‐carbon α‐dicarbonyl methylglyoxal in glycation reaction were investigated. Our results showed that these reactions generated yellow fluorescent products and several free radical species. From the reaction with alanine, three types of free radicals were identified by EPR spectroscopy: 1) the cross‐linked radical cation, methylglyoxal diaklylimine cation radical; 2) the methylglyoxal radical anion as the counterion; 3) the superoxide radical anion produced only in the presence of oxygen. Glycation of bovine serum albumin by methylglyoxal also generated the protein‐bound, cross‐linked free radical, probably the cation radical of the cross‐linked Schiff base as observed with alanine. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein‐bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen while the protein‐free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one‐electron oxidation‐reduction reactions. This active center, which exhibits enzyme‐like character, was suggested to be the cross‐linked Schiff base/the cross‐linked Schiff base radical cation of the protein. It mimics the characteristics of metal‐catalyzed oxidation system. These results together indicate that glycated proteins accumulated in vivo provide stable active‐sites for catalyzing the formation of free radicals.

Enhanced free radical generation of FALS-associated Cu,Zn-SOD mutants.

Familial amyotrophic lateral sclerosis (FALS) is an inherited disorder of motor neurons, which is associated with missense mutations in the Cu,Zn-super-oxide dismutase (Cu,Zn-SOD) gene. Mice from the G93A transgenic line was reported to develop a syndrome of FALS. The fact that the symptoms occurred against a background of normal mouse Cu,Zn-SOD activity suggests that dominant, gain-of-function mutations in SOD play a role in the pathogenesis of FALS. We investigated the nature of this gain-of-function of FALS mutants. We have previously reported that Cu,Zn-SOD has the free radical-generating function in addition to normal dismutation activity. These two enzymic activities were compared by using mutants (G93A and A4V) and the wild-type Cu,Zn-SOD prepared by recombinant method. Our results showed that the wild-type, G93A, and A4V enzymes have identical dismutation activity. However, the free radical-generating function of the G93A and A4V mutants, as measured by the spin trapping and EPR method, is enhanced relative to that of the wild-type enzyme (wild-type < G93A < A4V), particularly at lower H2O2 concentrations. This is due to the decrease in the Km value for H2O2, wild-type > G93A > A4V. The catalytic activity to generate free radicals is correlated to the clinical severity of the disorder induced by these mutant enzymes. Furthermore, we found that intact FALS mutants failed to enhance tyrosine nitration. Together, our results indicate that the amyotrophic lateral sclerosis symptoms are not caused by the reduction of Cu,Zn-SOD dismutation activity with the mutant enzymes; rather, it is induced in part by enhancement of the free radical-generating function.

Effect of CCS on the Accumulation of FALS SOD1 Mutant-containing Aggregates and on Mitochondrial Translocation of SOD1 Mutants: Implication of a Free Radical Hypothesis

Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H(2)O(2), mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation.