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Understanding the toxicological properties of MnIII-porphyrins (MnTPPS, MnTMPyP, or MnTBAP) can provide important biochemical rationales in developing them as the therapeutic drugs against protein tyrosine nitration-induced inflammation diseases. Here, we present a comprehensive understanding of the pH-dependent redox behaviors of these MnIII-porphyrins and their structural effects on catalyzing bovine serum albumin (BSA) nitration in the presence of H2O2 and NO2-. It was found that both MnTPPS and MnTBAP stand out in catalyzing BSA nitration at physiologically close condition (pH 8), yet they are less effective at pH 6 and 10. MnTMPyP was shown to have no ability to catalyze BSA nitration under all tested pHs (pH 6, 8, and 10). The kinetics and active intermediate determination through electrochemistry method revealed that both the pH-dependent redox behavior of the central metal cation and the antioxidant capability of porphin derivative contribute to the catalytic activities of three MnIII-porphyrins in BSA nitration in the presence of H2O2/NO2-. These comprehensive studies on the oxidative reactivity of MnIII-porphyrins toward BSA nitration may provide new clues for searching the manganese-based therapeutic drugs against the inflammation-related diseases., (© The Author(s) 2021. Published by Oxford University Press.)

Irreversible aggregation can extremely limit the bioavailability and therapeutic activity of peptide-based drugs. There is therefore an urgent demand of effective strategy to control peptide aggregation. Recently, we found that tyrosine nitration at certain sites of peptide can effectively inhibit its aggregation. This minor modification may be an ideal strategy to the rational design of peptide-based drugs with low aggregation propensity yet without loss of bioactivity. Human calcitonin (hCT) is such a peptide hormone known for its hypocalcaemic effect but has limited pharmaceutical potential due to a high tendency to aggregate. In this study, by using multiple techniques including Fluorescence, TEM, Nu-PAGE and CD, we demonstrated that Y12 nitration of hCT would significantly inhibit its self-assembles, and we also found that this modification would not only reduce the cytotoxicity induced by peptide aggregation, but also had little effect on its potency. This finding may provide a novel strategy for clinically application of hCT instead of sCT., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Flavonoid, as a potent antioxidant, exerts many beneficial effects in type 2 diabetes, whereas the prooxidative property may be also important in vivo if copper is involved. Here, we chose an insulin receptor kinase domain fragment (KK-1, residues 1126-1165), containing the A-loop of the receptor as well as three key autophosphorylation sites (Tyr 1158 , Tyr 1162 , and Tyr 1163 ) associated with receptor signal transduction to investigate the roles and the structure-activity relationship of three antidiabetic flavonoids (kaempferol, luteolin, and apigenin) and two others with a similar structure (diosmetin and genistein), on modulation of Cu(II)-mediated tyrosine nitration and the corresponding effect on its functional phosphorylation in the Cu 2+ /H 2 O 2 /NO 2 - system. We found that both properties of flavonoid played roles on inhibition of Cu(II)-mediated protein nitration in the H 2 O 2 system: (1) on the one hand, flavonoid scavenged free radicals as antioxidants, inhibited tyrosine nitration, and thus inhibited the reduction of tyrosine phosphorylation caused by tyrosine nitration; and (2) on the other hand, flavonoid promoted 2 - -induced tyrosine nitration and thus interfered with its phosphorylation. This study confirms that the weight relationship between antioxidation and prooxidation of a flavonoid needs to be studied clearly before nutritional and medical applications.• OH production as a prooxidant, which increased 3,3'-dityrosine formation. The formation of 3,3'-dityrosine decreased Cu 2+ -induced tyrosine nitration and thus interfered with its phosphorylation. This study confirms that the weight relationship between antioxidation and prooxidation of a flavonoid needs to be studied clearly before nutritional and medical applications.

Water-soluble iron porphyrins, such as FeTPPS (5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III)), FeTMPyP (5,10,15,20-tetrakis (N-methyl-4'-pyridyl) porphyrinato iron (III) chloride) and FeTBAP (5,10,15,20-tetrakis (4-benzoic acid) porphyrinato iron (III)), are highly active catalysts for peroxynitrite decomposition and thereby have been suggested as therapeutic agent for inflammatory diseases that implicate the involvement of nitrotyrosine formation. Here, we systemically investigated catalytic properties of FeTPPS, FeTMPyP and FeTBAP on protein nitration in the presence of hydrogen peroxide and nitrite. We showed that FeTPPS, FeTBAP and FeTMPyP all exhibited higher peroxidase activity in compared with hemin. As to protein nitration, the catalytic effect of FeTPPS and FeTBAP are effective in the presence of hydrogen peroxide and nitrite, while negligible BSA nitration was observed in the case of FeTMPyP. Moreover, the underlying mechanism of the oxidation of FeTPPS, FeTBAP and FeTMPyP was further studied. Collectively, our results suggest that, compound I and II species are involved in as the key intermediates in FeTMPyP/H 2 O 2 system as similar as those in FeTPPS/H 2 O 2 and FeTBAP/H 2 O 2 system. As compared to weak antioxidants, TPPS and TBAP, however, TMPyP scavenges oxo-Fe (IV) intermediates of FeTMPyP at a faster rate by significant self-degradation; results in the shortest lifetimes of OFe IV -TMPyP and the lowest catalytic activity on oxidizing tyrosine and nitrite; and therefore, attributes to inactivation of FeTMPyP in protein nitration. In addition, association of FeTMPyP to BSA was found weak, while strong binding of FeTPPS and FeTBAP were observed. The weak binding keeps away of target residue of BSA from the center of FeTMPyP where the RNS is generated, which might be attributed as additional factors to the inactivation of FeTMPyP in protein nitration., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Serum albumin (SA) has been shown to act as a heme scavenger in hemolysis and can protect cell against the toxic effect of heme. However, the mechanism of SA in heme detoxification is not well understood. Interestingly, increasing studies indicate that heme/H 2 O 2 -dependent reaction is unlikely to be the principal cause of heme toxicity in excessive intravascular hemolysis conditions. Moreover, high levels of NO 2 - and NO 3 - were also found in patients with severe hemolytic diseases, which seem to involve in heme toxic effect as well. Therefore, we proposed that studying the protection mechanism of SA against the heme/H 2 -induced cytotoxicity may be more consistent with free heme-associated disorder pathologies. In this study, we tested the hypotheses that tyrosine residues of bovine serum albumin (BSA) play a prominent role in detoxifying heme in SH-SY5Y cells. Both BSA and tyrosine modified BSA (BSA-T) were used to explore this protective mechanism. Most of cellular injury (oxidative and nitrative damage) induced by heme/H 2 /NO 2 - -induced cytotoxicity may be more consistent with free heme-associated disorder pathologies. In this study, we tested the hypotheses that tyrosine residues of bovine serum albumin (BSA) play a prominent role in detoxifying heme in SH-SY5Y cells. Both BSA and tyrosine modified BSA (BSA-T) were used to explore this protective mechanism. Most of cellular injury (oxidative and nitrative damage) induced by heme/H 2 O 2 , but by trapping radical through special residues such as tyrosine to render other important protein less damaged.2 - were prevented by pretreatment with an equimolar concentration of BSA or BSA-T, and BSA was found more efficient than BSA-T. Meanwhile, BSA or BSA-T binding to heme is not accompanied by a decrease of heme's peroxidase activity. Collectively, these data suggest that the protecting effect of BSA against heme-induced damage in the intravascular hemolysis diseases is not accomplished by preventing the primary reactivity of heme with H 2 O 2 , but by trapping radical through special residues such as tyrosine to render other important protein less damaged.

Protein tyrosine nitration is implicated in the occurrence and progression of pathological conditions involving free radical reactions. It is well recognized that hemin can catalyze protein tyrosine nitration in the presence of nitrite and hydrogen peroxide. Generally, the catalytic efficiency is positively correlated to its peroxidase activity. In this study, however, it is found that the efficiency of hemin in catalyzing protein tyrosine nitration is largely suppressed after functionalization with graphene derivatives, even though its peroxidase-like activity is more than quadrupled. Further studies show that the oxidation of tyrosine is still observed for these composites; dityrosine formation, however, is greatly inhibited. Furthermore, these composites also exhibit strong effects on the oxidation of nitrite into nitrate. Therefore, we propose a mechanism in which hemin-graphene derivatives facilitate the oxidation of tyrosine and nitrite to produce tyrosyl radicals and nitrogen dioxide radicals in the presence of hydrogen peroxide, but graphene interlayers serve as barriers that hinder radical-radical coupling reactions; consequently, protein tyrosine nitration is restrained. This property of hemin-graphene derivatives, by which they catalyze substrate oxidation but suppress radical-radical coupling reactions, shows their great potential in selective oxidation procedures for byproduct removal., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Peroxynitrite and heme peroxidases (or heme)-H 2 O 2 -NaNO 2 system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways-induced protein nitration and oxidation are controversial. Both nitrating systems can dose-dependently induce triosephosphate isomerase (TIM) nitration, however, heme-H 2 O 2 -NaNO 2 was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3-morpholinosydnonimine hydrochloride (SIN-1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme-H 2 O 2 -NaNO 2 and SIN-1 and protein oxidation induced by SIN-1, but promoted heme-H 2 O 2 -NaNO 2 -induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN-1-induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme-H 2 O 2 -NaNO 2 -induced nitration. Our results indicated that peroxynitrite and heme-H 2 O 2 -NaNO 2 -induced protein nitration was different, and the later one could be a better model for anti-nitration compounds screening., (© 2017 Wiley Periodicals, Inc.)

Amyloid-β plaques and oxidative stress are the major hallmarks of Alzheimer's disease. Our previous study found that the heme-Aβ complex enhanced the catalytic effect of free heme on protein tyrosine nitration in the presence of hydrogen peroxide (H 2 O 2 ) and nitrite (NO 2 - ). Y10 in Aβ could be the first target to be nitrated. We also found that nitration of Aβ 1-40 significantly decreased its aggregation. However, a contrary report showed that nitration of Aβ 1-42 by peroxynitrite enhanced its aggregation. To rule out the interference of peroxynitrite caused Aβ oxidation, we used synthetic Y10 nitrated Aβ 1-42 to study the influence of Y10 nitration on Aβ 1-42 's aggregation and cytotoxicity in this study. We confirmed that Aβ 1-42 could be nitrated in the presence of H 2 O 2 , NO 2 - , and heme by dot blotting. CD spectroscopy showed an increase of β-sheet structure of Aβ 1-42 and its mutants. The thioflavin T (ThT) flourescence assay revealed that both nitration and chlorination significantly inhibited Aβ 1-42 fibril formation. TEM and AFM observations of Aβ peptide aggregates further confirmed that Y10 modification inhibited Aβ 1-42 fibril formation. The cytotoxicity study of native and modified Aβ peptides on SH-SY5Y cells revealed that nitration of Aβ 1-42 remarkably decreased the neurotoxicity of Aβ 1-42 . On the basis of these results, we hypothesized that nitration of Y10 may block the π-π stacking interactions of Aβ 1-42 so that it inhibit its aggregation and neurotoxicity. More importantly, considerable evidence suggested that the levels of nitrite plus nitrate significantly decreased in the brain of AD patients. Thus, we believe that these findings would be helpful for further understanding the function of Aβ in AD.

Amyloid β peptide (Aβ) aggregation is considered to be a crucial pathological biomarker of Alzheimer's disease (AD). It was found that Aβ and heme can form an Aβ-heme complex, which results in increased heme pseudoperoxidase activity. Recently, we found that increasing pseudoperoxidase activity induces elevated tyrosine nitration on Aβ in the presence of nitrite and hydrogen peroxide. However, the nature of tyrosine nitration of Aβ and its physiologic significance are still unknown. In this study, we revealed that Aβ1-40 can be nitrated in vitro by binding to heme in the presence of nitrite and hydrogen peroxide. Moreover, we found that tyrosine nitration had little effect on Aβ1-40's binding activity with heme. A TMB assay also revealed that the peroxidase activity of the heme-Aβ1-40Y10(3N)T (tyrosine 10 was replaced with 3-nitrtotyrosine in Aβ1-40) complex was moderately increased compared with that of the heme-Aβ1-40 complex. Furthermore, Thioflavin T fluorescence and transmission electron microscopic characterization indicated that tyrosine nitration significantly decreased the aggregation of Aβ1-40. In addition, a cytotoxicity test verified that wild-type Aβ1-40 was more cytotoxic than that of Aβ1-40Y10(3N)T. These results suggest that nitration of Aβ1-40 might be an Aβ detoxicant process and a compensatory reaction to nitrative stress. Our findings may lead to a detailed understanding of the function of Aβ1-40 and may be helpful in preventing and curing AD.

Background: Serum albumin binds avidly to heme to form heme-serum albumin complex, also called methemalbumin, and this binding is thought to protect against the potentially toxic effects of heme. However, the mechanism of detoxification has not been fully elucidated., Methods: SDS-PAGE and Western blot were used to determine the efficiency of methemalbumin on catalyzing protein carbonylation and nitration. HPLC was used to test the formation of heme to protein cross-linked methemalbumin., Results: The peroxidase activity of heme increased upon human serum albumin (HSA) binding. Methemalbumin showed higher efficiency in catalyzing tyrosine oxidation than free heme in the presence of H2O2. Methemalbumin catalyzed self-nitration and significantly promoted the nitration of tyrosine in coexistent protein, but decreased the carbonylation of coexistent protein compared with heme. The heme to protein cross-linked form of methemalbumin suggested that HSA trapped the free radical accompanied by the formation of ferryl heme. When tyrosine residues in HSA were modified by iodination, HSA lost of protection effect on protein carbonylation. The low concentration of glutathione could effectively inhibit tyrosine nitration, but had no effect on protein carbonylation., Conclusion: HSA protects against the toxic effect of heme by transferring the free radical to tyrosine residues in HSA, therefore protecting surrounding proteins from irreversible oxidation, rather than by direct inhibiting the peroxidase activity. The increased tyrosine radicals can be reduced by endogenic antioxidants such as GSH., General Significance: This investigation indicated the important role of tyrosine residues in heme detoxification by HSA and suggested a possible novel mechanism., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Protein tyrosine nitration is a post-translational modification associated with numerous pathological conditions. The biological consequences of this modification strongly depend on the site selectivity. Unfortunately, to date there is still no reliable model for predicting the selectivity of protein tyrosine nitration. Previously, we found that amyloid beta (Aβ) changed the selectivity of enolase tyrosine nitration upon binding to heme. It seemed that there was a link between the hydrophilicity of Aβ and the site-specific tyrosine nitration. We further investigated the role of the hydrophilicity of the molecules that bind to heme in the selectivity of protein tyrosine nitration. We found that Aβ(1-16), Aβ(1-20), and Aβ(1-40), upon binding to heme and interacting with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a site-specific manner, differently modulated the site selectivity of heme-catalyzed GAPDH tyrosine nitration. The modulation is associated with the hydrophilicity of the Aβ peptides, which changed the surrounding environment of the heme. At the same time, the Aβ-heme complexes were found to be more effective at inactivating GAPDH than heme alone, and the selective tyrosine nitration that was catalyzed by Aβ-heme played an important role. These findings suggest an alternative mechanism for the selectivity of protein tyrosine nitration, which may lead to a better understanding of the factors that influence protein tyrosine nitration selectivity and the important roles of Aβ and heme in the pathogenesis of Alzheimer's disease, where Aβ accumulation and Aβ-dependent protein nitration play central roles.

Baicalin has been reported to protect against liver injury in iron-overload mice, however, the mechanisms underlying the hepatoprotective properties of baicalin are poorly understood. In this study, we systematically studied the protective effect of baicalin on iron overload induced liver injury, as well as the underlying mechanism based on nitrative stress in rat model. We found that when iron overload rats (500mgiron/kg) were fed baicalin-containing diet (0.3% and 1% w/w) for 45days, baicalin dose dependently protected against iron overload induced liver injury, including alleviation of hepatic pathological damage, decrease of SOD activity, iron content, carbonyl content, and the thiobarbituric acid-reactive substances level in hepatic tissues. It also increased serum iron content, SH content and GPx activity, decreased serum ALT and AST activities. Immunohistochemistry and immunoprecipitation analysis revealed that baicalin could also inhibit iron overload induced protein tyrosine nitration in liver. Moreover, in iron overload rat liver, we found that baicalin decreased the iron overload increased level of glutathione-S-transferases (GSTs) expression, oxidation and nitration. These results suggest that not only oxidative stress, but also nitrative stress, is involved in iron overload induced liver injury, and the underlying mechanism might partially relate to the involvement of GSTs expression and post-translational modification. Baicalin can effectively prevent iron overload caused abnormality and can be a candidate medicine for iron overload diseases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The dysfunction and further damage of endothelium play an important role in the development and progression of diabetic vascular complications. Protein tyrosine nitration is involved in endothelial cell injury induced by high glucose. Little is known about protein nitration in human umbilical vein endothelial cells (ECV304) induced by high glucose. In the present article, exposure of ECV304 to 30 mM high glucose (HG30) and 40 mM high glucose (HG40) or hemin-nitrite-H2O2 system for 72 h, the cell injury in ECV304 induced by high glucose and exogenous nitrating agent was studied. After 72 h treatment, it was found that high glucose stimulated ECV304 injury in a dose-dependent manner, including reducing cell viability, increasing malondialdehyde (MDA) content, decreasing glutathione (GSH) content, increasing intracellular reactive oxygen species (ROS), increasing the production of nitric oxygen (NO) (increased nitrite content in cell and nitrate content in medium) and generating protein tyrosine nitration. It was also found that protein tyrosine nitration could induce cell injury further. By comparison the protein tyrosine nitration induced by high glucose condition and extrinsic factors (hemin-nitrite-H2O2 system), it may be speculated that protein is nitrated selectively to generate nitrotyrosine in diabetic vascular complications.

Serum albumin (SA) has been shown to act as a heme scavenger in hemolysis and can protect cell against the toxic effect of heme. However, the mechanism of SA in heme detoxification is not well understood. Interestingly, increasing studies indicate that heme/H2O2-dependent reaction is unlikely to be the principal cause of heme toxicity in excessive intravascular hemolysis conditions. Moreover, high levels of NO2− and NO3− were also found in patients with severe hemolytic diseases, which seem to involve in heme toxic effect as well. Therefore, we proposed that studying the protection mechanism of SA against the heme/H2O2/NO2−-induced cytotoxicity may be more consistent with free heme-associated disorder pathologies. In this study, we tested the hypotheses that tyrosine residues of bovine serum albumin (BSA) play a prominent role in detoxifying heme in SH-SY5Y cells. Both BSA and tyrosine modified BSA (BSA-T) were used to explore this protective mechanism. Most of cellular injury (oxidative and nitrative damage) induced by heme/H2O2/NO2− were prevented by pretreatment with an equimolar concentration of BSA or BSA-T, and BSA was found more efficient than BSA-T. Meanwhile, BSA or BSA-T binding to heme is not accompanied by a decrease of heme's peroxidase activity. Collectively, these data suggest that the protecting effect of BSA against heme-induced damage in the intravascular hemolysis diseases is not accomplished by preventing the primary reactivity of heme with H2O2, but by trapping radical through special residues such as tyrosine to render other important protein less damaged. [ABSTRACT FROM AUTHOR]

Protein tyrosine nitration is implicated in the occurrence and progression of pathological conditions involving free radical reactions. It is well recognized that hemin can catalyze protein tyrosine nitration in the presence of nitrite and hydrogen peroxide. Generally, the catalytic efficiency is positively correlated to its peroxidase activity. In this study, however, it is found that the efficiency of hemin in catalyzing protein tyrosine nitration is largely suppressed after functionalization with graphene derivatives, even though its peroxidase-like activity is more than quadrupled. Further studies show that the oxidation of tyrosine is still observed for these composites; dityrosine formation, however, is greatly inhibited. Furthermore, these composites also exhibit strong effects on the oxidation of nitrite into nitrate. Therefore, we propose a mechanism in which hemin-graphene derivatives facilitate the oxidation of tyrosine and nitrite to produce tyrosyl radicals and nitrogen dioxide radicals in the presence of hydrogen peroxide, but graphene interlayers serve as barriers that hinder radical-radical coupling reactions; consequently, protein tyrosine nitration is restrained. This property of hemin-graphene derivatives, by which they catalyze substrate oxidation but suppress radical-radical coupling reactions, shows their great potential in selective oxidation procedures for byproduct removal. [ABSTRACT FROM AUTHOR]

Vasoactive intestinal peptide (VIP) is a neuropeptide that play an important role in immunoregulation and anti-inflammation. Numerous inflammatory/autoimmune disorders are associated with decreased VIP binding ability to receptors and diminished VIP activation of cAMP generation in immune cells. However, the mechanisms linking oxidative/nitrative stress to VIP immune dysfunction remain unknown. It has been reported that the elevated heme or Cu2+ in inflammatory diseases can cause oxidative and nitrative damage to nearby biological targets under high oxidative stress conditions, which affects the structure and activity of linked peptides or proteins. Thus, the VIP down-regulated immune response may be interfered by redox metal catalyzed VIP tyrosine nitration. To explore this, we systematically investigated the possibility of heme or Cu2+ to catalyze VIP tyrosine nitration. The results showed that Tyr10 and Tyr22 of VIP can both be nitrated in heme/H 2 O 2 /NO 2 − system as well as in Cu2+/H 2 O 2 /NO 2 − system. Then, we used synthetic mutant VIPs with tyrosine residues substituted by 3-nitrotyrosine to study the impact of tyrosine nitration on VIP activity in SHSY-5Y cells. Our findings demonstrated that VIP nitration dramatically decreased the content of its α-helix and random coil, suggesting that VIP nitration might reduce its affinity to the receptor. This was further confirmed in the cAMP assay. The results showed that 10 nM of these tyrosine nitrated VIPs could significantly (p < 0.01) decrease cAMP secretion compared to the wild type VIP. Our data reveal that the attenuation of the neuroprotective effect of VIP in inflammation-related diseases might be attributed to metal-catalyzed VIP tyrosine nitration. [Display omitted] • Vasoactive intestinal peptide (VIP) is prone to bind to heme or Cu2+. • Heme or Cu2+ can catalyze the nitration of Tyr10 and Tyr22 residues in VIP in the presence of H 2 O 2 and NO 2 −. • Tyrosine nitration of VIP reduced the content of its active monomer structure and influenced the production of cAMP in cells. [ABSTRACT FROM AUTHOR]

New studies raise the possibility that the higher glucagon (GCG) level present in type 2 diabetes (T2D) is a compensatory mechanism to enhance β-cell function, rather than induce dysregulated glucose homeostasis, due to an important role for GCG that acts directly within the pancreas on insulin secretion by intra-islet GCG signaling. However, in states of poorly controlled T2D, pancreatic α cell mass increases (overproduced GCG) in response to insufficient insulin secretion, indicating decreased local GCG activity. The reason for this decrease is not clear. Recent evidence has uncovered a new role of heme in cellular signal transduction, and its mechanism involves reversible binding of heme to proteins. Considering that protein tyrosine nitration in diabetic islets increases and glucose-stimulated insulin secretion (GSIS) decreases, we speculated that heme modulates GSIS by transient interaction with GCG and catalyzing its tyrosine nitration, and the tyrosine nitration may impair GCG activity, leading to loss of intra-islet GCG signaling and markedly impaired insulin secretion. Data presented here elucidate a novel role for heme in disrupting local GCG signaling in diabetes. Heme bound to GCG and induced GCG tyrosine nitration. Two tyrosine residues in GCG were both sensitive to the nitrating species. Further, GCG was also demonstrated to be a preferred target peptide for tyrosine nitration by co-incubation with BSA. Tyrosine nitration impaired GCG stimulated cAMP-dependent signaling in islet β cells and decreased insulin release. Our results provided a new role of heme for impaired GSIS in the pathological process of diabetes. The possible mechanism of intra-islet GCG activity impairment in diabetes. Since the majority of beta cells in human islets are in direct contact with alpha cells, there is a functional crosstalk between these two major islet cell types. Mechanistically, the positive effect of GCG on beta cell function is achieved by GCG binding to G protein-coupled receptors (GPCRs) (including GLP-1 receptor and GCG receptor), that are expressed in beta cells, and the activation of GPCRs signaling to stimulate the production of cAMP that acts as a necessary cofactor to support glucose-stimulated insulin secretion (GSIS) (the mechanism of GCG maintaining "β-cell tone" is shown as "Normal"). We proposed that tyrosine nitration of GCG may be one of the reason for a diminished intra-islet paracrine hormone action of GCG in diabetes. Heme bound to GCG promotes the probability of GCG tyrosine nitration in the presence of H 2 O 2 and NO 2 −. Tyrosine nitration may affect the structure of GCG, leading to impaired affinity for GPCRs, thereby decreased the stimulatory effect of GCG on insulin secretion from pancreatic beta cells (defective intra-islet GCG action due to that heme-induced GCG tyrosine nitration is shown as "Natrative stress"). [Display omitted] • Glucagon (GCG) is prone to bind to heme • Heme catalyzes GCG tyrosine nitration in the presence of H 2 O 2 and NO 2 − • GCG is a preferred target protein for tyrosine nitration • Tyrosine nitration impaired GCG's role in intra-islet paracrine signaling, which ultimately impaired insulin secretion [ABSTRACT FROM AUTHOR]

Abstract: Protein tyrosine nitration, protein oxidation and lipid peroxidation are nitrative/oxidative modification of protein and lipids. In this paper, a BSA (bovine serum albumin)–lecithin liposome system was used to study the nature of different forms of iron, including methemoglobin, hemin and ferric citrate, in catalyzing H2O2–nitrite system to oxidize protein and lipid as well as nitrate protein. It was found that in pH range of 5.0–9.0, in pure BSA solution or pure liposome solution, hemin and methemoglobin catalyzed protein tyrosine nitration and lipid peroxidation were decreased with the increasing of pH, while hemin and methemoglobin catalyzed protein oxidation was significantly and moderately increased, respectively. Lipid completely inhibited hemin catalyzed protein tyrosine nitration but only partially inhibited methemoglobin catalyzed protein tyrosine nitration, and its inhibitory effect on hemin induced protein oxidation was also more pronounced. In addition, BSA showed more efficient in inhibiting hemin and ferric citrate induced lipid peroxidation. At the same condition, ferric citrate was relatively ineffective in all tests. Considering protein tyrosine nitration, protein oxidation and lipid oxidation as overall oxidative damage, these results indicated that methemoglobin is more toxic than hemin and ferric citrate, the degradation procedure of heme containing macromolecules, e.g. hemoglobin to hemin and finally to low molecular weight bounded iron, is step by step detoxification. These results provide fundamental knowledge on oxidative/nitrative of biomolecules in lipid–protein coexistence system. [Copyright &y& Elsevier]

GAPDH was speculated to function as a transient trap to reduce the potential toxicity of free heme by a specific and reversible binding with heme. Up to now, there has been lack of studies focused on this effect. In this paper, the efficiency of GAPDH-heme complex on catalyzing protein carbonylation and nitration, the cross-linking of heme to protein formation, and cytotoxicity of GAPDH-heme were studied. It was found that the binding of GAPDH could inhibit H 2 O 2 -mediated degradation of heme. Peroxidase activity of GAPDH-heme complex was higher than that of free heme, but significantly lower than that of HSA-heme. Catalytic activity of heme corresponded complex toward tyrosine oxidation/nitration was decreased in the order of HSA-heme, heme and GAPDH-heme. GAPDH also inhibited heme-H 2 O 2 -NO 2 - induced protein carbonylation. No covalent bond was formed between heme and GAPDH after treated with H 2 O 2 . GAPDH was more effective than HSA on protecting cells against heme-NO 2 - -H 2 O 2 induced cytotoxicity. These results indicate that binding of GAPDH inhibits the activity of heme in catalyzing tyrosine nitration and protects the coexistent protein against oxidative damage, and the mechanism is different from that of HSA. This study may help clarifying the protective role of GAPDH acting as a chaperone in heme transfer to downstream areas. [ABSTRACT FROM AUTHOR]