Tyrosine nitration of glucagon impairs its function: Extending the role of heme in T2D pathogenesis.

New studies raise the possibility that the higher glucagon (GCG) level present in type 2 diabetes (T2D) is a compensatory mechanism to enhance ß-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 ß cells and decreased insulin release. Our results provided a new role of heme for impaired GSIS in the pathological process of diabetes.

Heme and Cu2+-induced vasoactive intestinal peptide (VIP) tyrosine nitration: A possible molecular mechanism for the attenuated anti-inflammatory effect of VIP in inflammatory diseases.

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/H2O2/NO2- system as well as in Cu2+/H2O2/NO2- 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.

Mussel inspired multifunctional bovine serum albumin (BSA) coatings loaded with Baicalein (BAI) to enhance osteogenesis and resist oxidative stress for potential application on implant.

Oxidative stress is associated with most traumatic or pathological bone defects, and seriously affects the effect of implantation. The construction of antioxidative and osteogenic coatings is of great significance to accelerate the bone regeneration of implants. In this study, baicalein (BAI), a nature flavonoid drug, was loaded in bovine serum albumin (BSA) by desolvent method to prepare BAI-BSA composite protein, and tannic acid (TA)/BAI-BSA coatings were further built via layer by layer self-assembly technology. BAI-BSA possesses good biocompatibility that showed no cytotoxicity to osteoblasts and erythrocytes, and helps to enhance the activity of alkaline phosphatase (ALP) and promote the formation of osteogenic mineralized calcium nodules. After assembled with TA, BAI-BSA coating significantly promoted cell adhesion and in vitro osteogenic mineralization of MC3T3-E1. Moreover, BAI drug loading improved the antioxidative function of BSA coatings effectively. The scavenging rates of (TA/BAI-BSA-10)4 for ABTS+• and DPPH• free radicals were 69.6 ± 16.1 % and 53.4 ± 2.4 %, respectively. At cellular level, the TA/BAI-BSA coating effectively inhibited the impact of oxidative stress on the oxidative damage of osteoblasts. The drug-loaded protein coatings possess both great antioxidative and osteogenic functions, which have important potential in the field of bone repair.

Inhibitory activities of flavonoids from Scutellaria baicalensis Georgi on amyloid aggregation related to type 2 diabetes and the possible structural requirements for polyphenol in inhibiting the nucleation phase of hIAPP aggregation.

Human islet amyloid polypeptide (hIAPP)-mediated cytotoxicity is identified as a potential target for developing new anti-diabetic molecules. Herein, we investigated the effect of the major bioactive compounds of Scutellaria baicalensis Georgi (S. baicalensis), including baicalein, baicalin, wogonin and oroxylin A, on hIAPP aggregation. We found that all of these compounds inhibited hIAPP fibril formation in a dose-dependent manner. But baicalein and baicalin, especially baicalein are more effective than wogonin and oroxylin A in stabilizing hIAPP monomers and eliminating toxic hIAPP assembly, suggesting that flavonoids with ortho-hydroxyl group on the A-ring exhibited higher anti-hIAPP nucleation potential than those without this structure. This stimulated our interest in further studying the possible structure-activity relationship between polyphenol and hIAPP aggregation inhibition. Our results demonstrated that flavonoids with ortho-hydroxyl group on the B-ring are also more effective against hIAPP nucleation than those without this structure. These results suggest that the ortho-hydroxybenzene structure is a key structural feature required for polyphenols to effectively inhibit hIAPP nucleation. This was further confirmed by the effects of polyphenol and phenols in inhibiting hIAPP nucleation. The conclusion that pyrogallol-type polyphenols are potential lead inhibitors may provide a valuable structural template for the further development of polyphenol-based inhibitor of amyloid peptides.

Poly(tannic acid) nanocoating based surface modification for construction of multifunctional composite CeO2NZs to enhance cell proliferation and antioxidative viability of preosteoblasts.

Ceria (CeO2) based materials possess many antioxidant enzyme-like activities and unique properties for bone repair, but their free radical scavenging function is still insufficient. In order to deal with the complex oxidative stress environment in bone repair, multifunctional composite CeO2 nanozymes (CeO2NZs), featuring multiple antioxidative properties, were constructed via surface modification on CeO2NZs with nanoscale poly(tannic acid) (PTA) coatings. Moreover, we adjusted pH conditions (ranging from 4 to 9) to effectively control the formation and antioxidative properties of PTA coatings on CeO2NZ surfaces. Here, the physical properties of this novel inorganic and organic composite antioxidant, such as surface morphology, particle size, crystal structure, surface charge and element composition, were thoroughly characterized. The PTA/CeO2NZs showed obvious coating morphology under weak acid conditions (pH = 5-6), and the PTA layer at pH = 5 is about 1 nm in thickness. Compared with untreated CeO2NZs, the PTA/CeO2NZs showed stronger SOD-like activity and obviously higher free radical scavenging rate (for both ABTS+˙ and DPPH˙).Notably, this composite antioxidative nanozyme not only exhibited favorable cell proliferation of preosteoblasts (MC3T3-E1) but also provided strong antioxidative property to maintain cell vitality against H2O2 induced oxidative damage. In particular, this study provides new insights into the designing of surface polyphenolic coatings at the nanoscale, and these multiple antioxidative properties shown by PTA coated CeO2NZs make them suitable for protecting cells under the oxidative stress environment.

Insights Into the Mechanism of Tyrosine Nitration in Preventing ß-Amyloid Aggregation in Alzheimer's Disease.

Nitration of tyrosine at the tenth residue (Tyr10) in amyloid-ß (Aß) has been reported to reduce its aggregation and neurotoxicity in our previous studies. However, the exact mechanism remains unclear. Here, we used Aß1-42 peptide with differently modified forms at Tyr10 to investigate the molecular mechanism to fill this gap. By using immunofluorescent assay, we confirmed that nitrated Aß was found in the cortex of 10-month-old female triple transgenic mice of Alzheimer's disease (AD). And then, we used the surface-enhanced Raman scattering (SERS) method and circular dichroism (CD) to demonstrate that the modification and mutation of Tyr10 in Aß have little impact on conformational changes. Then, with the aids of fluorescence assays of thioflavin T and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS), we found that adding a large group to the phenolic ring of Tyr10 of Aß could not inhibit Aß fibrilization and aggregation. Nitration of Aß reduces its aggregation mainly because it could induce the deprotonation of the phenolic hydroxyl group of Tyr10 of Aß at physiological pH. We proposed that the negatively charged Tyr10 caused by nitration at physiological pH could interact with the salt bridge between Glu11 and His6 or His13 and block the kink around Tyr10, thereby preventing Aß fibrilization and aggregation. These findings provide us new insights into the relationship between Tyr10 nitration and Aß aggregation, which would help to further understand that keeping the balance of nitric oxide in vivo is important for preventing AD.

The oxidative reactivity of three manganese(III) porphyrin complexes with hydrogen peroxide and nitrite toward catalytic nitration of protein tyrosine.

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.

Structure relationship of metalloporphyrins in inhibiting the aggregation of hIAPP.

Metalloporphyrins (FeTBAP, MnTBAP, FeTMPyP and MnTMPyP) have been proposed as effective therapeutic agents in ONOO--related disease including type 2 diabetes (T2D). As these metalloporphyrins share the structural similarities of the planar aromatic conjugation with a valuable class of inhibitors against amyloids fibrillation, they might be effective inhibitors via aromatic π-π stacking interactions with amyloid peptides. Here, we found that the anionic metalloporphyrins (FeTBAP and MnTBAP) are effective inhibitors against hIAPP fibrillation, while, the cationic metalloporphyrins (FeTMPyP and MnTMPyP) only have limited inhibitory effects. Besides, the porphyrin with iron center is more effective than the one with manganese center. Our results favor the electrostatic attraction contributes the main reason to the inhibitory effect between the anionic porphyrins and hIAPP, followed by the π-π stacking interactions between aromatic ring of porphyrins and hIAPP and the stronger coordination ability of iron center to hIAPP. Additionally, by comparison with FeTBAP, which can completely inhibit cytotoxicity induced by hIAPP via stabilizing hIAPP monomers, MnTBAP fails to reverse the cytotoxicity due to that it can only delay the transition of hIAPP from α-helix to ß-sheet rich oligomers. Our results provide theoretical significance for further designing or screening of metalloporphyrins as bifunctional antidiabetic drugs.

Near-Infrared Light-Induced Self-Powered Aptasensing Platform for Aflatoxin B1 Based on Upconversion Nanoparticles-Doped Bi2S3 Nanorods.

A light source plays a pivotal role in a photofuel cell (PFC)-based self-powered biosensor. Although a visible light source has been extensively employed to drive a PFC, it still has some drawbacks for biosensing due to its relatively high energy. Herein we constructed a PFC-based aptasensor using near-infrared (NIR) light as the irradiation source. To achieve an efficient absorption of the NIR light, NaYF4:Yb,Er upconversion nanoparticles (UCNPs) that could convert low-energy incident light into high-energy radiation were combined with Bi2S3 nanorods (UCNPs/Bi2S3) to serve as the photoactive materials. The PFC was comprised of a UCNPs/Bi2S3 photoanode and a Pt cathode, which could generate electrical output under NIR light irradiation to provide the self-powered sensing signal without the supply from an external power source. The aflatoxin B1 (AFB1) binding aptamer was immobilized on the photoanode to serve as the recognition element. The detection of AFB1 was based on the competition between the interaction of aptamer with AFB1 analyte and the hybridization of aptamer with Au nanoparticles-labeled DNA sequence (AuNPs-cDNA). Under optimum conditions, the proposed aptasensor presented good sensitivity and high specificity for AFB1 detection in the concentration range from 0.01 to 100 ng·mL-1, with a detection limit of 7.9 pg·mL-1. Moreover, the developed sensor was applied to an assay of AFB1 in flour samples with a desirable accuracy and precision.

Y12 nitration of human calcitonin (hCT): A promising strategy to produce non-aggregation bioactive hCT.

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.

Peroxynitrite scavenger FeTPPS effectively inhibits hIAPP aggregation and protects against amyloid induced cytotoxicity.

Type 2 diabetes (T2D) is associated with pancreatic ß-cell dysfunction, which can be induced by oxidative stress or/and the aggregation of human islet amyloid polypeptide (hIAPP). Therefore, ONOO- and hIAPP become the crucial targets of T2D treatment. Previously, we found heme could be an effective inhibitor of hIAPP aggregation. However, heme causes serious toxic effects on cells, tissues and organs through oxidative stress, which block it as a potential drug candidate for T2D treatment. 5,10,15,20-tetrakis(4-sulfonatophenyl) porphyrinato iron(III) chloride (FeTPPS), a water-soluble derivative of heme, is recognized as a high-efficient ONOO- decomposition catalyst, which is reported to have a great therapeutic potential in ONOO- -related diseases, including T2D. Here, we explored the potentiality of FeTPPS to be an inhibitor of hIAPP aggregation and the protective effects on cytotoxicity of hIAPP aggregation. It was found that the interaction between FeTPPS and hIAPP remarkably affected hIAPP fibrillation by both stabilizing hIAPP monomers and disaggregating the long fibrils into small oligomeric species. Furthermore, unlike heme, the addition of FeTPPS completely reversed the cytotoxicity and ROS level induced by hIAPP, which was consistent with its strong inhibitory activity. These results implied that FeTPPS could be a promising agent for the treatment of T2D.

Dual Anti-/Prooxidant Behaviors of Flavonoids Pertaining to Cu(II)-Catalyzed Tyrosine Nitration of the Insulin Receptor Kinase Domain in an Antidiabetic Study.

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 (Tyr1158, Tyr1162, and Tyr1163) 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 Cu2+/H2O2/NO2- system. We found that both properties of flavonoid played roles on inhibition of Cu(II)-mediated protein nitration in the H2O2/NO2- 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 •OH production as a prooxidant, which increased 3,3'-dityrosine formation. The formation of 3,3'-dityrosine decreased Cu2+-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.

Hemin-catalyzed biomimetic oxidative phenol-indole [3 + 2] reactions in aqueous media.

A hemin/H2O2 catalytic system for oxidative phenol-indole [3 + 2] coupling in aqueous solution has been developed, enabling benign synthesis of valuable benzofuroindolines under sustainable conditions. Mechanistic studies revealed the dual role of iron porphyrin responsible for both phenol oxidation and Lewis acid activation, which differs from the well-explored chemistry of hemin in carbene and nitrene insertion reactions. A preliminary experiment with cytochrome c showed that the turnover of iron porphyrin was amenable for a macromolecular setting with remarkable efficiency (ca. 13 300 TON).

Synthesis of a CdS-decorated Eu-MOF nanocomposite for the construction of a self-powered photoelectrochemical aptasensor.

A composite of CdS nanoparticles and a europium metal organic framework (Eu-MOF) (CdS/Eu-MOF) was synthesized. The unique properties of MOFs help to improve the photoelectrochemical (PEC) properties of CdS by reducing charge carrier recombination and utilizing a broader spectrum for light harvesting. Under visible light illumination, the photocurrent of the CdS/Eu-MOF composite modified electrode was about 2.5-fold higher than that of the CdS modified electrode. When an ampicillin (AMP)-binding aptamer was immobilized on the CdS/Eu-MOF modified electrode as a recognition element, a self-powered PEC aptasensor exhibiting a specific photocurrent response to AMP was constructed. Several experimental conditions such as the ratio of CdS to MOF, the coating amount of the CdS/Eu-MOF suspension and the concentration of the aptamer were studied. Under optimum conditions, the photocurrent of the developed sensor was linearly related to the logarithm AMP concentration in the range of 1 × 10-10 to 2 × 10-7 M, with a detection limit (3S/N) of 9.3 × 10-11 M. Moreover, this sensor exhibited excellent selectivity, good repeatability and desirable stability. It was successfully applied to the detection of AMP in lake water and milk samples.

Structure effect of water-soluble iron porphyrins on catalyzing protein tyrosine nitration in the presence of nitrite and hydrogen peroxide.

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/H2O2 system as similar as those in FeTPPS/H2O2 and FeTBAP/H2O2 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 OFeIV-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.

Heme prevents highly amyloidogenic human calcitonin (hCT) aggregation: A potential new strategy for the clinical reuse of hCT.

Irreversible aggregation can extremely limit the bioavailability and therapeutic activity of peptide-based drugs. Thus, peptide fibrillation is an excellent challenge for biotechnological drug development. 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. hCT is therefore not widely used preparation in clinical practice. Nonetheless, hCT seems to be still an ideal target for clinical therapy when fibrillation is effectively inhibited, because the alternatives of hCT can stimulate undesirable immune responses in patients and cause side effects. Interestingly, heme is an essential component for many livings and has been shown a strong inhibitory effect on some amyloidogenic peptides aggregation. Here we demonstrate that it may be a most suitable, safe, biocompatible small molecule inhibitor on hCT aggregation, and thereby improving its activity when guiding the drug peptide in clinical therapeutics. In this work, we found that heme was able to reversibly bind with hCT to form a heme-hCT complex with a moderate binding constant (9.17 × 106 M-1) and significantly suppress the aggregation of hCT probably accomplished by heme binding to it, blocking the ß-sheet structure assembly which is essential in hCT fibril aggregation. Meanwhile, the heme-hCT complexes showed enhanced bioactivity compared to hCT itself after a 24 h incubation time in reducing blood calcium levels in mice. This study may develop a new strategy to reuse the wild-type hCT in clinical therapeutics.

Nitration of hIAPP promotes its toxic oligomer formation and exacerbates its toxicity towards INS-1 cells.

Amyloid formation of human islet amyloid polypeptide (hIAPP) is one of the most common pathological features of type 2 diabetes (T2D). Increasing evidences have shown that the overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an important role in the development of the T2D. Interestingly, our previous studies indicated that heme could bind to hIAPP, and the complex might induce the nitration of tyrosine residue (Y37) of hIAPP in the presence of hydrogen peroxide and nitrite. However, it remains unclear about effect of the nitration on the implicated function of hIAPP in the development of T2D. In this study, fluorescent assays, transmission electron microscopy (TEM), atomic force microscope (AFM) were used to demonstrate that nitration of hIAPP significantly decreased its fibril formation. But the decreased fibril formation was not through the diminished aggregation of hIAPP monomer as suggested by the results of circular dichroism spectroscopy (CD) and gel electrophoresis assay. Surface-enhanced raman spectroscopy (SERS) indicated that nitration of hIAPP impaired the intermolecular hydrogen bonding. On the basis of these results, we hypothesize that nitration of hIAPP may block the intermolecular hydrogen bonding, leading to the inhibition of its fibril formation. In addition, cytotoxicity study of native and modified hIAPP was also performed on INS-1 cells, which revealed exacerbated toxicity of hIAPP by its nitration. The findings in this study that nitration of hIAPP promotes its oligomer formation and thus exacerbates its cytotoxicity suggests a possible link between the nitrite (or the sum of nitrite and nitrate) levels and T2D, and ameliorated nitration of hIAPP by diminishing nitrative stress might be a promising therapeutic strategy for T2D.

Tyrosine residues of bovine serum albumin play an important role in protecting SH-SY5Y cells against heme/H2O2/NO2--induced damage.

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.

Nitration of amyloid-ß peptide (1-42) as a protective mechanism for the amyloid-ß peptide (1-42) against copper ion toxicity.

It is known that copper ion (Cu(II)) binds to amyloid-ß peptide (Aß), induces Aß oligomer formation and ultimately exacerbates Aß-aggregation neurotoxicity in Alzheimer's disease (AD). It becomes interesting to know that how this chemical modification of Aß would affect interaction of Aß and Cu(II) and their roles in the development of AD. In this work, we investigated the interaction of Aß1-42 nitration with the toxic Cu(II). It showed that Cu(II)induced Aß1-42 nitration in the presence of nitrite and hydrogen peroxide. Circular dichroism studies also revealed significant conformational change of Aß1-42 and Tyr10 nitrated amyloid-ß peptide(1-42) (Aß1-42NT) when interacting with Cu(II). Even though nitration did not alter the binding of Aß1-42 to Cu(II) or the peroxidative activity of Aß1-42-Cu(II) complex, nitration ameliorated the aggregation and neurotoxicity of Aß1-42 induced by Cu(II), which was also further confirmed by the cell study. Given our previous findings that Aß nitration dramatically inhibited its aggregation and thus reduced its toxicity, we speculated that nitration of Aß1-42 altered its intermolecular interaction, which protected itself against the toxicity of Cu(II). Based on this hypothesis, we propose that nitration of Aß1-42 may be an important protective mechanism for normal function of Aß1-42 and deserves more attention in AD drug development.

Study on the detoxification mechanisms to 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) chloride (FeTPPS), an efficient pro-oxidant of heme water-soluble analogue.

5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinato iron(III) chloride (FeTPPS) is a water-soluble analog of heme and widely employed as peroxynitrite scavenger in vivo. However, previous studies have showed that like heme, FeTPPS could also act as an effective pro-oxidant towards appreciable substrates in vitro in the presence of oxidant. The reason that FeTPPS did not show any pro-oxidative damage in previous studies when it was used as peroxynitrite decomposition catalyst in vivo, has not been studied. Herein, the effects of two main detoxification mechanisms of heme, i.e., serum albumin (SA) binding and heme oxygenase-1 (HO-1) induction, were examined on FeTPPS in vitro. Fluorescence quenching studies showed bovine serum albumin (BSA) could bind to FeTPPS with high affinity (Kb ~ 109 M-1). Molecular docking studies presented us the details of the binding site that is not a heme pocket. Furthermore, the intrinsic pro-oxidative activity of FeTPPS was found effectively inhibited by forming BSA-FeTPPS complex of low reactivity, which could be thought to protect against the potentially toxic effects of FeTPPS on blood components. In addition, this binding could protect FeTPPS against oxidative degradation. In albumin-free cell system, cell viability results indicated FeTPPS was innoxious to living cells and could protect cells against the oxidative impairment of H2O2 effectively rather than promoting damage. Using western blot, we illustrated that HO-1 expression could not be induced by FeTPPS, which suggested that HO-1 was not related to the protective capacity of FeTPPS. Our results provide a better understanding of FeTPPS and lead to a new guidance to its application.