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 (H2O2) and nitrite (NO2–). Y10 in Aβ could be the first target to be nitrated. We also found that nitration of Aβ1–40 significantly decreased its aggregation. However, a contrary report showed that nitration of Aβ1–42 by peroxynitrite enhanced its aggregation. To rule out the interference of peroxynitrite caused Aβ oxidation, we used synthetic Y10 nitrated Aβ1–42 to study the influence of Y10 nitration on Aβ1–42’s aggregation and cytotoxicity in this study. We confirmed that Aβ1–42 could be nitrated in the presence of H2O2, NO2–, and heme by dot blotting. CD spectroscopy showed an increase of β-sheet structure of Aβ1–42 and its mutants. The thioflavin T (ThT) flourescence assay revealed that both nitration and chlorination significantly inhibited Aβ1–42 fibril formation. TEM and AFM observations of Aβ peptide aggregates further confirmed that Y10 modification inhibited Aβ1–42 fibril formation. The cytotoxicity study of native and modified Aβ peptides on SH-SY5Y cells revealed that nitration of Aβ1–42 remarkably decreased the neurotoxicity of Aβ1–42. On the basis of these results, we hypothesized that nitration of Y10 may block the π–π stacking interactions of Aβ1–42 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.

Neuropeptide Y (NPY) is a member of the pancreatic peptide family of neuropeptides that play a crucial role in numerous central and peripheral nervous system responses. Recently, it has been shown that NPY protects cells against neurotoxic damage from β-amyloid peptides (Aβ) in Alzheimer's disease (AD). Heme is a common factor linking several metabolic perturbations in AD and altered heme metabolism has been shown to be related to the pathologies of AD. Thus, heme may have a chance to act on NPY and potentially counteract its function. To explore this, UV-visible spectroscopy, fluorescence spectroscopy and differential pulse voltammetry (DPV) were used to demonstrate that NPY can bind with heme to form a NPY–heme complex and the binding enhances the peroxidase activity of heme. Dot blotting results indicate that NPY is easily nitrated upon binding with heme when H2O2 and NO2− are present. Furthermore, LC-MS/MS results confirm that tyrosine36 (Tyr36), an important amino acid residue of NPY in binding and activating neuropeptide receptors, can be nitrated during the nitration process. Thereafter, we used mutant peptide NPY(3N) (Tyr36 replaced by 3-nitrotyrosine) to investigate the impact of nitration on the structure and bioactivity of the peptide. Our results show that Tyr36 nitration destabilizes the α-helix conformation of the peptide, and counteracts NPY-induced inhibition of cAMP accumulation in SK-N-MC cells. Collectively, these data imply that the self-association of NPY with heme potentially induces tyrosine nitration, destroys the active monomeric conformation of the peptide and thereby counteracts its bioactivity.

•Binding of GAPDH could inhibit H2O2-mediated degradation of heme.•GAPDH inhibited heme-H2O2-NO2− induced oxidation of protein.•The order of catalytic activity toward tyrosine oxidation/nitration was HSA-heme > heme > GAPDH-heme.•No covalent bond was formed between heme and GAPDH.•GAPDH was more effective than HSA on protecting cells against heme-NO2−-H2O2 induced cytotoxicity.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 H2O2-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-H2O2-NO2- induced protein carbonylation. No covalent bond was formed between heme and GAPDH after treated with H2O2. GAPDH was more effective than HSA on protecting cells against heme-NO2--H2O2 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.