| Alzheimer's disease (AD), the main reason for elderly dementia, is currently the most common neurodegenerative disease. Amyloid beta (Aβ) deposition and senile plaque (SP) formation have been demonstrated to be the pathological hallmarks of AD. The Aβcascade hypothesis indicated that deposition of Aβin the brain parenchyma is a critical step that eventually leads to AD. Protein oxidation and nitration were found significantly increased, while Aβis considered to be directly associated. However, the molecular mechanism is unclear. Meanwhile, the metabolism of heme in the brain of AD patients is disturbed. Interestingly, it is newly found that heme binds to Aβ, inhibiting Aβaggregation and even dismantling the Aβaggregates, which may be associated with impaired heme metabolic and mitochondrial function in AD. In addition, the Aβ-heme complex showed higher peroxidase activity than heme. Peroxidases can catalyze peroxides to oxidize many biological molecules. Therefore, the interaction of heme and Aβin AD may play an important role. In this paper, we focused on the interaction of Aβand heme, studied the potentially physiological effects. The main results are as follows:(1) The binding properties of Aβand heme and the effects of which on the peroxidase activity of heme. Aβ1-40 and Aβ1-42 was selected to study the properties of the interaction of Aβand heme and determine the number of binding sites and binding constants using capillary electrophoresis-frontal analysis (CE-FA). The results showed that Aβ1-42 has higher affinity for heme than Aβ1-40, with another more primary binding site. Meanwhile, Aβ1-40-heme showed higher peroxidase activity than Aβ1-42-heme. Results also showed that the aggregation state of Aβhas lower effect on the peroxidase activity of heme, which imply that the effects of Aβon promoting the peroxidase activity of heme is not depend on its aggregation. Furthermore, different truncated and mutated Aβpeptides were incubated with heme to form complexes and the peroxidase activities were determined. Results showed that the hydrophilic N-terminal of Ap involved in heme binding, while the F19 and/or F20 were essential for the peroxidase activity of Aβ-heme. These results further revealed the active site environment of the Aβ-heme complex and may help to clarify different physiological functions of the different Aβpeptides in vivo.(2) The mechanism of heme inhibits Aβaggregation and dismantles Aβaggregates. Different truncated Aβpeptides were chosen to study the roles of the hydrophilic and hydrophobic terminal of Aβin interacting with heme. Results showed that Aβ1-16 and Aβ1-40 has different effects on the peroxidase activity of heme. Meanwhile, the effect of Aβ1-16 on the absorption spectrum of heme is relatively small, whereas, Aβ1-40 significantly enhanced the absorption around 410 nm and 530 nm. These results imply that the hydrophobic terminal of Aβ1-40 affects the coordination between Aβand heme. F19 and/or F20 in the hydrophobic core of Aβare essential for Aβ-heme peroxidase activity, at the same time, the UV-Vis absorption spectrum of the Aβ-heme complex was significantly changed when F19 and/or F20 were mutated, indicating the interaction of F19 and/or F20 with heme. Further study showed that heme and PP are both effective in inhibiting Aβ1-40, Aβ1-40F19L and Aβ1-40F20L aggregation, but different in dismantling the aggregates of these peptides. Meanwhile, in dismantling Aβ17-40 aggregates, the effects of heme and PP are not significantly different. These results imply that heme inhibits Aβaggregation at least through binding to the histidine that locates in the hydrophilic N-terminal of Aβwith the iron center at one hand, and conjugating with the hydrophobic phenylalanine with the porphyrin ring on the other hand, and one is enough for inhibiting Aβaggregation, but two is necessary for dismantling Aβaggregates.(3) The catalytic activity of Aβ-heme in protein nitration and oxidation. The activity of Aβ-heme in catalyzing H2O2-NO2- system to oxidize and nitrate protein was investigated. Results showed that the binding of Aβto heme significantly enhanced the nitration of enolase and synaptic proteins, whereas, decreased the oxidation of these proteins. These results imply that Aβhas changed the pattern of heme-catalyzed protein oxidative damage. Meanwhile, data showed that the enzyme activity of Aβ-heme-H2O2-NO2--treated enolase is higher than that of heme-H2O2-NO2--treated, which imply that the change of protein oxidative damage pattern could be protective in a certain sense. And the different effects of Aβ1-40-heme and Aβ1-42-heme in promoting protein nitration and inhibiting protein oxidation reveal possible different physiological effects of Aβ1-40 and Aβ1-42 in vivo.(4) The site selectivity of Aβ-heme catalyzed protein nitration. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was utilized to investigate the effects of Aβ1-40 and Aβ1-42 on the site selectivity of heme-catalyzed enolase nitration. Results showed that heme-catalyzed enolase nitration occurs mainly in the hydrophobic environment (Y12), whereas, Aβ1-40-heme and Aβ1-42-heme selectively catalyze the nitration of tyrosine in the hydrophilic environments (Y191 and Y426). These results imply that the binding of Aβto heme can change the site selectivity of heme-catalyzed protein nitration. To further study the relationship between Aβhydrophilicity and the nitration site selectivity, three Aβpeptides with different hydrophilicity were designed. Results showed that the nitration sites of GAPDH are closely related to the hydrophilicity of the Aβ-heme complex that catalyzes the nitration reaction. Meanwhile, the secondary structural perturbations of GAPDH upon incubating with heme or Aβ-heme revealed that these compounds interact with GAPDH in different manners. This site-specific interaction might lead to site-specific generation of reactive nitrogen species (RNS) and the preferential nitration of the nearby tyrosine. These studies may help clarify the roles of Aβ-heme in the selective protein nitration and the factors that influence protein nitration selectivity.
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