| Alzheimer's disease (AD), a kind of neurodegenerative disease, the pathological pattern of Alzheimer's disease (AD) is characterized by the progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. AD'S clinical manifestations include cognitive decline, behavioral abnormalities and reduced activities of daily living. The mechanism of AD is very complicated and May be the result of the interaction of multiple factors, However, the exact mechanism of AD remains elusive.In 1990s, Uchida and coworkers, while investigating the pathogenesis of AD, established that brain extracts from AD patients stimulate the neuron survival and neurite extension of rat neuronal cultures to a greater degree than normal brain extracts, resulting in neuron outgrowth and death. This activity was proposed to be associated with the loss of a human neuronal growth inhibitory factor (hGIF), which is subsequently identified as a member of the human metallothionein (MT) gene family and renamed as hMT3 because of the high similarity in primary structure with MT family member. Neuronal growth inhibitory factor (GIF) impairs the survival and neurite formation of cultured neurons. It is known that theα-βdomain-domain interaction of hGIF is crucial to the neuron growth inhibitory bioactivity although the exact mechanism is not clear. Herein, theβ(MT3)-β(MT3) mutant and the hGIF truncated A33-35 mutant(A33-35 mutant, the fragment of 33SCC35 in hGIF was removed, and it contains two M3S9 clusters as same as sGIF (sheep neuronal growth inhibitory factor)) were constructed, and their biochemical properties were characterized by pH titration, EDTA and DTNB reactions. Their inhibitory activity toward neuron survival and neurite extension was also examined. We found the A33-35 mutant a-domain containingβ-domain-like M3S9 cluster exhibits the function ofα-domain with M4S11 cluster in hGIF. These results showed that the stability and solvent accessibility of the metal-thiolate cluster in P-domain is very significant to the neuronal growth inhibitory activity of hGIF, and also indicated that the particular primary structure ofα-domain is pivotal to domain-domain interaction in hGIF. Probably the particular amino acid residues of the a-domain containing, which influence the stability and solvent accessibility of the metal-thiolate cluster inβ-domain through the hydrogen bond or hydrophobic interaction, that play an important role in the Physiological function of hGIF. Amyloid beta peptide is the major component of the senile plaques in the AD brain and occupies a central position in the pathogenesis of AD. The balance among Aβ, APP, MT3, metals and metal transporter in nerve cells is the key to its normal function. In addition to amyloid peptide aggregation in the brain, the pathological features of AD cells include GIF going down and heme metabolic disorders. Although scientists have already realized the GIF and heme can effectively inhibit the aggregation of amyloid peptides, and in vitro experiments the chemical synthesis of Aβbinding with heme and forming a complex with peroxidase activity have been reported, but as for Aβand heme interaction mechanism and physiological significance of the binding remains elusive. In this paper, we first got biosynthesis amyloid peptide Aβ1-40 without any extra amino acid residues using prokaryotic expression system, at the same time, the combination of heme and Aβ1-40 and the changes of Aβ1-40 secondary structure during the combination process were monitored, and the biochemical properties of the complex were tested. Moreover, the impact of the Aβ1-40-heme complex to the cytotoxicity caused by amyloid beta and copper ions was also studied. The results show that heme is able to inhibit the aggregation of amyloid peptide duing to heme binding with amyloid peptide, and makes amyloid beta peptide maintainingα-helix stably; also show that the cytotoxicity caused by amyloid peptide must be accompanied by changing fromα-helix toβ-sheet of the amyloid peptide. When adding an appropriate amount of heme into the mixture of amyloid peptide and copper ions, the cytotoxicity caused by Aβand copper ions decreases significantly. If we considered the special peroxidase activity of the AP-heme complex, the reason of this phenomenon may be due to the intracellular superfluous hydrogen peroxide which generated by amyloid peptide and copper ions could be decomposed through the complex's catalysis, and reduce downstream oxygen free radicals from the hydrogen peroxide and protect cells.The latest research indicated that usually APP is fixed on the cell membrane, which is digested by the two proteases related with the AD after growth factor is deprived, then followed by cell proapoptosis induced by DR6 (death receptor 6). The N-APP, which has previously been overlooked, maybe the culprit in AD and may become a new target for the treatment of AD. Therefore, the focus of treatment strategy for AD may shift from the Aβto N-APP or its downstream proteins controlling the apoptosis. In this paper, through kinds of expression system we have successfully constructed and expressed several truncated APP and the separation and purification methods in some of them have been established, which laid a good foundation of the research in the field of APP. Next, we will use those proteins to study the structure, character and function of some APP functional domains, as well as the physiological function caused by the interaction between APP and others. |
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