| Alzheimer’s disease, also known as senile dementia disease, being one of the neurodegenerative disease of central nervous system, its main pathological features include extracellular senile plaques (Senile plaques, SPs) and intracellular neurofibrillary tangles (Neurofibrillary tangles, NFTs). The Aβ hypothesis, which states that Aβ aggregates are the major pathogenic factors, is still the main pathogenesis of AD. Clusterin, as the extracellular molecule companion of Aβ, has been proved to have direct interaction with Aβ both in vivo and in vitro. Not only the mechanism of the interaction between CLU-His and Aβ is still not clear, but also there are many contradictory results reported in the literatures. So, to study the interaction between CLU-His and Aβ oligomer at single molecule level will help to reveal the pathogenesis of AD.Dual color-fluorescence cross correlation spectroscopy (DC-FCCS) is a single molecule fluorescence detection technology by measuring the fluorescence intensity fluctuations of fluorescence signals. As DC-FCCS can analysis the cross-correlation between two separate detecting channels, thus, it’s very suitable for studying the interactions among bio macromolecules. In this paper, we performed a new method for studying the interaction between CLU-His and Aβ42 by using DC-FCCS at a single molecular level. This study was investigated based on our previous work, but it was much easier when compared with previous method. Recombinant CLU-His was cloned, expressed and purified to detect the interaction of Aβ42 oligomers and CLU-His. CLU-His has no any fluorescent tag and it does not need to be labeled in vitro. We only need to incubate CLU-His protein with two different fluorescent labeled Aβ42 together and then observe the dynamic behavior of the interaction between CLU-His and Aβ42 by using DC-FCCS.After the coding sequences of the CLU gene and the His gene were amplified, they were inserted into the pFastBacl plasmid from the Bac-To-Bac Baculovirus expression system. Recombinant baculovirus DNA was then obtained from E. coli DH10Bac through transformation. The recombinant baculovirus was cultured in Sf9 cells after transfected with Cellfectin II reagent. When a high-titer baculoviral stock was obtained, it was used to infect Sf9 cells for the expression of CLU-His. CLU-His were then purified with two steps by Ni-affinity chromatography and gel filtration. The Aβ42 monomers were labeled with Bodipy FL and ATTO 647N respectively and then fluorescence labeled Aβ42 oligomers were prepared. At last, the interactions between the Aβ42 oligomers and CLU-His were investigated by DC-FCCS at single molecule level.The results showed that Aβ42 oligomers had a direct interaction with CLU-His and the binding efficiency was gradually increasing within 0 to 4 h. The results also showed CLU-His could accelerate the aggregation of Aβ42 monomers over a period of time. The established single-molecule detection method could be used for the interactions between AP42 oligomers and non-fluorescent labeled proteins as detected by DC-FCCS, thus providing additional insight into the molecular mechanism of Aβ hypothesis in AD. |
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