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Interaction And Binding Mechanism Of Human Heavy Chain Ferritin(HFn) With Asexual Blood Stages Of Plasmodium Falciparum

Posted on:2024-09-28Degree:MasterType:Thesis
Country:ChinaCandidate:Y YinFull Text:PDF
GTID:2544306917457554Subject:Pathogen Biology
Abstract/Summary:PDF Full Text Request
Malaria caused by the apicomplexan protozoan of the Plasmodium genus remains one of the most prevalent parasitic diseases in the world.Plasmodium has a complex life cycle,with the erythrocyte stages of malaria parasites being the main period that causes clinical symptoms and is also an important stage for antimalarial drugs play.Plasmodium-infected red blood cell(iRBC)that are under severe oxidative stress,and iron homeostasis is critical for the survival of parasites.The erythrocyte stages of malaria parasites isolate a portion of iron through their own detoxification process,and capture and store excess iron in cells through the ferritin in the protozoan cytoplasm to control ferrous ion oxidation to achieve balance.At the same time,the expression of human heavy chain ferritin(HFn)also controls the host’s metabolism of iron overload in tissues.However,Plasmodium falciparum(P.falciparum)neither express ferritin or other known iron storage proteins homologues not known ferritin receptors,and the mechanisms by which it stores and detoxifies excess iron are unknown.Therefore,our research group collaborated with Yan Xiyun’s team from the Institute of Biophysics of the Chinese Academy of Sciences to simulate and synthesize HFn,which is a diameter of 12 nm in vitro for in-depth research.First,flow cytometry and immunofluorescence were used to determine the binding rate and potential mechanism of HFn with iRBC.Then,the proteins that interact with HFn were then initially screened by co-immunoprecipitation and mass spectrometry techniques.Finally,performed on these proteins to further clarify the interaction proteins and provide a scientific basis for refining the mechanism of interaction between host HFn and erythrocyte stages of P.falciparum.The specific results of the study are as follows:(1)HFn is dependent on new permeation pathways(NPPs)to enter iRBC.The binding of HFn to iRBC was observed by flow cytometry and immunofluorescence,followed by polystyrene fluorescent microspheres and Nitrogen-Doped Carbon Dots(NCDs)to investigate the formation time of NPPs on the surface of the Erythrocyte plasma membrane(EPM)of iRBC and nanomolecule passage condition to determine whether HFn is dependent on NPPs to enter iRBC.The results showed that HFn could bind specifically to iRBC and cluster around the cytoplasm of erythrocyte stages of P.falciparum.Flow cytometry results showed that the binding rate of nanomaterials of different particle sizes to P.falciparum at different stages of development was variable,which verified the biological characteristic of NPPs to reach peak permeability at 28 h after synchronization of P.falciparum and allow the passage of small molecules below 80 nm.Before the formation of NPPs,there was no significant difference in the binding rate of HFn to iRBC compared to NCDs of the similar size of HFn.However,the binding rate increased significantly after the formation of NPPs and showed a higher binding rate.These results confirmed that HFn is dependent on NPPs to enter iRBC,and that there might exist proteins interacting with HFn in the erythrocyte stages of P.falciparum.(2)Twenty-eight candidate proteins interacting with HFn were screened by coimmunoprecipitation and mass spectrometry.Three different distributions of P.falciparum proteins were extracted by the Percoll-sorbitol synchronization,the saponin membrane extracting method and the tetanolysin membrane extracting method and the P.falciparum proteins interacting with HFn were captured and identified by co-immunoprecipitation and mass spectrometry,respectively.The results showed that the plasmodium which outer membrane is EPM,the parasitophorous vacuolar membrane(PVM)and the parasite plasma membrane(PPM)membrane were respectively extracted.Distinctly different bands were identified by comparing the Input and IP bands in coimmunoprecipitation.A total of 28 P.falciparum proteins that might interact with HFn were successfully screened based on multiple mass spectrometry.(3)Eight candidate proteins interacting with HFn were further screened using protein microarray technology.Primers were designed and full-length gene sequences of the 28 candidate proteins were amplified,the recombinant vectors were constructed,and the candidate proteins were obtained by the prokaryotic protein expression system,and finally the binding strength of the candidate proteins to HFn was observed by protein microarray.The results showed that 27 candidate protein sequences were successfully amplified.24 candidate proteins were obtained,of which 15 proteins were expressed by pET-2 8a(+)vector and 9 proteins were expressed by pET-32a(+)vector.Among the 24 candidate proteins,8 candidates with strong binding strength to HFn were selected by protein microarray.(4)Three P.falciparum proteins interacting with HFn were identified by protein interaction experiments.After expression and purification of eight His-tagged candidate proteins,the interaction between the two proteins was conducted by Pull-down and protein array technology.The results showed that 3 proteins were expressed as soluble proteins,5 proteins were expressed as inclusion bodies,and only 7 purified target proteins were obtained after dialysis or renaturation,respectively.In Pull-down,No.3,5 and 16 proteins could be successfully pulled down by HFn and could be detected by His antibody,indicating that No.3,5 and 16 proteins could interact with HFn.By protein array technology,it was found that with the increase of the concentration of No.3,5 and 16 proteins,the binding strength with HFn also increased,showing that their binding was dose-dependent.The above results indicate that No.3,5 and 16 proteins were proteins interacting with HFn in the erythrocyte stages of P.falciparum.In conclusion,our research verified that HFn is dependent on NPPs to enter the erythrocyte stages of P.falciparum,and discovered for the first time that proteins No.3,5 and 16,interacting with HFn,exist in the cytoplasm of P.falciparum,providing a new research direction for understanding iron metabolism and its regulatory mechanism in erythrocyte stages of P.falciparum.
Keywords/Search Tags:Plasmodium falciparum, Host-pathogen interaction, Protein-protein interactions, Human heavy chain ferritin, New permeation pathways
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