The Mechanism Of Nuclease Function Of MIF In Parthanatos

Parthanatos is a form of programmed cell death,which is considered to be the main cause of many neurodegenerative diseases.During the occurrence of Parthanatos,PARP1 is overactivated by severe DNA damage,causing the nuclear translocation of AIF.When AIF enters into nucleus,resulting in chromatin condenses and DNA fragmentation,eventually cell death.However,AIF does not have the nuclease property,and how AIF leads to the degradation of DNA has not been well elucidated.Recently a study identified that MIF actd as a nuclease in Parthanatos and AIF was required for MIF go into the nucleus,moreover MIF was more inclined to cleave 3 ’single stranded DNA(ss DNA)in vitro.However,what region of the genome exists specific 3’ss DNA which can be the substrates of MIF and how MIF is recruited to this site,the mechanism is still unclear and needs to be further explored.It is generally think that the damage is too serious to repair is the reason of the cell’s insistence on going to death.Well Parthanatos begins with severe DNA damage,DNA as an important genetic material when he damages the repair system will be initiated immediately.Therefore,we cannot ignore the influence of DNA damage repair’s process on cell fate decision.It has been demonstrated that AIF can interact with H2 AX after he enters into nucleus,and γH2AX(the phosphorylated form of H2AX)is a key factor in DNA double-strand breaks(DSBs)repair.DSBs is a severe form of DNA damage,in the process of repair of DSBs,it happens to be formed 3’ss DNA with sticky end as the intermediate products of DSBs repair,which is consistent with the binding substrate of MIF in vitro.So we speculate that by interacting with AIF/H2 AX,MIF locates to 3 ’ss DNA sites which generated in the process of DSBs repair and then cleaves them,which leads to failure of DSBs repair,and ultimately promotes cell death.In this study,a Parthanatos model was established by stimulating He La cells with MNNG,and Asi-SI-U2 OS cells were stimulated with 4OHT to simulate the environment of DSBs.The main results are as follows:(1)We proved that MIF’s nuclease function depended on DSBs by experiments such as TUNEL.(2)We also proved that the target of MIF in Parthanatos was ss DNA produced during the repair of DSBs by immunofluorescence experiments.(3)By immunofluorescence,co-IP,and PLA experiments,we found that MIF would interact with γH2AX,the lack of AIF would significantly affect the combination of the them.(4)By experiments such as co-ip we proved that MIF and γH2AX interact with each other under the DSBs,by GST-Pull down experiment we further verified that MIF has specific interaction with H2AX/γH2AX(especially γH2AX),These resuits reminded us that MIF could locate the site of DSBs repair through its own specific interaction with H2AX/γH2AX.Taken together,this study further revealed the molecular mechanism of Parthanatos,and proposed that MIF blocking DSBs repair is the key to the occurrence of Parthanatos,and it also introduce the DNA damage repair failure to cell death,providing the new clues for the study of cell death.