Study Of Promyelocytic Leukemia Protein Structure

Objective: Promyelocytic leukemia protein(PML)is the main component of PML nuclear body(PML-NBs),and is a tumor suppressor protein first found in patients with acute promyelocytic leukemia(APL).The PML-RARα gene produced by the PML gene and RARα gene due to chromosomal ectopic is a key factor in the pathogenesis of APL.Due to differential cleavage,PML proteins form seven major isomers,named PMLI.-VII.,and the seven subtypes of PML function differently within cells.Differential cleavage of these seven isomers occurs only at the C-terminus of the PML protein.All seven isomers share the same RBCC domain at the N-terminus,which consists of RING,B1-box,B2-box,and a spiral-coiled CC protein domain.RBCC domains play an important role in SUOMIZATION of PML,the formation of PML nucleosomes,and the maintenance of the normal function of PML proteins.PML-NBs with PML as the bone framework mediate a variety of cell biological processes in cells,including transcriptional regulation,mediating post-translational modification of specific target proteins,immune response to viral infection,cell cycle regulation,aging,apoptosis,etc.The PML protein itself also plays a vital role in the human body.After years of research,PML protein not only plays an important role in the occurrence and development of APL,but also plays a non-negligible role in the occurrence and development of triple-negative breast cancer,glioblastoma,liver cancer and other cancers.As a well-known tumor suppressor protein,PML also plays a role in promoting tumorigenesis and development in some tumors.Not only that,PML also plays a role in viral infection.However,little is known about the structure and function of PML.In this paper,we mainly try to analyze the three-dimensional structure of PML protein,and try to find new targets for the development of anticancer drugs and the prevention of viral infection.Methods: The target protein with high purity was purified by affinity chromatography and molecular sieve chromatography,and then crystallized by the obtained protein or observed by cryo-EM to analyze the structure of different truncated fragments of PML,in order to further understand the function and function of PML by analyzing the three-dimensional structure of PML protein.Results:(1)Although PML(47-184)obtained a protein with high purity,it did not obtain crystals after a large number of crystallization conditions screening.(2)PML(1-256)obtained a protein with higher purity,and the negative staining results were also very good,but the cryo-EM protein was frozen and crushed,which may be related to the lack of protein stability,and subsequent experiments need to continue to be optimized.(3)Tried to express PML(1-256)-SUMO-UBC9 complex through eukaryotic cells,but from the experimental results did not have the ideal complex,but unexpectedly found that compared with SUMO3,SUMO1 can promote the high polymerization of PML.Conclusion: PML protein is not crystallized,cryo-EM will freeze and crush may be because PML protein is not stable enough,and it is necessary to try to obtain more stable PML protein by adding tags and forming complexes.SUMO1 can significantly promote the high polymerization of PML,and we will also improve relevant experiments to explore what physiological functions this phenomenon causes,and whether the promotion effect of SUMO1 on PML high polymerization is related to the occurrence and development of cancer.