The Role Of The Oligomerization Of PML_B-box In PML Nuclear Body Assembly

The gene encoding promyelocytic leukemia protein(PML)was first identified in the genes of patients with acute promyelocytic leukemia.PML fuses with the RAR? gene on chromosome 17 to form the PML/RAR? fusion gene,eventually leads to the occurrence of APL.The PML protein is packaged in the body to form a nucleosome structure with a diameter of 0.1-2 micrometers.In PML nuclear bodies,PML has been shown to be able to directly or indirectly interact with more than 270 partner proteins.It affects the biological functions of PML and it’s partner proteins through the post-translational modification and then regulates many cellular biological processes,including gene transcription regulation and inducing apoptosis.PML can at least generate seven different protein variants,which are called PMLⅠ-Ⅶ,according to the variable splicing of the C-terminus.Interestingly,all spliceosomes contain four conserved domains at the N-terminus,namely the RBCC domain(a RING domain,two zinc finger-like B-box domains,and a predicted coiled-coil domain).The conserved domains are not only the basis for inhibition of infinite cell proliferation,but it is also speculated that it plays a key role in PML nuclear body assembly.Starting from the structure of PML protein,we explored the assembly mechanism of PML nuclear bodies and PML-mediated oligomerization.Based on this,we first purified a PML large fragment containing RBCC domains with a purity of more than 90%.Based on gel filtration chromatography experiments,cross-linking experiments,and analytical ultracentrifugation experiments,we observed the presence of monomers,dimers,tetramers,and N-mers in PML proteins.Which proves that the oligomerization of the RBCC domains is the basis of PML nuclear body assembly.In addition,we analyzed the crystal structure of the PML-B1-box domain,and combining with the biological small-angle experiments,we found an unprecedented oligomerization network mediated by the W157-,F158-and the SD1-interfaces.These key interfaces play an important role in the aggregation of PML proteins.From the perspective of structural biology,we revealed that B1 domain-mediated oligomerization is very important for the normal oligomerization of PML protein,the correct assembly of PML nuclear body,the SUMO modification of PML,and the colocalization of PML protein with other proteins.After treatment with arsenic agent ATO,the damaged PML nuclear body and SUMO modification caused by B1 key interface mutations can be restored to a certain extent.Combining with the previous research,we proposed a preliminary assembly mechanism of PML nuclear body,which is based on the N-terminal RING tetramer,and then undergoes regular dynamic assembly of B-box oligomerization and CC polymerization to form PML nuclear body.At the same time,we tried to purify and crystallize the B2 domain in order to understand the resistance of arsenic at the atomic and molecular level.