The Molecular Mechanism And Function Of Phase Separation Of Cancer-related Proteins P53 And Annexin A1

Cancer is one of the leading reasons for death in the world,which seriously endangers human life and health and restricts social and economic development.Currently,the incidence and mortality of cancer is increasing rapidly,and it has become urgent to explore new mechanisms of carcinogenesis and develop new therapeutic modalities and drugs.Liquid-liquid phase separation（LLPS）drives the formation of membrane-free compartments,which is one of the important ways of cellular compartmentalization and is involved in regulating various cellular life activities,and more and more studies have shown that LLPS is associated with cancer.Mutations in the tumor suppressor p53 are present in more than half of all cancers,making it extremely significant in the cancer field and have been a hot spot for research.Recent studies have found that p53 undergoes LLPS under specific conditions in vitro,however,the molecular mechanism of p53 phase separation,its physiological function as well as its relationship with cancer are not yet clear and need to be explored.Annexin A1,a member of the Annexins family,is a potential cancer diagnostic marker and therapy target that has received much attention.It has been found that Ca²⁺-induced Annexin A1 forms micron-sized particles on the cell surface with reversible nature,similar to the droplet formation by phase separation,indicating the possibility of LLPS of Annexin A1.In this thesis,the molecular mechanisms and physiological functions of phase separation of p53 and Annexin A1 were investigated to explore the relationship between their phase separation and cancer,with the following main findings:（1）Research on the molecular mechanism of p53 phase separation in vitro.The relevant prokaryotic expression plasmids were constructed and recombinant proteins p53-FL（p53 full-length）,fragment p53-UBR,p53-△UBR（fragment of excised UBR）,and p53-UBR mutant R337H,R337C,R337P,L344P with green fluorescent tags were purified.The UBR was identified for the first time as a crucial region for p53 phase separation through turbidity experiments,fluorescence imaging,and fluorescence bleaching recovery experiments,and the UBR underwent LLPS driven by electrostatic and hydrophobic interactions.Comparing the phase separation behavior and oligomerization status of wild-type UBR with its tetrameric region（TD）oncogenic mutation,it was found that the oncogenic mutation of TD inhibited the phase separation of p53 by preventing the formation of the tetramer,which provided a basis for studying the phase separation and function of p53 at the cellular level.（2）Research on the characterization and functions of intracellular phase separation of p53.Eukaryotic expression plasmids of p53-FL and its TD oncogenic mutations R337H,R337C,R337P,and L344P were constructed,and the above plasmids were transfected into He La cells,a classical tumor model,and H1299 cells,which do not express p53,to express the protein.Dox was used to simulate intracellular DNA damage,and cellular p53 was found to phase segregate and TD oncogenic mutation inhibited p53 phase segregation by confocal imaging,FRAP assay,and stress removal assay,which were consistent with the in vitro results.Immunofluorescence experiments showed that Ser15phosphorylated p53 was highly co-localized with the cohesion formed by p53phase separation,indicating that p53 phase separation was closely related to the activation of p53.In addition,oncogenic mutants R337C,R337P,and L344P significantly attenuated the expression of p21,a gene downstream of p53,and improved cell survival.We reveal the physiological function of p53 phase separation and its relationship with cancer:TD oncogenic mutations inhibit p53phase separation and attenuate p53 activation,which in turn reduces the expression of p53 downstream target genes and leads to cancer development and progression.（3）Research on the molecular mechanism of Annexin A1（ANXA1）phase separation and its facilitation of signal transduction.We expressed and purified the recombinant proteins ANXA1-FL and ANXA1-CTD with green fluorescent tags.We found that Ca²⁺mediates the phase separation of ANXA1 at three levels:in vitro solution,cell surface,and artificial membrane and N-terminal is the core region of ANXA1 phase separation,and ANXA1 phase separation is driven by electrostatic and hydrophobic interactions using confocal observation and FRAP experiments.Moreover,phase separation of ANXA1 was associated with the ANXA1-FPR1 complex,thus enhancing the phosphorylation activity of ERK downstream of the FPR1 signaling pathway and promoting cancer cell migration.The results in this chapter suggest that ANXA1 phase separation is related to its oncogenic mechanism:ANXA1 undergoes liquid-liquid phase separation on the surface of cancer cells and participates in the formation of the outer membrane ANXA1-FPR1 complex,which regulates the signaling pathway within cancer cells and thus promotes cancer.In summary,this thesis identifies the UBR as a critical region for p53 phase separation,and oncogenic mutations in TD inhibit the phase separation of p53and thus hypothesize a new mechanism of p53 oncogenesis;the N-terminal region was identified as the core region of ANXA1 phase separation,and the phase separation of ANXA1 was found to be involved in the formation of ANXA1-FRR1 complex,linking ANXA1 phase separation to signal transduction and explicating an oncogenic mechanism of ANXA1.This thesis further revealed the relationship between p53 and ANXA1 and carcinogenesis,broadened the research field of cancer and phase separation and provided new ideas for the treatment and drug development of p53 and ANXA1-related cancers.