| BackgroundNucleophosmin(NPM1)is a multifunctional nucleolar protein.Although NPM1 is mainly located in the nucleolus,it can also rapidly shuttle between the nucleolus,nucleus,and cytoplasm,assisting in the transport of substances within and outside the nucleus.Early studies have shown that NPM1 is involved in the processing of ribosomal RNA(rRNA)through its nucleic acid binding and nuclease activity,as well as the transport of ribosome particles.Therefore,NPM1 is considered to be an important participant in ribosomal biogenesis.Subsequent studies revealed that the chaperone activity of NPM1 serves as the molecular basis for its function.NPM1 can not only prevent the abnormal aggregation of viral proteins and metabolic enzyme molecules in vitro,but also act as a molecular chaperone for canonical histones.NPM1 can bind to core histones H3,H4,H2A/B and assist in nucleosome assembly of histone octamers.Recent studies have shown that NPM1,which located in the granular compartment(GC)of the nucleolus,is a key molecule in maintaining the separation of nucleolar compartments and plays an important role in the process of repairing misfolded proteins in cells.More and more studies show that the imbalance of protein homeostasis is closely related to aging and human disease process.Molecular chaperones can help the peptide fold correctly,restore the conformation of abnormal agglutinin,and assist protein transmembrane transport.Therefore,molecular chaperones are essential for maintaining protein homeostasis in cells and ensuring normal life activities.The molecular chaperone function of NPM1 is crucial for maintaining genomic stability.Knockdown of NPM1 in cultured cells can lead to abnormal chromosome association,spindle formation and microtubule-centromeric association,but the mechanism is unknown.Additionally,NPM1 may be regulated by mitotic kinases.Aurora A and B mediate the phosphorylation of NPM1,and overexpression of non-phosphorylated NPM1 mutants results in an abnormal number of centrosomes.NPM1 knockout mice exhibit significantly delayed embryo development and died,while isolated Mouse Embryonic Fibroblasts(MEFs)display an abnormal number of centrosomes,multipolarized cells,and a high proportion of abnormal karyotype cells.This suggests that NPM1 is essential for maintaining the genomic stability of stem cells and is a necessary gene for early embryonic development.Subsequent studies have suggested that NPM1 phosphorylation mediated by the cycle-dependent kinase CDK2/cyclin E may be a key mechanism in regulating centrosome replication and ensuring centrosome number.However,it has also been suggested that there is no direct evidence linking NPM1 or phosphorylation of NPM1 to the number of centrosomes,and proteomic analysis of centrosomes has not shown significant enrichment of NPM1.NPM1 is also an important effector molecule in cellular stress response.In normal cells,both environmental changes and drug treatment can induce NPM1 to exit the nucleus and regulate important downstream molecules,such as stabilizing the tumor suppressor p53 to activate p53-dependent transcriptional regulation.Although the function of NPM1 within the nucleolus is clear,its interaction molecules and mechanisms outside the nucleolus,as well as in different phases of the cell cycle,remain unci ear.To explore more unknown functions of molecular chaperone NPM1,In this study,the sequence encoding the Stag-Flag tandem tag was first knocked into the Npml locus to obtain a cell line stably stably expressing endogenous NPM1-Stag-Flag.Then affinity tandem purification was used to enrich the binding proteins of NPM1 during mitosis,and identification was performed by mass spectrometry.We found that HJURP(Holliday Junction Recognition Protein)was significantly enriched by NPM1 in the mitotic group.In human cells,HJURP is a centromere protein A(CENP-A)specific chaperone and a key factor mediating CENPA centromere localization and nucleosome assembly.Loss of HJURP leads to CENP-A misintegration in centromeres,resulting in abnormal chromosome segregation,cell division defects,and genomic instability.The effect of NPM1 on human centromere has not been reported.Although HJURP is a specific chaperone protein of CENP-A,NPM1 is likely to play a significant role as a co-chaperone molecule in its upstream regulation.We propose that NPM1 may be an important regulatory factor for histone variant CENPA.NPM1 may regulate the stability of HJURP-CENP-A as a molecular chaperone,affecting the function of human centromeres.This study clarified the effects of NPM1 knockout on centromere function and chromosome segregation in the human Tlymphoma cell line Jurkat E6-1,and focused on the molecular mechanism of NPM1 regulating the HJURP-CENP-A complex to elucidate the molecular chaperone function of NPM1.Methods1.The NPM1-Stag-Flag stable expression cell line was constructed by CRISPR/Cas9 technology,and tandem affinity purification and mass spectrometry were used to find the protein that binds NPM1 during the mitosis stage.2.The interaction between HJURP and NPM1 was verified by co-immunoprecipitation.3.Phosphatase treatment was used to detect the effect of phosphorylation modification on HJURP binding to NPM1.4.NPM1 homozygous knockout cell line was constructed and immunofluorescence was used to observe whether the cell line exhibited genomic instability.The effects of NPM1 deletion on HJURP protein level and cellular localization were detected by western blot and immunofluorescence.5.Use cell survival curve,flow cytometry and other techniques to explore the effect of NPM1 knockout on cells.6.Real-time was used to detect whether NPM1 deletion affects the mRNA expression level of HJURP.7.The interaction relationship between HJURP and NPM1 was verified by GST pull-down and prokaryotic co-expression,and the influence of the functional domain of NPM1 on binding was explored.Results1.NPM1 costituted a complex with HJURP and CENP-A,and phosphorylation modification may affect the interaction between NPM1 and HJURP.2.Micronucleus appeared after NPM1 knockout,cells growth slowed down,and cell arrested in G1 phase.3.After NPM1 knockout,the content of HJURP and CENP-A proteins decreased,the nucleolus localization of HJURP was reduced.Additionaly,the localization signal of CENP-A on the centromeres was diminished.4.The mRNA level of HJURP was not affected by the absence of NPM1.5.NPM1 interacted with HJURP directly to enhance its stability.6.Deletion N-terminal or IDR of NPM1 caused it lost binding activity with HJURP.ConclusionThe molecular chaperone NPM1 may function as a stabilizer of the HJURP-CENP-A complex during cell division in Jurkat E6-1,ensuring functional integrity of centromere and genomic stability. |
PDF Full Text Request