Studies Of NPM1 Interacting Mitotic Proteins

Object:Nucleophosmin1(NPM1)is a rich nucleolar phosphorylated protein,with a wide range of biological functions,such as participating in ribosomal RNA processing,embryo development,stimulation response to stress,etc.In recent years,it has been found that NPM1 is not only overexpressed in many solid tumors such as ovarian cancer and prostate cancer,but also one of the most common targets of gene modification in hematopoietic tumors.Therefore,the research on NPM1 in the field of biomedical science has become a focus of attention in recent years.Further elucidation of the biological function and regulatory mechanism of NPM1 is helpful to understand the occurrence and development of diseases,and is a key breakthrough to improve the therapeutic effect of cancer,which has far-reaching clinical significance.Studies on the function of NPM1 have been reported,but the main biological function of NPM1 during cleavage has not been elucidated.Therefore,in order to discover the other gene regulation NPM1 and small molecule compounds,find the split phase of potential control method and mechanism,this study applies CRISPR/Cas9 gene editing techniques in endogenous NPM1 gene locus type Stag-Flag label,establishes a stable NPM1-Stag-Flag fusion protein expression of cell lines,and through the Stag-Flag tandem affinity purification experiment,mass spectrum analysis to find in a divided period of specific binding molecules,This study lays a foundation for further analyzing the biological function of NPM1 in mitosis and understanding the process of tumor genesis and development.Methods:1.Construction of stably cell line expressing NPM1 fusion protein this study combined with CRISPR/Cas9 gene editing techniques and use the homologous recombination mechanisms respectively on endogenous NPM1 N(prior to the start codon)and end(prior to the termination codon)type C coding Stag-Flag sequences,through the separation of drug screening,and further by biochemistry and molecular biology method to identify monoclonal cell lines.2.NPM1 binding molecules were analyzed by mass spectrometry during the cleavage phase In this study,cell cycle synchronization was adopted to conduct immunoprecipitation in the two groups of cells in the dividing phase and the non-dividing phase respectively.The specific binding molecules of NPM1 in the cleavage phase were detected by biochemistry and mass spectrometry.Results:1.Two homozygous human T lymphoma Jurkate 6-1 cell lines with Stag-flag knockout were established successfully.2.The efficiency of NPM1 expression in C-terminal insertion of Stag-flag tags was significantly better than that in N-terminal insertion3.Twenty-six NPM1-interacting proteins were screened by mass spectrometry,including CDK1 and CDK6 involved in cell cycle regulation,NUDC,NAP1L1,Mki67 and HSPA9 related to cell proliferation,PGK1 and GPI related to glucose metabolism pathway,and PTBP1,FRG1,DAZAP1 and HNRNPU related to protein synthesis.Conclusion:1.NPM1 interacts with intracellular CDK1,CDK6,NUDC,NAP1L1,Mki67,and HSPA9,suggesting that NPM1 may be involved in a variety of physiological processes such as glycolysis,molecular chaperones,cell cycle regulation,and cell proliferation during division.2.Jurkat E6-1 cell line with endogenous expression of NPM1-Stag-flag is a powerful tool to search for unknown NPM1-interacting molecules and carry out functional studies.