Identification Of Novel Pml-nb Localized Proteins And Its Involvement And Mechanism In Regulation Of Dnadamage Responses

The integrity of the genome is fundamental to the propagation and metabolism of all living beings. To maintain the integrity of the genome, cells have evolved the ability to detect and propagate an initial DNA damage signal to elicit cellular responses that include cell cycle arrest, DNA repair, and apoptosis, which collectively have been termed the DNA damage response (DDR). DNA damage response machinery, as an important mechanism to keep cell homeostasis, is one of the core contents in research area of genetic disease, ageing, and cancer. Recent evidence suggests that PML NBs, a multi-protein complex associated with nuclear matrix, play crucial roles in maintaining genomic stability, and are dynamic sensors of DNA damage. After irradiation, PML NBs dynamically recruit or release important proteins involved in cell cycle regulation, DNA repair, and apoptosis.Although it is reported that PML NBs play an important role in the DNA damage responses of mammalian cells, the molecular mechanisms underlying the biological processes occurring within this nuclear domain in DNA damage responses are not yet fully elucidated. Therefore, to understand the function of PML in the DDR, it is essential to characterize its dynamic interactions during specific stresses. As such, in this current study, using a combination of immunoprecipitation and mass spectrometry, we analyzed the composition of PML NBs in irradiation-treated MCF-7 cells. We identified a total of 124 proteins, which may localize to PML NBs after irradiation. Four proteins, NPM1, MVP, G3BP1, and DHX9, were verified to co-localize with PML differentially before and after IR treatment. Based on these results, NPM1, identified as a novel PML-associating protein, was selected for further investigation. The primary results of this study are as follows:I. Functional proteomics analysis of PML NBs1. We first surveyed the expression of PML in 13 cell lines and chose MCF-7 cells with higher PML expression for further study. To obtain results that could reveal the dynamic components in PML NBs in irradiation responses, we monitored the number and subcellular distribution of PML NBs before and after irradiation by immunofluorescence. Finally, un-irradiated cells and cells irradiated for 6 h were collected for co-immunoprecipitation and mass spectrometry (MS).2. A proteomic approach, using CoIP, mono-dimensional electrophoresis, and tandem mass spectrometry, allowed us to identify a total of 124 proteins that may associate with PML after irradiation, of which 15 were previously reported PML-NB proteins and their homologues. Bioinformatic analysis of the identified proteins showed that most of them were related to characterized PML functions, such as transcriptional regulation, cell cycle regulation, cell death regulation, and response to stress, confirming the plurifunctional nature of PML NBs.3. Four proteins, NPM1, MVP, G3BP1, and DHX9, were verified to co-localize with PML differentially before and after IR treatment. We also visualized the colocalization of MVP, G3BP1, and NPM1 with PML after irradiation in MCF-7 cells using fluorescence microscopy. The proteome of PML NBs presented in this study will significantly improve our understanding of the dynamic organization and multiple functions of PML NBs in DDR.Based on above results, NPM1, identified as a novel PML-associating protein, was selected for further investigation.II. The function and molecular mechanisms that the interaction of NPM1 and PML had in DNA damage response.1. Function of NPM1 in DSBs damage response. The protein level of NPM1 in MCF-7 cells increased afterγirradiation. We observed the translocation of NPM1 from nucleolus to nucleus 24h afterγirradiation. Correspondingly, cells in G2/M phase increased significantly. Collectively, these data implied a role for NPM1 in G2/M arrest.2. The knockdown of NPM1 expression delayed the repair of DSBs along with slower disappearance ofγ-H2AX focies. Decrease in G2/M arrest and increase in apoptosis were noted in NPM1 siRNA-transfected MCF-7 cells compared with negative control siRNA-transfected cells after irradiation induction. These data indicated that transfection with NPM1 siRNA made MCF-7 cells more sensitive to irradiation-induced cell-killing. In irradiation-induced DDR, NPM1 played a role of promoting the repair of DNA damage and G2/M arrest, inhibiting apoptosis.3. Double-knockdown of NPM1 and PML confirmed that NPM1 regulated G2/M arrest and apoptosis through PML protein. On one hand, knockdown of NPM1 induced increased PML expression, Caspase 3 activation, and apoptosis increase; double-knockdown of NPM1 and PML partly inhibited apoptosis with unactivated Caspase 3; these data indicated that down-regulation of PML inhibited the knockdown of NPM1 induced increase of apoptosis through activating Caspase 3. On the other hand, knockdown of NPM1 induced increased PML expression, decreased p21 expression, and G2/M arrest; double-knockdown of NPM1 and PML partly recovered G2/M arrest with samely downregulated p21 expression; these data indicated that down-regulation of PML recovered the knockdown of NPM1 induced decrease of G2/M arrest through p21-independent pathways.4. The immunofluorescence staining and confocal observations were performed on MCF-7 cells transfected with GFP-NPM1 using antibodies against PML, and demonstrated a co-localization of PML and NPM1 in the nucleolus. We conformed the interaction of PML and NPM1 in vivo and in vitro using co-immunoprecipatition and GST-pull down. Binding sites of NPM1 was mapped within the domain of 1-117 in its N terminus. Binding sites of PML was mapped within the domain of 1-360 amino acids and 394-433 amino acids.5. Over-expression of NPM1 inhibited the expression of PML significantly, likewise, the knockdown of NPM1 expression up-regulated the expression of PML, indicating NPM1 could negatively regulate the expression of PML. Further research revealed that NPM1 regulated PML protein level through ubiqutin/protesome parthway.Taken together, the proteome of PML NBs presented here will help to draw a more detailed picture about the role of PML NBs in DNA damage response regulation and tumor suppression. The identification of new NPM1-PML pathway has further provided the mechanistic explanation for PML NBs-regulated cell cycle and apoptosis responses. Keywords: DNA damage response; proteome; cell cycle; apoptosis; PML NBs; PML; NPM1...