Functional analysis of P53 negative regulators in Bombyx mori

概要

Chapter 1 Genera l introduction
　Apoptosis serves as one of the crucial intracellular antiviral immune responses in insects that lack the adaptive immune systems mediated by B- and T-cells found in vertebrates. Apoptosis is induced in several insect cell lines following infection with various species of nucleopolyhedroviruses (NPVs) - insect-pathogenic, large DNA viruses that belong to the family Baculoviridae. A recent study from our laboratory demonstrated that apoptosis induction is mediated by the Bombyx mori ortholog of p53 (bm-p53) in B. mori cells infected with vBm∆p35, a recombinant B. mori NPV (BmNPV) lacking the functional anti-apoptotic gene p35. In mammals and in Drosophila melanogaster, P53 protein is a multi-functional transcription factor responsible for various cellular responses, including apoptosis. In normal cells, the P53 protein is tightly regulated by multiple post-translational modifications, including ubiquitination. In this study, to provide a basis for the Bm-P53 regulation mechanism in normal and NPV-infected cells, I identified and characterized P53 negative regulators in B. mori.

Chapter 2 Cloning and characterization of putative P53 negative regulators in B. mori
　In this chapter, B. mori homologs of negative regulators of P53 in mammals and/or D. melanogaster, Bonus, Corp, Mdm2, Rad6, Sce, and Synoviolin, which have a direct interaction with P53, were searched using a tblastn search (search translated nucleotide databases using a protein query) against the NCBI nucleotide collection (nr/nt) database. The search revealed that B. mori possessed homologs of the mammalian and Drosophila E2 ligase Rad6, and E3 ligases Bonus, Mdm2, Sce, and Synoviolin, while not that of the Drosophila regulator, Corp. Among the B. mori homologs examined here, only the Mdm2 homolog of B. mori (Bm-Mdm2) is clearly involved in the negative regulation of Bm-P53 levels. RNAi- mediated knockdown of bm-mdm2 resulted in increased endogenous Bm-P53 accumulation in B. mori cells, whereas transient overexpression of Bm-Mdm2 significantly reduced Bm-P53 accumulation. Furthermore, co-expression assay in B. mori cells demonstrated that Bm-Mdm2 attenuated Bm-P53-induced apoptosis and effector caspase activity and that exogenous Bm- P53 protein was possibly ubiquitylated by Bm-Mdm2. Interestingly, despite its capacity for ubiquitylation of Bm-P53 and attenuation of Bm-P53-induced apoptosis, Bm-Mdm2 lost the N-terminal P53-binding domain and underwent several extensive deletions of the acidic region (AR), both of which are crucial for P53 binding and subsequent ubiquitylation of P53 in mammals. Overexpressed Bm-Mdm2 was localized in the nucleolus which differs from mammalian Mdm2 that is localized in the nucleus and cytoplasm. These results suggest that B. mori has a different P53 negative regulation mechanism from mammals, as well as D. melanogaster, which has Corp instead of Mdm2.

Chapter 3 Regulation mechanism of Bm-P53 by Bm-Mdm2
　Motif alignment of Mdm2 and Corp from various organisms identified five highly conserved motifs, M1, M2, M3, M5, and M6 (aa270–328, 125-151, 102-124, 39-58, and 18-37 of Bm-Mdm2, respectively), among insects and invertebrates. M1 and M2 corresponded to RING and zinc finger (Zn-F) of Bm-Mdm2, respectively, M3 and M5 include a portion of predicted AR, and M6 exists N-terminal of Bm-Mdm2. To gain knowledge on the mechanism of Bm-Mdm2- mediated regulation of Bm-P53 via ubiquitination, transient co-expression analysis of Bm- P53 and Bm-Mdm2 motif deletion mutants were conducted. As a result, all Bm-Mdm2 motif deletion mutants could not inhibit apoptosis induced in B. mori cells transfected with Bm- P53, indicating that all the deleted regions of Bm-Mdm2 contain the functional region for Bm-P53 negative regulation. This analysis also demonstrates that M1 includes the signal responsible for Bm-Mdm2 localization to the nucleolus. Furthermore, to determine the ubiquitinated residues of Bm-P53, Bm-P53 protein bearing C-terminal deletion was examined for ubiquitination by Bm-Mdm2. I found that B. mori cells co-expressing Bm-Mdm2 and C- terminal deletion mutant of Bm-P53 resulted in apoptosis, and ubiquitinated Bm-P53 was not observed. These results suggest that Bm-Mdm2 inhibits Bm-P53-triggered apoptosis through functional interaction with the Bm-P53 C-terminal region. This Bm-P53 C-terminal region has five lysine residues which are suspected to be ubiquitinated by Bm-Mdm2. However, single, or double lysines to arginine mutation within each domain did not affect ubiquitylation of Bm-P53 by overexpressing Bm-Mdm2. These results suggest that Bm-Mdm2 can alternatively use other available lysine residues, and it will take complete mutations of all possible attachment sites to stop ubiquitination as reported in mammalian P53.

Chapter 4 Analysis of Bm-Mdm2 response in B. mori cells infected with NPV
　The previous study demonstrates that RNAi-mediated knockdown of bm-p53 expression abolishes apoptosis of B. mori cells induced by infection with vBmΔp35 and that levels of both bm-p53 mRNA and Bm-P53 protein were maintained at a basal level during BmNPV infection. These results suggest the complexity of Bm-P53-mediated apoptosis induction in NPV-infected B. mori cells. In this chapter, to understand the function of Bm-Mdm2 in BmNPV-infected B. mori cells, changes in endogenous Bm-Mdm2 mRNA and protein levels were examined during NPV infection. The infection of B. mori cells with either BmNPV or vBmΔp35 resulted in a drop of bm-mdm2 mRNA levels. In contrast, endogenous Bm-Mdm2 protein levels increased from 6 to 24 h post infection (p.i.) and seemed to be degraded at 48 h p.i. Further analyses using fluorescence microscopy and a knockdown assay of viral genes suggest that this increase of Bm-Mdm2 protein levels was not due to localization change of Bm-Mdm2 during infection and triggered by the onset of viral early gene expression and viral DNA replication. However, since overexpression of Bm-Mdm2 has no influence on apoptosis induction and virus protein expression in B. mori cells infected with BmNPV or vBm∆p35, I could not elucidate the function of Bm-Mdm2 in NPV-infected B. mori cells in this study.

Chapter 5 Conclusion
　In this study, I identified and characterized the B. mori homologs of Bonus, Mdm2, Rad6, Sce, and Synoviolin, which are crucial for the ubiquitylation and negative regulation of P53 in D. melanogaster and/or mammals. Among the identified homologs, Bm-Mdm2 is the prime negative regulator for Bm-P53, and this regulation is mediated through ubiquitination of Bm- P53 and suggested to occur in the nucleolus. Mdm2, which is a primary negative regulator of P53 levels in mammals, is also widely conserved in the arthropods, including insects, but not encoded by the D. melanogaster genome. This study is the first demonstration of the functionality of an insect Mdm2 using the Mdm2 homolog of the lepidopteran insect B. mori. I have shown that Bm-Mdm2 also serves as a negative regulator of P53 though Bm-Mdm2 possessed many differences with the mammalian Mdm2, including its localization and conserved domains. Taken together, these findings suggest that lepidopteran insects use a different P53 regulation mechanism from those used in dipteran insects, such as D. melanogaster, which lost Mdm2 and acquired a new regulator, Corp, and those used in mammals. Further analysis using P53 and its negative regulators from various insects will give insight into the conservation and diversification of the P53 negative regulation mechanism.