Molecular Mechanism Underlying The Interaction Between Hsp70 And Fzr During Endoreplication In Insects

Endoreplication,also known as endocycling,is a unique variant of the cell cycle in which cells undergo multiple rounds of genome replication and lack mitosis and chromosome segregation,resulting in polyploidy.The silk glands of silkworm and salivary glands of Drosophila are the most studied endoreplication organs in insects.Previous studies showed that the upregulation of scaffold protein Fzr is essential for the mitosis-to-endoreplication transition during the embryo stage and the stable expression of Fzr maintains the endoreplication progression during the larva stage.Although the key functions of Fzr protein in regulating the transition of mitosis-to-endoreplication and the maintaining of endoreplication progression is largely understand,the regulatory mechanism for the stability of Fzr remains unclear.In our previous study,we identified that potential candidates interacting with Fzr in human embryonic kidney cells（HEK293-FT cells）and focused on heat shock protein（Hsp70）.Hsp70 is regarded as a molecular chaperone to regulate the conformation and stability of target proteins.In the present study,we analysed that Hsp70 interact with Fzr in silkworm and Drosophila,explored the mechanism underlying the effects of Hsp70 on maintaining the stability of Fzr and the functions of Hsp70 in endoreplication.The main results were as follows:1.The interaction between Hsp70 and Fzr in silkworm and DrosophilaBased on a sequence homology analysis using the amino acid sequence of human Hsp70 as a query,we identified the silkworm and Drosophila Hsp70 gene sharing a high identity to human Hsp70.Furthermore,we constructed the overexpression vectors of silkworm,Drosophila and human species Hsp70 and Fzr genes.Then transfected the vectors into silkworm BmE cells,Drosophila S2 cells and human HEK293-FT cells respectively.Subcellular analysis showed that both Hsp70 and Fzr can be located in nucleus.Further co-immunoprecipitation（Co-IP）analysis demonstrated that Hsp70 and Fzr can interact with each other in silkworm,Drosophila and human species,suggesting that this interaction is conservative.We treated the cells co-overexpressing Hsp70 and Fzr gene with the inhibitor PES that target Hsp70 interacting proteins.The following Co-IP analysis showed that the PES treatment abolished the interaction between Hsp70and Fzr.The Fzr protein contains 6 repeated WD40 domains,which was involved in mediating protein-protein interactions.Using a deletion mutation,we analyzed the exact WD40 domains in Fzr interacting with Hsp70.We first performed a two domains deletion successively and constructed overexpression vectors.Co-IP analysis showed that the simultaneous deletion of the first and the second or the third and the fourth domains had no effect on the interaction between Hsp70 and Fzr.However,the simultaneous deletion of the fifth and sixth domains disturbed this interaction.Then,we deleted the single domain of the fifth and sixth domain and set the single domain deletion of the the third and the fourth domain as a positive control.The results showed that both the fifth and sixth domain deletion inhibited the interaction between Hsp70and Fzr.Therefore,we concluded that the fifth and sixth domain in Fzr mediated Hsp70-Fzr interaction.2.Hsp70 participates in regulating the stability of Fzr in silkworm and DrosophilaIt was considered that Hsp70 protein could be involved in maintaining the stability of the interacting protein.We further analyzed the regulatory effect of Hsp70 on the stability of Fzr protein.Based on the principle of RNA interference（RNAi）technology,we designed and synthesized double strandsRNA（dsRNA）according to the silkworm and Drosophila Hsp70 encoding gene.The exogenous Fzr protein level was detected with a downregulation of endogenous Hsp70 gene in silkworm BmE cells and Drosophila S2 cells using the Hsp70 dsRNA.RT-PCR showed that Hsp70 RNAi had no effect on Fzr gene transcription.Western blot results showed that RNAi of Hsp70 gene significantly reduced the protein level of Fzr,while the protein level of Cyclin B,the target protein involved in cell division and can be ubiquitin modified and depredated by Fzr,was significantly accumulated.Comprehensive analysis showed that Hsp70 was involved in maintaining the stability of Fzr protein.Further,we studied how Hsp70 regulates the stability of Fzr protein.Considering that Hsp70 gene downregulation reduced Fzr protein level in silkworm and Drosophila,and ubiquitination of protein is one of the important pathways of protein degradation,we hypothesized that this reduction might related with ubiquitination and degradation of Fzr.Therefore,we carried out in vivo ubiquitination experiment at cellular level.The Co-IP experiment results suggested that Hsp70 RNAi increased the ubiquitination level of Fzr and decreased Fzr protein level.On the contrary,Hsp70 overexpression inhibited Fzr ubiquitination and elevated Fzr protein level.These data indicate that the interaction between Hsp70 and Fzr is likely involved in maintaining the stability of Fzr by inhibiting Fzr ubiquitination.In view of the proteasomal and lysosomal pathways involved in the ubiquitination degradation of silkworm Fzr protein,we next evaluated the possible pathway of ubiquitination degradation of Fzr.We used the CHX which inhibits protein synthetization treated BmE cells co-expressing Hsp70 and Fzr gene.Further western blot showed that Fzr protein level was decreased following the CHX treatment,indicating that Fzr was degraded.Based on the CHX treatment,the BmE cells were then treated with proteasome inhibitor MG132 and lysosome inhibitor NH₄Cl,respectively.The results demonstrated that MG132 treatment significantly inhibited the reduction of Fzr protein level,and NH4Cl treatment had no effect on Fzr protein level.Therefore,we conclude that the ubiquitination degradation of Fzr should be through the proteasome pathway in silkworm.3.The function of Hsp70 in endoreplication of salivary glands in DrosophilaIn view of the fact that Fzr is a key regulator of the endoreplication and Hsp70 is involved in maintaining the stability of Fzr protein.We further analyzed the function of Hsp70 in the salivary gland using the mature Gal4/UAS genetic operating system in Drosophila.Drosophila salivary gland specific Sg-Gal4 was used to drive Hsp70 gene to produce tissue-specific RNAi in salivary glands .The results of EdU staining showed that Hsp70 RNAi abrogated DNA synthesis and decreased the salivary gland size.Immunostaining and western blot analysis confirmed that Hsp70 knockdown reduced Fzr protein level and also induced an accumulation of Cyclin B.The comprehensive analysis showed that Hsp70 interacted with Fzr protein to maintain its stability by inhibiting the ubiquitination modification and degradation of Fzr protein,thus participating in the regulation of the process of endorelication.