Study On The Mechanism Of The Interaction Of DHCR24 And Its Interaction Protein MDM2 And The Influence To Cell Function

The ubiquitin system has been identified as a key regulator of many cellular regulatory processes,including protein degradation,DNA repair,gene transcription,cell cycle control,endocytosis and autophagy.Ubiquitin often changes in cancer cells,and some tumor suppressors and oncogenes are ubiquitin binding and disintegration enzymes.The ubiquitin is mainly linked to the lysine residue and can also bind to the N-terminal amino group of other substrate proteins.Ubiquitination is mediated by E1 ubiquitin activator,E2 ubiquitin-binding enzyme,E3 ubiquitin ligase.MDM2 protein is an E3 ubiquitin ligase.DHCR24 plays an important role in cholesterol biosynthesis,oxidative stress response,neuroprotection,anti-apoptosis and anti-inflammatory effects.DHCR24 is also involved in a variety of disease regulation processes,such as cardiovascular disease,cancer(prostate cancer)and hepatitis C.DHCR24 functions in many important biological processes might be through its interaction with other proteins.In our previous experiment,bioinformatics was used to predict some candidate proteins those could interact with DHCR24.MDM2 is one of the candidate proteins.In order to explore the molecular mechanism of the interaction between DHCR24 and E3 ligase MDM2,we used immunoprecipitation experiments to confirm the effect of MG132 on the ubiquitination of DHCR24,then the possible binding pattern of the interaction between DHCR24 and MDMD2 was analyzed by bioinformatics and protein docking techniques.After treatment with MG132,the whole protein of PC12 cells transfected with recombinant adenovirus expressing DHCR24 was collected and then subjected to immunoprecipitation method.Experimental results showed that MG132 could enhance the ubiquitination of DHCR24 in nervous cells.Moreover,the increase of the ubiquitination of DHCR24 induced by MG132 could significantly enhance its neuroprotective effect.Then,we used the websites UbiPred,UbPred and CKSAAP_Ub Sites to predict the ubiquitination sites of the DHCR24 protein,respectively.Based on a comprehensive analysis of the results of three sites,K301,K306,K367,K427 of DHCR24 might be the ubiquitination sites which were induced by MDM2 protein.In order to further study the interaction mechanism of DHCR24 with its interacting protein MDM2,we docked DHCR24 and MDM2 protein under two different docking strategies by using protein docking online serve ZDOCK.The results showed that the interactions of DHCR24 and MDM2 protein were in the similar way.DHCR24 and MDM2 protein were identical to bind to each other and mainly through the interaction between the helix Y298-K312 and helix T332-D341 in DHCR24 protein,indicating that Y298-K312 and T332-D341 in the DHCR24 sequence plays a crucial role in the binding of DHCR24 to the MDM2 protein.In this study,the possible molecular mechanism of the interaction between DHCR24 and MDM2 protein was studied.It was confirmed that MG132 could enhance the ubiquitination level of DHCR24.Moreover,we found that the enhancement of ubiquitination could improve the neuroprotective effect of DHCR24.We also predicted the ubiquitination sites of DHCR24 and the molecular mechanism of DHCR24 and MDM2.This study not only provides a new theoretical basis and new ideas for the interaction between DHCR24 and MDM2 protein,but also provides a new idea for the development of DHCR24 as a neuroprotectivc gene therapy drug.