Study On The Screening Of DHCR24 (24-Dehydrocholesterol Reductase) Interacting Proteins

It is widely accepted that Alzheimer disease (AD) is associated with the abnormal cholesterol metabolism.3β-hydroxysterol Δ24-reductase (DHCR24) catalyzing the conversion of desmosterol to cholesterol, is a multiple function enzyme that possesses anti-apoptotic activities, however, its position and function in cell signal transduction is still unclear. The present studyis aimed to screen and identify the interaction proteins with DHCR24 to explore the mechanisms of its affecting cell fate. The combination of chuman brain cDNA library T7 phage display system and Co-IP technology was used to screen the DHCR24-binding proteins.First of all, we constructed the plasmid pT7CFE1-His-hDHCR24 and obtained overexpressed fusion protein His-hDHCR24 (DHCR24) using aan in vitro-protein expression.This vector contais an EMCV IRES element which is critical for obtaining high expression leves.The function of DHCR24 has been confirmed by the data obtained from the immunorecipitational analysis using the antibody against DHCR24 as the IP antibody and the antibody against MDM2 as the Western Blotting antibody, because there is known interaction between DHCR24 and MDM2. After that, we performed the immunoprecipetational experiment using the antibody against DHCR24 and subject the gel band samples which is expected to contain DHCR24-binding protein to perform the mass spectra (MS) analysis. About 300 candicate proteins were identified as the DHCR24-binding proteins from this experiment with the combination of IP and MS analysis. TheT7 cDNA phage display system was also performed using the overexpressed and purified DHCR24 protein, and thenmore than 100 peptides expressed in human brain were identified as DHCR24-binding candicate proteins.Finally, more than 20 proteins were identified and firstly selected to be confirmed by futher experiments like co-immunoprecipitational analysis and functional study.Taken together, our results provide a fundamental function for futher understanding the mechanism of multi-function of DHCR24 in neuronal system.