The Study On Topology Of 3 Beta-hydroxysteroid-delta 24 Reductase

DHCR24 encodes 3-β-hydroxysterol△-24-reductase, which catalyzes the synthesis of cholesterol from desmosterol and belongs to a family of FAD-dependent oxidoreductases. DHCR24 mRNA comprises an ORF of 516 amino acid with a calculated molecular weight of 60.1 kD, with a potential N-terminal secretory signal sequence, and with at least one putative transmembrane helix. In recent years, it has been demonstrated that DHCR24 is an endoplasmic reticulum-resident, multifunctional enzyme that possesses anti-apoptotic and cholesterol-synthesizing activities. DHCR24 inhibits the activation of caspase-3,a key modulator of apoptosis. Moreover,it functions also as a mediators of hormones, like estrogen, insulin like growth factor (IGF) and thyroid hormone(TH) in their function on neuronprotection and neuron development. Obviously, further investigation is significant in order to fully elucidate both the role of DHCR24 in maintaining nervous cells homeostasis and the alterations leading to pathological conditions affecting the nervous system, aiming to possibly identify new areas of pharmacological intervention.The present work is based on the DHCR24 (Genebank Acession NO: BC011669. 2) sequence information and its transmembrane domain predicted by bioinformatics. Specific primers were designed for polymerase chain reaction(PCR) amplification. The full-length and the transmembrane domain deletion(TM minus) cDNAs encoding human DHCR24 gene were subcloned to cloning vector pGEM-T-easy after amplified by PCR. The recombinant pGEM-T-easy-DHCR24 were transformed into E.coli.JM109, which were identified by double digestion with restriction endonucleases and sequencing. Then the full-length and the TM minus DHCR24 cDNAs were respectively ligated into eukaryotic expression vectors pDsred-C1 and pEGFP-N1. The constructed plasmids pDsred-C1-DHCR24, pEGFP -N1-DHCR24 and pDsred-C1-DHCR24 TM (-) were transfected into mouse neuroblastoma cell line N2A cells by lipofectamin tranfection method after identification by double digestion with restriction endonucleases. The expression and subcellular localization of DHCR24 fusion proteins in N2A cells were assayed by fluorescent immunocytochemistry.To study DHCR24 topology, we used a new fluorescence-based technique, fluorescence protease protection (FPP). We transfected three versions of plasmids expressing DHCR24 fusion proteins into N2A cells. One of them expressed DHCR24 fused with enhanced green fluorescence protein (EGFP) attached to its C terminus (DHCR24-EGFP), the second expressed the DHCR24 with DsRed attached to its N terminus (DsRed-DHCR24), and another expressed TM deleted DHCR24 with DsRed attached to its N terminus (DsRed-DHCR24 TM minus).Finally we examined whether subcellular localization of FAD binding domain could influence its H2O2-scavenging activity through the reactive oxygen species Detection Kit (DCFH-DA).The conclusion of the study: three versions of plasmids expressing tagged DHCR24s were constructed successfully and these fusion proteins were expressed highly in N2A cells. Immunofluorescence results show that full-length DHCR24 fusion proteins located in the endoplasmic reticulum (ER), the DHCR24 transmembrane domain deletion fusion protein located in the cytoplasm. And the FPP results suggest that DHCR24 is a single transmembrane protein with its N terminus facing the ER lumen and its C terminus facing the cytoplasm. So we come to a conclusion: FAD binding domain located in the cytoplasm. Finally, using a cell-permeant indicator, H2DCFHDA, it was found that DHCR24 TM minus exhibit significant H2O2-scavenging activity, which does not depend on its location. These results are important to clarify the molecular basis of its anti-apoptotic through its anti-ER stress and anti- oxidation stress function.