The membrane topological analysis of 3β-hydroxysteroid-Delta24 reductase (DHCR24) on endoplasmic reticulum.
DHCR24 encodes 3β-hydroxysteroid-Δ24 reductase, catalyzing the conversion of desmosterol to cholesterol. Our previous study demonstrated that DHCR24 exerts an anti-apoptotic function as a reactive oxygen species (ROS) scavenger, for which it needs its FAD-binding domain. The membrane topology of DHCR24 on endoplasmic reticulum (ER) and the functional significance of its FAD-binding domain are not completely understood. Based on the structure predicted by bioinformatics, we studied the membrane topology of DHCR24 in murine neuroblastoma cells (N2A), using the fluorescent protease protection (FPP) technique. We showed that full-length DHCR24 is localized to the membrane of ER, whereas the predicted transmembrane (TM) domain-deleted DHCR24 mutation is localized to the cytoplasm. The change of DHCR24 localization suggests that the N-terminal TM domain is essential for the ER membrane targeting of DHCR24. The FPP assay demonstrated the membrane topology of DHCR24 with an N-terminal luminal/C-terminal cytoplasmic orientation. Measurement of intracellular ROS using H(2)DCFDA revealed that the ROS levels of cells infected by plasmids driving expression of full-length DHCR24 or the TM domain-deleted DHCR24 mutation after H(2)O(2) exposure were lower than those of control cells, suggesting that the ER membrane targeting of DHCR24 is not required for its enzymatic ROS scavenging activity. Confocal fluorescence microscopy revealed that the DHCR24-overexpressed cells were protected from apoptosis in response to oxidative stress, which was accompanied by a decrease in DHCR24 content on the ER and activation of caspase-3, suggesting that the anti-apoptotic function of DHCR24 is associated with its cleavage by caspase.
Department of Biochemistry and Cell Biology, School of Life Science, Liaoning University, Huang-Gu-Qu, Chong-Shang-Zhong-Lu No. 66, Shenyang 110036, China.
Xiuli
X
Lu
Yang
Y
Li
Jianli
J
Liu
Xiangyu
X
Cao
Xude
X
Wang
Delong
D
Wang
Hisao
H
Seo
Bing
B
Gao
D020169:Caspases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D023061:Gene Order; D006801:Humans; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D009419:Nerve Tissue Proteins; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; C499055:DHCR24 protein, human; D020169:Caspases