The membrane topological analysis of 3β-hydroxysteroid-Δ24 reductase (DHCR24) on endoplasmic reticulum
| Autor: | Xiuli Lu, Yang Li, Xiangyu Cao, Jianli Liu, Hisao Seo, Bing Gao, Delong Wang, Xude Wang |
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| Rok vydání: | 2011 |
| Předmět: |
Oxidoreductases Acting on CH-CH Group Donors
biology Recombinant Fusion Proteins Endoplasmic reticulum Gene Expression Nerve Tissue Proteins Reductase Endoplasmic Reticulum Transmembrane protein Transport protein Cell biology Oxidative Stress Protein Transport Endocrinology Cytoplasm Caspases Membrane topology Gene Order biology.protein Humans Reactive Oxygen Species Molecular Biology Caspase Intracellular |
| Zdroj: | Journal of Molecular Endocrinology. 48:1-9 |
| ISSN: | 1479-6813 0952-5041 |
| DOI: | 10.1530/jme-11-0132 |
| Popis: | DHCR24encodes 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 H2DCFDA revealed that the ROS levels of cells infected by plasmids driving expression of full-length DHCR24 or the TM domain-deleted DHCR24 mutation after H2O2exposure 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. |
| Databáze: | OpenAIRE |
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