title: OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle.
abstract: We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle.
authors.affiliation: Department of Biotechnology, Jinan University, Guangzhou 510632, China.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Zhong
authors.affiliation: Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: You
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Biotechnology, Jinan University, Guangzhou 510632, China.
authors.forename: Jiwei
authors.initials: J
authors.lastname: Li
authors.affiliation: Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Raghavendra
authors.initials: R
authors.lastname: Mysore
authors.affiliation: Department of Biotechnology, Jinan University, Guangzhou 510632, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Luo
authors.affiliation: Department of Biotechnology, Jinan University, Guangzhou 510632, China.
authors.forename: Shiqian
authors.initials: S
authors.lastname: Li
authors.affiliation: Institute of Biochemical Sciences, National Taiwan University, No. 1, Taipei, Taiwan.
authors.forename: Mau-Sun
authors.initials: MS
authors.lastname: Chang
authors.affiliation: Minerva Foundation Institute for Medical Research, Helsinki, Finland.
authors.forename: Vesa M
authors.initials: VM
authors.lastname: Olkkonen
authors.affiliation: Department of Biotechnology, Jinan University, Guangzhou 510632, China. Electronic address: tydg@jnu.edu.cn.
authors.forename: Daoguang
authors.initials: D
authors.lastname: Yan
mesh_terms: D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects* / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D056945:Hep G2 Cells; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D011987:Receptors, Steroid / Q000378:metabolism*; D013347:Subcellular Fractions; D020798:Two-Hybrid System Techniques; D000098825:Oxysterol Binding Proteins
chemical_list: D018797:Cell Cycle Proteins; D006888:Hydroxycholesterols; D011987:Receptors, Steroid; C486663:SPAG5 protein, human; D000098825:Oxysterol Binding Proteins; C007997:25-hydroxycholesterol
keywords: 25-Hydroxycholesterol; Astrin; Cell cycle; ORP8; Oxysterol-binding protein; SPAG5