OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

Journal Article · Tue Apr 01 04:00:00 EDT 2014 · Experimental Cell Research

DOI:https://doi.org/10.1016/J.YEXCR.2014.01.002· OSTI ID:22395879

Zhong, Wenbin ^[1]; Zhou, You ^[2]; Li, Jiwei ^[1]; Mysore, Raghavendra ^[2]; Luo, Wei; Li, Shiqian ^[1]; Chang, Mau-Sun ^[3]; Olkkonen, Vesa M. ^[2]; Yan, Daoguang ^[1]

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1. Department of Biotechnology, Jinan University, Guangzhou 510632 (China)
2. Minerva Foundation Institute for Medical Research, Helsinki (Finland)
3. Institute of Biochemical Sciences, National Taiwan University, No. 1, Taipei, Taiwan (China)

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. - Highlights: • The oxysterol-binding protein ORP8 was found to interact with the mitotic regulator SPAG5/Astrin. • Treatment of HepG2 cells with 25-hydroxycholesterol caused cell cycle retardation in G2/M. • ORP8 overexpression caused a similar G2/M accumulation, and ORP8 knock-down reversed the 25-hydroxycholesterol effect. • Reduction of cellular of SPAG5/Astrin reversed the cell cycle effects of both 25-hydroxycholesterol and ORP8 overexpression. • Our results suggest that ORP8 mediates via SPAG5/Astrin the oxysterol interference of HepG2 cell cycle.

• OSTI ID: 22395879
• Journal Information: Experimental Cell Research, Journal Name: Experimental Cell Research Journal Issue: 2 Vol. 322; ISSN 0014-4827; ISSN ECREAL
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ANTIGENS
CELL CYCLE
ENDOPLASMIC RETICULUM
HOMEOSTASIS
HYBRIDIZATION
INTERFERENCE
LIPIDS
MICROTUBULES
PHENOTYPE
PROTEINS
REDUCTION
SPERMATOZOA
YEASTS