Molecular and cellular dissection of the OSBP cycle through a fluorescent inhibitor
Posted on: 24 February 2020 , updated on: 26 February 2020
Preprint posted on 20 November 2019
Article now published in JBC at https://www.jbc.org/content/early/2020/02/19/jbc.RA119.012012
An intrinsically fluorescent, natural product specifically inhibits the lipid transport protein OSBP
Selected by Berrak Ugur
Categories: biochemistry, bioengineering, cell biology, plant biology
Background:
A group of naturally occurring products collectively called ORPhilins inhibit growth of several cancer cell lines. These structurally diverse bioactive molecules work by inhibiting certain lipid transport proteins (LTPs), namely Oxysterol-binding protein (OSBP) and OSBP related protein 4 (ORP4). OSBP is a cytosolic protein that is able to contact and connect the Endoplasmic Reticulum (ER) to the trans Golgi Network (TGN) through its N-terminal domain (Ridgway et al., 1992, Levine and Munro S., 2002). The C-terminal OSBP related domain (ORD) has been shown to counter exchange PI4P and cholesterol (Mesmin et al., 2013) and therefore ORD is the region responsible for lipid transfer activity of OSBP.
Cephalostatin, OSW-1, ritterazine B, schweinfurthin family and stellettin are among some of the ORPhilins described to date (Burgett et al., 2011). Among these members, Schweinfurthin family members have higher affinities for OSBP compared to ORP. Schweinfurthins are isolated from the African plant Macaranga schweinfurthii (Figure 1) and have been shown to affect the nervous system as well as certain cancer cells (Harmalkar et al., 2018). However, the molecular mechanisms of how Schweinfurthins function is poorly studied.
Key Findings:
The more OSBP you have in your system, the less sensitive you become to SWG
Previously it was shown that the sensitivity of cells to Schweinfurthins is affected by the cellular levels of OSBP (Burgett et al., 2011). To test if the sensitivity of the drug Schweinfurthin G (SWG) is correlated with OSBP levels in different cells, the authors check OSBP levels in various cell lines and show that OSBP levels vary up to 5-fold between these cell lines. In addition, they check sensitivity of these cell lines to SWG and report that sensitivity to SWG is inversely proportional to the cellular level of OSBP. Moreover, SWG caused OSBP to become predominantly localized to Golgi in the short term but it caused OSBP’s dissociation from the Golgi in the long term. These results indicate that SWG specifically targets OSBP.
SWG specifically inhibits lipid transfer activity of OSBP
Due to SWG’s effect on OSBP Golgi localization, the authors ask whether SWG affects the secretory pathway. Through RUSH assay, the authors show that SWG regulates the formation of post-Golgi (but not pre-Golgi) transport intermediates. Because OSBP regulates lipid transfer between Golgi and ER, SWG may function through inbition of OSBP’s lipid transfer activity. To test this hypothesis, the authors perform in vitro liposome-based reconstitution experiments and show that SWG inhibits the lipid transport domain of OSBP. Moreover, SWG is capable of blocking both (1) ER to Golgi Cholesterol Transfer and (2) Golgi to ER PI(4)P Transfer. Finally, the authors show that lipid transfer region of OSBP is able to bind to SWG by using FRET (Fluorescence Resonance Energy Transfer).
Take home messages
- SWG is a naturally florescent bioactive molecule
- SWG inhibits specifically the lipid exchange cycle of the lipid transport protein OSBP
- SWG affects post-Golgi trafficking, membrane cholesterol levels and PI(4)P turnover
- Intermolecular FRET shows direct binding of SWG into OSBP lipid-binding cavity
What I liked about this study:
There are a number of natural OSBP inhibitors, however in this manuscript the authors show an inhibitor that is fluorescent. The fluorescence of SWG allows an easy detection of its intracellular localization. Following the “seeing is believing” motto, this study documents a visible bioactive molecule that permits us to study OSBP and/or Golgi lipid transport.
Open Questions:
- It is very interesting that there is a number of natural occurring molecules that inhibit OSBPs function. Is there any evidence indicating that these molecules are evolutionary conserved, is there a specific structure shared by all of these ORPhilins?
- How do other schweinfurthins differ from SWG, do any of them have intrinsic fluorescence?
References:
Burgett, A. W. G., Poulsen, T. B., Wangkanont, K., Anderson, D. R., Kikuchi, C., Shimada, K.,Okubo, S., Fortner, K. C., Mimaki, Y., Kuroda, M., Murphy, J. P., Schwalb, D. J., Petrella, E. C.,Cornella-Taracido, I., Schirle, M., Tallarico, J. a, and Shair, M. D. (2011) Natural products reveal cancer cell dependence on oxysterol-binding proteins. Nat. Chem. Biol. 7, 639–47.
Harmalkar, D. S., Mali, J. R., Sivaraman, A., Choi, Y., & Lee, K. (2018). Schweinfurthins A-Q: Isolation, synthesis, and biochemical properties. RSC Advances, 8(38), 21191-21209.
Levine TP, Munro S. (2002) Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr Biol.,12(9):695–704.
Ridgway ND, Dawson PA, Ho YK, Brown MS, Goldstein JL.(1992)Translocation of oxysterol binding protein to Golgi apparatus triggered by ligand binding. J Cell Biol.,116(2):307–319
Mesmin, B., Bigay, J., Moser von Filseck, J., Lacas-Gervais, S., Drin, G., and Antonny, B.(2013) A four-step cycle driven by PI(4)P hydrolysis directs sterol/PI(4)P exchange by the ER-Golgi tether OSBP. Cell.,155, 830–43
doi: https://doi.org/10.1242/prelights.17168
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