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Molecular and cellular dissection of the OSBP cycle through a fluorescent inhibitor

Tiphaine Péresse, David Kovacs, Mélody Subra, Joëlle Bigay, Meng-Chen Tsai, Joël Polidori, Romain Gautier, Sandy Desrat, Lucile Fleuriot, Delphine Debayle, Marc Litaudon, Van-Cuong Pham, Jérôme Bignon, Bruno Antonny, Fanny Roussi, Bruno Mesmin

Preprint posted on 20 November 2019 https://www.biorxiv.org/content/10.1101/844548v1

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

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.

Fig1. Macaranga schweinfurthii. M. schweinfurthii cultivated in Yaounde by John Beutler https://www.flickr.com/photos/103233542@N02/9938871953

 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: 

  1. 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? 
  1. 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

 

Posted on: 24 February 2020 , updated on: 26 February 2020

doi: https://doi.org/10.1242/prelights.17168

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Author's response

Bruno Mesmin and Fanny Roussi shared

To address your first question, we know that ORPphilins are synthesized by various organisms, such as plants or marine organisms like sponges, and they are all evolutionary conserved because they are made for the self-defense of these organisms against various predators. Nevertheless, all these natural OSBP inhibitors have very different chemical structures. This is unexpected at first sight, because the chemical similarities between molecules that interact with a specific protein are usually more obvious. However, the binding site for Schweinfurthin G (and most probably for all ORPphilins) is the lipid-binding cavity of the ORD domain of OSBP, which is already adapted for two very different lipids: the cholesterol, a rather small, uncharged and bulky lipid, and the PI(4)P, which has two long acyl chains and a large charged polar head. This might explain why there is great structural diversity in OSBP inhibitors.

 Regarding your second question, we think that all Schweinfurthins are fluorescent because they possess a trans-stilbene moiety (which is responsible for the fluorescence of the molecule). However, their bioactivity depends on the hexahydroxanthene moiety (HHX moiety, left part of the molecule), and not all schweinfurthins have an HHX.

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