Membrane-lipid therapy in operation: the HSP co-inducer BGP-15 activates stress signal transduction pathways by remodeling plasma membrane rafts - PubMed Skip to main page content
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. 2011;6(12):e28818.
doi: 10.1371/journal.pone.0028818. Epub 2011 Dec 12.

Membrane-lipid therapy in operation: the HSP co-inducer BGP-15 activates stress signal transduction pathways by remodeling plasma membrane rafts

Imre Gombos  1 , Tim Crul, Stefano Piotto, Burcin Güngör, Zsolt Török, Gábor Balogh, Mária Péter, J Peter Slotte, Federica Campana, Ana-Maria Pilbat, Akos Hunya, Noémi Tóth, Zsuzsanna Literati-Nagy, László Vígh Jr, Attila Glatz, Mario Brameshuber, Gerhard J Schütz, Andrea Hevener, Mark A Febbraio, Ibolya Horváth, László Vígh
Affiliations

Membrane-lipid therapy in operation: the HSP co-inducer BGP-15 activates stress signal transduction pathways by remodeling plasma membrane rafts

Imre Gombos et al. PLoS One. 2011.

Abstract

Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP) expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO) cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1) acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in 'membrane-lipid therapy' to combat many various protein-misfolding diseases associated with aging.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts. AH is affiliated with LipidArt Ltd, which company does not gain or lose financially from this publication. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Molecular dynamics simulation of BGP-membrane interaction.
A) Position of atoms and molecules in SM/Chol membrane after 5 ns of simulation. SM is blue, Chol is yellow, all other atoms of the components and BGP-15 are colored using the Yasara CPK scheme. B) The position of BGP-15 molecules (in red) and the structural changes of SM/Chol membranes after addition of BGP-15. The control membrane is indicated with −; the membrane upon addition of BGP-15 is indicated with +.
Figure 2
Figure 2. Effect of BGP-15 on DChol desorption from lipid monolayers to MBCD.
A) MBCD-mediated removal of DChol from lipid monolayers at constant lateral surface pressure. When indicated 10 µM BGP-15 was injected into the subphase underneath SM/DChol monolayer 5 min before MBCD was administered. Surface area was measured before (A0) and at the indicated time points after (At) MBCD injection. B) Effect of BGP-15 concentration on MBCD-mediated DChol desorption from SM/DChol monolayers. The monolayers were equilibrated with BGP-15 for 5 min before MBCD was injected into the subphase.
Figure 3
Figure 3. The effect of BGP-15 on the temperature-induced change in cluster fraction of mGFP-GPI.
CHO cells stably expressing mGFP-GPI were subjected to 39.5°C with or without 10 µM BGP-15. TOCCSL experiments were performed and at indicated times the cluster fraction (two or more fluorophores per domain) was calculated [n = 2 error bars represent standard error of the mean (SEM)].
Figure 4
Figure 4. The effect of BGP-15 on cholesterol-rich membrane domains and the heat shock inducibility of hsp25 in B16-F10 cells with different cell density.
A) Fluorescence intensity of fPEG-Chol in B16-F10 cells seeded with low (LCN, 1.5×106/10 cm plate) or high cell number (HCN, 6×106/10 cm plate with or without 10 µM BGP-15). Student's t-test was used for statistical analyses, and p = 0.05 was set as a significance threshold. B) The size distribution profile of membrane microdomains labeled by fPEG-Chol in HCN (with or without 10 µM BGP-15) and LCN cells and imaged by TIRF microscopy. C) HCN (with or without 10 µM BGP-15) and LCN cells were heated at 42°C for 1 hour and the expression of hsp25 was tested by RT-PCR.
Figure 5
Figure 5. Hsp25 mRNA expression in B16-F10 cells modified by Rac1 inhibitor and BGP-15 administration.
B16-F10 cells were pretreated or not by Rac1 inhibitor NSC23766 for 2 hours before 1 h 41.5°C heat shock with or without 10 µM BGP-15. RNA was isolated and the expression of hsp25 was tested by RT-PCR. [n = 3, error bars represent standard error of the mean (SEM)].
Figure 6
Figure 6. Effect of BGP-15 treatment on heat-induced HSF1 acetylation in HEK293T cells and on in vitro SIRT1 activity.
A) HEK293T cells transiently co-transfected with mouse HSF1-FLAG and p300 were heat shocked for different lengths of time at 42°C or for 60 min at 42°C followed by 60 min recovery (R). After immunoprecipitation by FLAG, samples were probed for acetylated lysin by western blotting [n = 3, error bars represent standard error of the mean (SEM)]; B) Samples were or were not treated with 10 µM BGP-15 for 60 min before and during 60 min heat shock or 60 min recovery following heat shock (R) Acetylated HSF1 was determined as above. [n = 4, p<0.05, error bars represent standard error of the mean (SEM)]; C) In vitro activity of SIRT1 using activators, inhibitors and BGP-15 [n = 3, error bars represent standard error of the mean (SEM)].
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