Membrane-Lipid Therapy in Operation: The HSP Co-Inducer BGP-15 Activates Stress Signal Transduction Pathways by Remodeling Plasma Membrane Rafts

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

Effect of BGP-15 treatment on heat-induced HSF1 acetylation in HEK293T cells and on <i>in vitro</i> SIRT1 activity.

<p><b>A</b>) 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>B</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)]; <b>C</b>) In vitro activity of SIRT1 using activators, inhibitors and BGP-15 [n = 3, error bars represent standard error of the mean (SEM)].</p

Effect of BGP-15 on DChol desorption from lipid monolayers to MBCD.

<p><b>A</b>) 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 (A_t) MBCD injection. <b>B</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.</p

<i>Hsp25</i> mRNA expression in B16-F10 cells modified by Rac1 inhibitor and BGP-15 administration.

<p>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 <i>hsp25</i> was tested by RT-PCR. [n = 3, error bars represent standard error of the mean (SEM)].</p

The effect of BGP-15 on the temperature-induced change in cluster fraction of mGFP-GPI.

<p>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)].</p

The effect of BGP-15 on cholesterol-rich membrane domains and the heat shock inducibility of <i>hsp25</i> in B16-F10 cells with different cell density.

<p><b>A</b>) 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>B</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. <b>C</b>) HCN (with or without 10 µM BGP-15) and LCN cells were heated at 42°C for 1 hour and the expression of <i>hsp25</i> was tested by RT-PCR.</p

Hydroximic Acid Derivatives: Pleiotrophic Hsp Co-Inducers Restoring Homeostasis and Robustness

According to the "membrane sensor" hypothesis, the membranes physical properties and microdomain organization play an initiating role in the heat shock response. Clinical conditions such as cancer, diabetes and neurodegenerative diseases are all coupled with specific changes in the physical state and lipid composition of cellular membranes and characterized by altered heat shock protein levels in cells suggesting that these "membrane defects" can cause suboptimal hsp-gene expression. Such observations provide a new rationale for the introduction of novel, heat shock protein modulating drug candidates. Intercalating compounds can be used to alter membrane properties and by doing so normalize dysregulated expression of heat shock proteins, resulting in a beneficial therapeutic effect for reversing the pathological impact of disease. The membrane (and lipid) interacting hydroximic acid (HA) derivatives discussed in this review physiologically restore the heat shock protein stress response, creating a new class of "membrane-lipid therapy" pharmaceuticals. The diseases that HA derivatives potentially target are diverse and include, among others, insulin resistance and diabetes, neuropathy, atrial fibrillation, and amyotrophic lateral sclerosis. At a molecular level HA derivatives are broad spectrum, multi-target compounds as they fluidize yet stabilize membranes and remodel their lipid rafts while otherwise acting as PARP inhibitors. The HA derivatives have the potential to ameliorate disparate conditions, whether of acute or chronic nature. Many of these diseases presently are either untreatable or inadequately treated with currently available pharmaceuticals. Ultimately, the HA derivatives promise to play a major role in future pharmacotherapy