Effects of NSC and 2-Brp on HSP25 and HSP70 protein levels under heat shock conditions.

<p>Cells were treated as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089136#pone-0089136-g005" target="_blank"><b>Figure 5</b></a> and were collected after an overnight recovery at 37°C. (<b>A</b>) Western blotting was performed for equal amounts of total protein samples. Membranes were probed with anti-HSP25, anti-HSP70 and anti-Gapdh antibodies. (<b>B</b>) The band intensities of protein expression levels were quantitated and normalized to Gapdh. In calculations of fold changes, the protein levels of 41.5°C samples were taken as 100. Data are means ± SD, n = 3.</p

Change in size of PM microdomains as functions of temperature and 2-Brp treatment.

<p>(<b>A</b>) 2-Brp was added/or not to samples for 30 min, and they were then heat-treated at 41.5°C or 43°C or kept at 37°C for 1 h. After BODIPY FL C12-sphingomyelin labeling and TIR microscopy, the domain distribution was analyzed with ImageJ and CellProfiler software and the average domain sizes are shown. (<b>B</b>) From the experiment shown in (<b>A</b>), the PM microdomains were separated into five classes, depending on their sizes. The data shown are means ± SEM, n = 3.</p

Effects of NSC and 2-Brp on <i>hsp25</i> and <i>hsp70</i> gene expressions under heat shock conditions.

<p>B16F10 cells were either treated/or not with NSC or 2-Brp for 2 h or 30 min, respectively, and then exposed/or not to the indicated heat shock temperatures for 1 h. Total RNA was isolated immediately after the heat shock, and the expression levels of <i>hsp70</i> and <i>hsp25</i> mRNAs were measured by quantitative RT-PCR and normalized to <i>gapdh</i>. If BGP-15 (HSP co-inducer) was applied, it was administered at 10 µM during heat shock. Data shown are fold changes compared to the mRNA levels measured in inhibitor untreated, heat shocked samples which was considered 1. Inserted graphs represent the fold changes compared to the mRNA levels measured in 37°C samples. Data are means ± SEM, n = 3.</p

Translocation of Rac1 to membranes as a result of Rac1 inhibitor and heat shock treatment.

<p>(<b>A</b>) Localization of Rac1 to the crude membrane fraction in response to heat shock treatment. B16F10 cells were subjected to heat shock at the indicated temperatures for 1 h. Immediately after this, the crude membrane fraction was isolated and solubilized in Laemmli buffer. Equal amounts of proteins were run for western blotting. Rac1 probing was performed for the membrane, and caveolin immunostaining was used for normalization. Changes in normalized Rac1 band intensities: 37°C = 100, 41.5°C = 132, 42°C = 310, 43°C = 255. (<b>B</b>) Visualization of the association of Rac1 to the PM. B16F10 cells in glass-bottomed plates were kept in a water bath at the indicated temperatures for 1 h. The cells were then fixed, permeabilized and immunoreacted with Rac1 mAb, and probed with Alexa488-labeled secondary antibody by confocal microscopy. Intensity profiles of regions of interest on confocal images (indicated with white lines) are shown. Black arrows indicate PM. The red curve refers to 43°C, the blue curve to 41.5°C and the black curve to 37°C. (<b>C</b>) The effects of Rac1 inhibitor administration on the PM binding of Rac1 under heat stress conditions. Cells were treated/or not with the Rac1 inhibitor NSC or 2-Brp, heat-shocked/or not, and then immunostained as above. Confocal images were taken and 15 cells/3 views for each treatment were quantified by ImageJ. The bars represent the fluorescence intensity of the PM versus the fluorescence intensity of the whole cell. Black bars relate to cells not treated with inhibitor, white bars to NSC-treated and gray bars to 2-Brp-treated cells. Data are means ± SEM, n = 3, Student’s t-test was used for statistical analysis. *: p<0.05; **: p<0.01; ***: p<0.001.</p

F-actin and cell morphology changes in B16F10 cells under heat shock conditions.

<p>(<b>A</b>) Measurement of heat stress-triggered F-actin alterations by using flow cytometry. After heat stress, cells were collected by trypsinization, fixed and labeled with Alexa 647 phalloidin. Flow cytometry measurements were made with a BD Accuri C 6 cytometer, and CFLow Plus 1.0.227.2. software was used for data analysis. (<b>B</b>) Surface area changes of B16F10 cells in response to heat shock and Rac1 inhibitor administration. B16F10 cells were subjected to stress conditions at 41.5°C or 43°C or kept at 37°C with or without inhibitor administration as indicated, and samples were fixed for SEM imaging. From the SEM images, 30 cells were chosen for the calculation of surface area with ImageJ software. Black bars relate to the lack of inhibitor treatment, while white and gray bars relate to NSC and 2-Brp administration, respectively. The data from three independent experiments are shown, along with the S.D. Student’s t-test was used for statistical analysis. *: p<0.05; **: p<0.01; ***: p<0.001. (<b>C</b>) Representative SEM photos of 37°C, 37°C+NSC, 37°C+2-Brp, 43°C, 43°C+NSC and 43°C+2-Brp-treated cells.</p

Effect of Rac1 inhibition on hyperphosphorylation level of HSF1.

<p>B16F10 cells were treated or not with inhibitors (NSC or 2-Brp) prior to the indicated heat shock conditions. Immediately afterwards, samples were harvested and equal amounts were used for western blotting. Membranes were probed with anti-HSF1 and anti-Gapdh antibodies.</p

[Dataset] The Crystal Structure of the Hsp90-LA1011 Complex and the Mechanism by which LA1011 may Improve the Prognosis of Alzheimer’s Disease

Data for paper published in Biomolecules 2023, 13(7), 105  The data sets are the results for the ITC experiments investigating the interactions between Hsp90, FKBP51 and LA1011. A data set constitutes an ITC file from a heat of dilution or interaction experiment. Data sets are processed by using a pair of experiments represented by a heat of dilution and interaction pair that have the same date stamp at the front of the file name. 'dil' in the filename denotes the heat of dilution. Heats of dilution are into buffer. After the date stamp the interacting partner proteins or small molecule is shown.  The data files require Origin software to access. Abstract    Functional changes in chaperone systems play a major role in the decline of cognition and contribute to neurological pathologies, such as Alzheimer’s disease (AD). While such a decline may occur naturally with age or with stress or trauma, the mechanisms involved have remained elusive. The current models suggest that amyloid-β (Aβ) plaque formation leads to the hyperphosphorylation of tau by a Hsp90-dependent process that triggers tau neurofibrillary tangle formation and neurotoxicity. Several co-chaperones of Hsp90 can influence the phosphorylation of tau, including FKBP51, FKBP52 and PP5. In particular, elevated levels of FKBP51 occur with age and stress and are further elevated in AD. Recently, the dihydropyridine LA1011 was shown to reduce tau pathology and amyloid plaque formation in transgenic AD mice, probably through its interaction with Hsp90, although the precise mode of action is currently unknown. Here, we present a co-crystal structure of LA1011 in complex with a fragment of Hsp90. We show that LA1011 can disrupt the binding of FKBP51, which might help to rebalance the Hsp90-FKBP51 chaperone machinery and provide a favourable prognosis towards AD. However, without direct evidence, we cannot completely rule out effects on other Hsp90-co-chaprone complexes and the mechanisms they are involved in, including effects on Hsp90 client proteins. Nonetheless, it is highly significant that LA1011 showed promise in our previous AD mouse models, as AD is generally a disease affecting older patients, where slowing of disease progression could result in AD no longer being life limiting. The clinical value of LA1011 and its possible derivatives thereof remains to be seen.</p

Schematic representation of TG synthesis in <i>S</i>. <i>pombe</i>.

<p>Dga1p uses FA-CoA and diacylglycerol (DG) to produce TG, while Plh1p catalyses the direct FA transfer between a glycerophospholipid (GPL) and DG to generate TG and a lysophospholipid (LPL).</p

Proposed mechanism of membrane rigidization in response to HS for the <i>dga1Δ</i> strain.

<p>Net changes expressed as lipid/prot_{(after HS–before HS)} (nmol/mg/h) values (left; average data from n = 3 independent experiments are shown), and schematic representation of TG-supported membrane rigidization (right) in the <i>dga1Δ</i> strain. DG, diacylglycerol; GPC/GPE/GPI, phosphorylated headgroups; GPL, glycerophospholipid; LPL, lysophospholipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; TG, triacylglycerol.</p

Growth arrest of heat-stressed DKO cells correlates with enhanced signalling lipid generation.

<p>Changes in the amounts of Cer and DG by lipid class and species levels are shown. Cells were untreated (30°C) or stressed at 40°C for 1 h. Values are expressed as mean ± SD of lipid/protein values (nmol/mg), n = 3 for <i>plh1Δ</i> and <i>dga1Δ</i>, n = 4 for DKO, and n = 7 for WT; * p<0.05 (30°C vs 40°C), # p<0.05 (WT vs mutants at 30°C), $ p<0.05 (WT vs mutants at 40°C).</p