text:
Carlos H. Ramos
State University of Campinas, Brazil
Jason C. Young
McGill University, Canada
Brian Blagg
University of Notre Dame, United States
ORIGINAL RESEARCH article
Front. Mol. Biosci., 07 June 2018
Sec. Protein Folding, Misfolding and Degradation
Volume 5 - 2018 | https://doi.org/10.3389/fmolb.2018.00051
Dihydropyridines Allosterically Modulate Hsp90 Providing a Novel Mechanism for Heat Shock Protein Co-induction and Neuroprotection
Mark S. Roe1, Ben Wahab2, Zsolt Török3, Ibolya Horváth3, László Vigh3 and Chrisostomos Prodromou1*†
1Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
2Sussex Drug Discovery Centre, University of Sussex, Brighton, United Kingdom
3Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences (HAS), Szeged, Hungary
Chaperones play a pivotal role in protein homeostasis, but with age their ability to clear aggregated and damaged protein from cells declines. Tau pathology is a driver of a variety of neurodegenerative disease and in Alzheimer's disease (AD) it appears to be precipitated by the formation of amyloid-β (Aβ) aggregates. Aβ-peptide appears to trigger Tau hyperphosphorylation, formation of neurofibrillary tangles and neurotoxicity. Recently, dihydropyridine derivatives were shown to upregulate the heat shock response (HSR) and provide a neuroprotective effect in an APPxPS1 AD mouse model. The HSR response was only seen in diseased cells and consequently these compounds were defined as co-inducers since they upregulate chaperones and co-chaperones only when a pathological state is present. We show for compounds tested herein, that they target predominantly the C-terminal domain of Hsp90, but show some requirement for its middle-domain, and that binding stimulates the chaperones ATPase activity. We identify the site for LA1011 binding and confirm its identification by mutagenesis. We conclude, that binding compromises Hsp90's ability to chaperone, by modulating its ATPase activity, which consequently induces the HSR in diseased cells. Collectively, this represents the mechanism by which the normalization of neurofibrillary tangles, preservation of neurons, reduced tau pathology, reduced amyloid plaque, and increased dendritic spine density in the APPxPS1 Alzheimer's mouse model is initiated. Such dihydropyridine derivatives therefore represent potential pharmaceutical candidates for the therapy of neurodegenerative disease, such as AD.
The chaperone network helps maintain protein homeostasis (proteostasis) by aiding protein folding, by clearing aggregated and damaged protein from cells and by maintaining proteins in an active state (Hartl, 1996; Kim et al., 2013; Penke et al., 2018). However, with age there is a decline in the capacity of cells to maintain proteostasis and the prevalence of neurodegenerative diseases such as Alzheimer's- (AD) and Parkinson's-disease increases (Valastyan and Lindquist, 2014; Toth et al., 2015; Hartl, 2017). Heat shock proteins have been shown to decrease age-related proteotoxicity, while simultaneously increasing the life span in organisms such as Caenorhabditis elegans (Murshid et al., 2013).
The current model of AD suggests that the overexpression of mutant forms of β-amyloid precursor protein (APP) lead to amyloid-β (Aβ) plaque and neurofibrillary tangle formation by Tau (Choi et al., 2015). It is thought that Aβ peptide triggers the hyperphosphorylation of Tau, a Hsp90 dependent process (Jinwal et al., 2011), that subsequently leads to neurofibrillary tangles and neurotoxicity (Hardy and Selkoe, 2002; Selkoe, 2002; Oddo et al., 2003; Tanzi and Bertram, 2005; Annaert and De Strooper, 2010; Karran et al., 2011; Benilova et al., 2012). Maintenance of healthy homeostasis of proteins by modulation of protein processing and folding systems by chaperone induction represents a prime focus for drug discovery with AD and neurodegenerative diseases as key therapeutic targets (Hamos et al., 1991; Alavez et al., 2011). The accumulation and aggregation of Tau is actually facilitated by Hsp90, thus increasing its toxicity (Blair et al., 2013). Hence, there has been a drive to develop Hsp90 inhibitors that promote the degradation of Tau (Dickey et al., 2007; Luo et al., 2007; Blair et al., 2014). Small molecules that interfere with the normal function of Hsp90 are known to induce the heat shock response (HSR) and increase the degradation of Hsp90 dependent client proteins such as Tau by a Hsp90-CHIP E3 ubiquitin ligase dependent pathway (Dickey et al., 2007).
The mechanism of neuroprotective action of dihydropyridine (DHP) derivatives remains elusive (Kasza et al., 2016), but it was recently reported that they could activate the HSR (Kasza et al., 2016). The HSR response was only seen in diseased cells and consequently these compounds were defined as co-inducers since they upregulate chaperones and co-chaperones only in the context of a pathological state (Kasza et al., 2016). Noteworthy, is that Hsp27 and Hsp70 levels are lower in the brain of AD patients and consequently the induction of heat shock proteins represents a potential strategy for the treatment of neurodegenerative disorders (Klettner, 2004). The induction of heat shock proteins, such as Hsp27 and Hsp72, that promote cell survival by preventing protein aggregation and promoting protein refolding and by eliciting protein degradation of aggregated proteins (Ehrnsperger et al., 1997), might represent a novel tool in the treatment of neurodegenerative disease (Söti and Csermely, 2002; Franklin et al., 2005; Morimoto, 2008; Manaenko et al., 2010; Tóth et al., 2013; Bobkova et al., 2014; Eroglu et al., 2014; Kalmar et al., 2014; Wang et al., 2014). This is all the timelier since the announcement of a variety of clinical trial failures including those for idalopirdine and intepirdine, selective 5-hydroxytryptamine-6 receptor antagonists, and solanezumab, an Aβ antibody (Atri et al., 2018; Honig et al., 2018; Simpson, 2018).
The inhibition of Hsp90 is best characterized by binding of small molecules to its N-terminal ATP binding domain, and include geldanamycin, radicicol, and synthetic molecules such as AUY922 (Roe et al., 1999; Brough et al., 2008). Less well characterized are the C-terminal Hsp90 binding compounds, including Novobiocin, Coumermycin and KU-32 (Marcu et al., 2000; Donnelly and Blagg, 2008; Kusuma et al., 2012), and recently some compounds have emerged as activators of the ATPase activity of Hsp90. Such compounds include rhamnoside that are thought to modulate the conformation of Hsp90, lowering transition states that favor N-terminal dimerization and therefore ATPase activity (Sattin et al., 2015). Rhamnoside, and its derivatives (Sattin et al., 2015), were selected initially based on the stereoelectronic properties displayed by an allosteric site in the C-terminal domain of Hsp90, which itself was identified using molecular dynamic simulations (Vettoretti et al., 2016). Rhamnoside derivatives, which have a benzofuran scaffold at their core, were later shown to accelerate the Hsp90 cycle and to stimulate the ATPase activity up to six-fold and to be cytotoxic against a variety of cancer cell lines (Sattin et al., 2015). Unlike N-terminal inhibitors, the benzofuran derivatives did not induce the HSR, but reduced the levels of Hsp90 dependent client proteins such as ErbB2, Akt, Cdk4, and Survivin (Sattin et al., 2015). In contrast to activators, a set of structurally-symmetrical chemical derivatives based on NSC145366, which contains a bisphenol A core, were shown to be allosteric inhibitors of the ATPase activity of Hsp90 (Goode et al., 2017). These derivatives compromised the chaperoning function of Hsp90, by inhibiting its ability to prevent the aggregation of citrate synthase and the refolding of luciferase. NSC145366 was particularly active in promoting androgen receptor and BRAC1 client protein degradation in the LNCaP androgen sensitive prostate adenocarcinoma cancer cell line. This inhibitor did not induce the classical HSR seen with N-terminal inhibitors. To date, and to the best of our knowledge, no experimentally determined structure of Hsp90 in complex with a C-terminal binding compound has been reported.
Here we show that DHP derivatives bind to the C-terminal- and middle-domain of Hsp90 and activate its ATPase activity, which in turn compromises the chaperone activity of Hsp90. Through docking studies and mutagenesis, we identify the C-terminal binding site of DHP derivatives. The molecular basis for the mechanism by which these DHP derivatives have a neuroprotective effect in the APPxPS1 Alzheimer's mouse model is proposed.
Protein Purification and Synthesis of Dihydropyridine Derivatives
pRSETA was used to express yeast full–length Hsp90, human full-length Hsp90α, Hsp90β and a variety of yeast fragments: [Hsp901−220 (N-terminal domain), Hsp901−560 (N and middle-domain), Hsp90273−709 (middle and C-terminal domain), Hsp90546−709 (C-terminal domain), monomeric yeast Hsp90 (Hsp90F675L, I679M), and human Hsp909−236 (N-terminal domain)]. Mutants of Hsp90 were generated by Genscript (860 Centennial Ave., Piscataway, NJ 08854, USA). Constructs were purified by Talon affinity, gel-filtration with an appropriate molecular mass exclusion, and ion-exchange chromatography as previously described (Panaretou et al., 1998). The synthesis of DHP derivatives was previously described (Kasza et al., 2016).
Isothermal Titration Calorimetry
Heat of interaction was measured on an ITC200 microcalorimeter (Microcal) under the same buffer conditions (20 mM Tris, pH 7.5, containing 1 mM EDTA, and 5 mM NaCl) at 30°C. Either 1.9 or 3.8 μL aliquots of the LA1011, LA1014, or LA1026 [provided by Gedeon Richter Ltd. (www.richter.hu)] at 1 mM were injected into 60 μM of Hsp90 protein or a variety of constructs, including mutants of Hsp90 and complexes of Hsp90 with 5 mM ADP or AMPNP, or Hsp90 complexed with 5 mM AMPPNP and 600 μM Sba1. 6 mM MgCl2 was included in the buffer in experiments containing nucleotide. Additionally, 3.8 μL aliquots of 90 μM Sti1 were injected into 6 μM Hsp90 containing 1 mM LA1011. 1 mM LA1011 was also injected into 60 μM human Hsp72. The concentration of components used in ITC experiments were based on previous experiments showing how Hsp90 interacts with nucleotide, Sba1 and Sti1 (Siligardi et al., 2004; Prodromou et al., 2009; Vaughan et al., 2009). Heats of dilution were determined by diluting injectant at 30°C into buffer. Data were fitted using a non-linear least squares curve-fitting algorithm (Microcal Origin) with three floating variables: stoichiometry, binding constant (KB = 1/KD) and change of enthalpy of interaction (ΔH°).
ATPase Assays and Gel-Filtration
ATPase assays using 2 μM Hsp90 were conducted as previously described (Prodromou et al., 1999) and displayed a turnover of 1.2 per min. Geldanamycin was prepared at 20 mM in DMSO, whereas 50 mM LA1011, LA1014, and LA1026 were soluble in 20 mM Tris pH7.5, 1 mM EDTA, and 5 mM NaCl. Estimation of the relative molecular mass of wild type Hsp90 and the monomeric yeast Hsp90F675L, I679M was conducted on a superpose 6 10/300 GL gel-filtration column equilibrated in 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, and 1 mM DDT.
Citrate Synthase Aggregation Assay
150 nM citrate synthase (2 mg/mL), was incubated at 45°C for 60 min in 1 mL of 90 mM HEPES buffer containing 20 mM KOH, 50 mM KCI, 10 mM (NH4)2SO4, 2 mM potassium acetate, pH 7.8, and Hsp90 at 0, 120, 240, 480, 600, or 1000 nM. Experiments using 0.1 or 0.2 mM LA1011 were conducted with 600 nM Hsp90. The change in optical density was monitored at 320 nm.
Refolding of Denatured Luciferase
QuantiLum Luciferase (Promega E170A) was diluted to 100 ug/mL in 25 mM Tricine pH 7.8, containing 10 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 10% glycerol, and 100 mg/mL BSA (sigma 05470) and denatured at 44°C for 30 min. 0.5 μL of denatured luciferase was then added to 20 μL of rabbit reticulocyte lysate (Promega, L415A) containing either buffer or LA1011 at 1 or 5 mM or geldanamycin at 2.5 or 5 μM and incubated at 30°C. Geldanamycin was prepared in DMSO at 0.5 and 2 mM and geldanamycin experiments contained a final DMSO concentration of 1%. Refolding of luciferase was determined by removing 2 μL aliquots of the rabbit reticulocyte mix at 0, 15, 30 45, 60, 90, and 120 min and adding this to 48 μL of Briglt-GloTM reconstituted reagent (Promega, E2610), which contains buffer, substrate and ATP required for luciferase activity, as instructed by the manufacturer. Luminescence was measured in a CLARIOstarR microplate reader using half area 96 well flat bottom plates.
Structural Modeling and Biomolecular Simulations
HSP90 crystal structures were analyzed and one, an Hsp90α structure, was selected for docking, based on the regions required for HSP90 homodimerization (PDB code 3Q6N, 3.05A) (Lee et al., 2011). The structure was prepared in Schrodinger Suite 2017-3 (Schrodinger LLC, Cambridge, MA 02142, USA) at pH 7.4 before running a site finding scan. Structure-Activity Relationship (SAR) and Site Directed Mutagenesis (SDM) data were used to narrow down the binding pocket, which was then used as a starting locus for Induced Fit Docking (Glide + Prime IFD, OPLS 3.0, XP). The Ligand (LA1011) was prepared with LigPrep at pH 7.4, and a single low energy conformer was presented for docking. The docking results were compared to experimental SAR and SDM data and the most reasonable pose was checked for stability in Molecular Dynamics (Desmond, 100 ns NPT, SPC, 300 K). Binding mode analyses were conducted on the outcomes of the molecular dynamics simulations. PyMol was used to create cartoon images of the structures1.
Hsp90 is the Molecular Target of Dihydropyridine Derivatives
The upregulation of Hsp27, Hsp40 and Hsp70 is a well-documented response to the perturbation of Hsp90 activity, resulting from the activation of HSF1 (Heimberger et al., 2013). We therefore used isothermal titration calorimetry (ITC) to investigate whether the water-soluble LA1011 drug candidate would bind Hsp90, thus providing a route by which such compounds are able to co-induce the HSR. We found that LA1011 bound to yeast Hsp90 (Kd = 13.5 ± 0.9 μM), human Hsp90α (Kd = 3.8 ± 0.7 μM), and human Hsp90β (Kd = 9.7 ± 0.7 μM) (Figures 1A–C). LA1011 bound Hsp90 with a small favorable enthalpic and entropic contribution in all cases (Table 1). The stoichiometry of binding appeared to be one molecule of LA1011 bound too one Hsp90 dimer, indicating that binding must occur at a symmetrically located position along the central long-axis of Hsp90, and it is noteworthy that LA1011 possesses a plane of symmetry (Figure 2). The degree of symmetrical interaction with each Hsp90 protomer is, however, not absolute since LA1014 lacks the symmetry of LA1011, but non-the-less still bound Hsp90.
Figure 1. Isothermal titration curves for the binding of LA1011 to a variety of intact chaperone constructs and gel-filtration analysis of Hsp90 monomeric mutant. (A) yeast full-length; (B) human Hsp90α full-length; (C) human Hsp90β full-length; and (D) yeast monomeric Hsp90F675L, I679M. (E) Gel-filtration profile of wild type dimeric yeast Hsp90 (blue trace, peak elution volume 13.8 ml) and the monomeric mutant Hsp90F675L, I679M (red trace, peak elution volume 14.4 ml), showing a decrease in relative molecular mass of the monomeric mutant Hsp90F675L, I679M mutant. (F) LA1011 binding to human Hsp72. Red lines in ITC figures represent heat of dilution, while the black fittings represent the corrected heat of interaction for the experiment.
Table 1. Thermodynamic terms for the binding of dihydropyridine derivatives to Hsp90 constructs.
Figure 2. Chemical structure of the synthesized 1,4-dihydropyridine derivatives.
To ascertain the stoichiometry of the reaction we investigated the binding of LA1011 to a monomeric mutant of yeast Hsp90, that contains two mutations, F675L and I679M that disrupt dimerization of the C-terminal domains, and no binding was detected (Figure 1D). We confirmed that this mutant was indeed monomeric by gel-filtration chromatography (Figure 1E). This confirmed that LA1011 only binds dimeric Hsp90 and that one molecule of LA1011 must simultaneously bind to both halves of the Hsp90 molecule, although the degree of interaction with each protomer remains unknown. Finally, we tested the ability of LA1011 to bind human Hsp72 and found that it did not interact with this chaperone, suggesting that DHP compounds specifically target the Hsp90 chaperone system (Figure 1F).
LA1011 Binds the C-Terminal Domain of Hsp90
To determine the exact Hsp90 domain responsible for the binding of LA1011 we investigated binding to a variety of Hsp90 constructs, including the N-terminal domain of yeast Hsp90 and Hsp90α, Hsp901−560 (representing the N- and middle-domain of yeast Hsp90), Hsp90273−709 (representing the middle- and C-domain of yeast Hsp90), and finally a yeast C-terminal construct, Hsp90546−709. LA1011 failed to bind the isolated N-terminal domains of yeast and human Hsp90α and a construct (Hsp901−560) representing the N- and middle-domain of yeast Hsp90 (Figures 3A–C). In contrast, LA1011 bound with the same stoichiometry and with a similar affinity to Hsp90273−709, representing the middle and C-terminal domain construct of yeast Hsp90 (Kd = 9.7 ± 0.7 μM; Figure 3D), as with that seen for the intact yeast Hsp90 (Kd = 13.5 ± 0.9 μM). The binding affinity was somewhat lower toward the yeast C-terminal domain alone (Hsp90546−709; Kd = 60.6 ± 7.9 μM) (Figure 3E). The results suggest a single binding site located predominantly in the C-terminus of Hsp90, which is sufficient for the binding of LA1011, but that binding is favored in the presence of the middle domain. Similarly, LA1014 and LA1026 (Figures 3F,G) bound yeast Hsp90273−709, but with slightly lower affinity (Kd = 20.2 ± 3.0 and 48.8 ± 4.7 μM, respectively). LA1014 and LA1026 also bound Hsp90273−709 displaying a small favorable enthalpy and entropy (Table 1).
Figure 3. Isothermal titration curves for the binding of DHPs to a variety of truncated Hsp90 constructs. Binding of LA1011 to (A) yeast Hsp90 N-terminal domain; (B) Hsp90α N-terminal domain; (C) Hsp901−560 (N and middle domain); (D) Hsp90273−709 (middle and C-terminal domain); and (E) Hsp90546−709 (C-terminal domain). (F) The binding of LA1014 and (G) LA1026 to the Hsp90273−709 (middle and C-terminal domain). The C-terminal domain appears to be the main site of interaction for all DHP compounds tested. Red lines in ITC figures represent heat of dilution, while the black fittings represent the corrected heat of interaction for the experiment.
Nucleotide Effects and Co-Chaperone Binding
We next tested the ability of LA1011 to bind intact yeast Hsp90 in the presence of ADP or AMPPNP, a non-hydrolysable analog of ATP. We found that both nucleotides compromised LA1011 binding, reducing the Kd to approximately 66.2 μM (Figures 4A,B). Interestingly, using a Hsp90-AMPNP-Sba1 complex slightly weakened the binding of LA1011 (Kd = 23.1 ± 1.9 μM; Figure 4C), relative to wild type yeast Hsp90. Finally, binding of the yeast co-chaperone Sti1 to Hsp90 was not compromised by LA1011 (Kd = 0.32 ± 0.01 μM) (Figure 4D), when compared to previous studies showing that Sti1 binds yeast Hsp90 with a similar affinity (Kd = 0.33 ± 0.03 μM) (Prodromou et al., 1999). These results indicate that both the closed (AMPPNP bound) and open (Sti1 bound) conformation of Hsp90 interacts with LA1011.
Figure 4. Effect of nucleotide on LA1011 binding and effects on or with co-chaperone interaction. Binding of LA1011 to (A) yeast Hsp90-ADP complex; (B) yeast Hsp90–AMPPNP complex, and (C) Hsp90-AMPPNP-Sba1 complex. (D) Binding of Sti1 to Hsp90-LA1011 complex. Nucleotide appears to decrease the affinity for LA1011, while LA1011 has little effect on Sti1 binding itself, based on previous studies (Prodromou et al., 1999). Sba1 weakens LA1011 binding slightly. Red lines in ITC figures represent heat of dilution, while the black fittings represent the corrected heat of interaction for the experiment.
LA1011 Is an Activator of the ATPase Activity of Hsp90
The inhibition of the ATPase activity of Hsp90 by naturally occurring antibiotics and synthetic molecules is well documented (Roe et al., 1999; Zhao et al., 2012) and is a known mechanism for the upregulation of the HSR. Previous attempts to look at the effects of DHP derivatives on the ATPase activity of Hsp90 suggested that such derivatives did not target Hsp90 (Kasza et al., 2016). However, the levels of DHP derivatives previously used were below the Kd values determined in this study. With this in mind we tested the effect of these compounds on their ability to modulate the ATPase activity of Hsp90. Control reactions with geldanamycin showed inhibition of Hsp90 ATPase activity (non-inhibited turnover 1.2 min), as expected (Figure 5A). In contrast, LA1011, LA1014, and LA1026 activated the ATPase activity of yeast Hsp90 (Figures 5B–D). These results suggest that the ATPase activity of Hsp90 can be allosterically modulated by the binding of a small molecule to the C-terminal domains of the chaperone.
Figure 5. ATPase assays showing the activation of Hsp90 by DHP derivatives. The turnover for Hsp90 was 1.2 per min. (A) control assay showing the inhibition of yeast Hsp90 by geldanamycin (Geld). Activation of Hsp90 ATPase activity by (B) LA1011; (C) LA1014, and (D) LA1026. The activated activity can be inhibited by geldanamycin showing that activation of Hsp90 is specific. Error bars represent standard deviation and assays were carried out in triplicate. Paired t-test was used for statistical difference. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.
LA1011 Compromises Chaperoning by Hsp90
It has been previously proposed that the C-terminal domain of Hsp90 is a potential interaction site for client proteins (Harris et al., 2004). Consequently, DHP derivatives have the potential to affect the ability of Hsp90 to chaperone such clients. We therefore investigated whether LA1011 would compromise Hsp90 in preventing the aggregation of citrate synthase (Figure 6). Based on the Kd of 13 μM for the binding of LA1011 to the yeast protein the fractional occupancy for the binding of LA1011 would be 88–93%, calculated as [ligand]/[ligand]+Kd, at 0.1–0.2 mM LA1011, respectively. At these concentrations, LA1011 disrupted yeast Hsp90's ability to protect citrate synthase from aggregation by around 50% at 45°C. This suggests that LA1011 might prevent access of client protein to Hsp90 or influence its ability to protect unfolding protein and so interferes with its chaperone activity.
Figure 6. Citrate synthase aggregation assays. Increasing concentrations of yeast Hsp90 prevent citrate synthase aggregation at 45°C and this activity is compromised in the presence of LA1011.
LA1011 Inhibits the Reactivation of Denatured Luciferase
To further confirm that LA1011 compromises Hsp90's ability to chaperone client protein we used luciferase refolding in rabbit reticulocyte lysates to investigate its ability to refold client protein. Based on the Kd of 66 μM for the binding of LA1011 to Hsp90 in complex with AMPPNP, the fractional occupancy for LA1011 would be 93%, calculated as [ligand]/[ligand] + Kd, at 1 mM LA1011. At this concentration, LA1011 inhibited Hsp90 by around 50% and 5 mM completely prevented refolding of luciferase confirming that LA1011 compromised the chaperoning function of Hsp90 (Figure 7A). In control assays using geldanamycin we found that 5 μM inhibited luciferase refolding by approximately 50% (Figure 7B). These results are consistent with LA1011 and geldanamycin disrupting the refolding of luciferase by either directly competing with luciferase binding to Hsp90 or through perturbation of the Hsp90 chaperone cycle.
Figure 7. Refolding of luciferase. Refolding of luciferase in rabbit reticulocyte lysate is compromised in the presence of (A) LA1011 and (B) geldanamycin. Red, control without LA1011, or geldanamycin, blue, 1 mM LA1011, or 2 μM geldanamycin and black 5 mM LA1011 or 5 μM geldanamycin. Errors bars represent standard error and the experiment was carried out in triplicate.
Structural Modeling by Docking and Molecular Dynamics Reveals the C-Terminal Binding Site
The stoichiometry for the binding of LA1011 was clearly 0.5:1 (Figure 1), representing one LA1011 molecule per Hsp90 dimer. We used docking studies to test whether this would locate LA1011 into a site upon the human Hsp90α dimer that was consistent with this stoichiometry. We found that LA1011 bound the symmetric dimer of Hsp90 asymmetrically occupying a single site within the dimer (Figure 8). This was consistent with the stoichiometric interaction with Hsp90 determined by ITC (Figure 1).
Figure 8. PyMol cartoons of LA1011 bound to Hsp90. (A) Surface representation of Hsp90α C-terminal and middle domain regions with LA1011 (yellow sticks) bound at the interface of the two Hsp90 protomers. The protomers of Hsp90 are green-magenta and cyan-gold, respectively. (B) PyMol cartoon representation showing the C-terminal and middle domain residues that were identified as potentially interacting residues with LA1011 through molecular dynamics simulations (residue numbers are for Hsp90α). LA1011, yellow sticks; cyan residues, C–terminus and middle domain of Hsp90 protomer 1 and green residues, C–terminus and middle domains of Hsp90 protomer 2. Blue dashes, potential hydrogen bonds.
During the molecular dynamic simulations, as was expected, the hydrophobic head group of the ligand buried itself into a hydrophobic ridge, over a hydrophobic cage motif, with the polar tails being more flexible around the polar side-chains, further from the interface (Figure 8). Overall, LA1011 sits in amongst loops connecting the two terminal helices that form the inherent C-terminal dimer interface of Hsp90, another loop that is formed by the C-terminal residues 601–632, much of which is disordered, and a middle domain loop formed by residues 495–499. The phenyl ring of LA1011 sits exactly at the center of the dimer between Ser 677 from each protomer (Figure 8B). This ensures that only one molecule of LA1011 can bind a single Hsp90 dimer. The trifluoromethyl group projects into a pocket lined by Leu 672, Leu 678, Pro 681, and the main chain of Phe 676. Potential hydrogen bonds are formed between the nitrogen atom of the two ethyldimethylamine groups of LA1011, with either the main-chain carbonyl of Gly 675 or the main-chain carbonyl of Leu 678. The hydroxyl of Ser 677, potentially, also hydrogen bonds the carbonyl group of the methyl methanonate. In contrast, the methyl substituent of the same methyl ethonate group projects into a hydrophobic pocket lined by the side chains of Leu 672 form each protomer, and those of Leu 678, Pro 681, and His 684.
Mutagenesis Supports the C-Terminal Binding Site Identified by Docking Studies
In order to validate the C-terminal binding site for LA1011 identified in docking studies we used mutants of Hsp90 that would interfere with the binding of LA1011. Our model did not reveal side chain interactions that if mutated would readily disrupt binding of LA1011. Consequently, we identified residues that could potentially impact on binding by providing slight steric hindrance during binding (Figure 9A). Our analysis suggested that the yeast mutation P661R (P681 in Hsp90α) would be more disruptive than E477R (E487 in Hsp90α) and S657R (S677 in Hsp90α). Using ITC, we found that the Kd for the binding of LA1011 to these mutants was 80.6 ± 19, 26.1 ± 0.9, and 28.6 ± 1.6 μM, respectively (Figures 9B–D). This is consistent with the predicted effects of these mutations based on our model and confirms that the C-terminal site identified by docking studies is the site for LA1011 binding.
Figure 9. Effect of Hsp90 mutants on LA1011 binding. (A) PyMol cartoon showing residues mutated close to the LA1011 binding site and their potential conformation within the structure. Residues mutated to arginine are shown in gold stick representation. Residue numbers in black are for Hsp90α, while those in red are for yeast Hsp90. Yellow sticks, LA1011. (B–D) ITC binding curves for LA1011 to the Hsp90 mutants S4657R, P661R, and E477R, respectively. Red lines in ITC figures represent heat of dilution, while the black fittings represent the corrected heat of interaction for the experiment.
Currently, it is thought that Aβ plaques, formed due to the aggregation of Aβ-precursor protein, trigger the hyperphosphorylation of Tau resulting in neurofibrillary tangle formation and neurotoxicity (Hardy and Selkoe, 2002; Selkoe, 2002; Oddo et al., 2003; Tanzi and Bertram, 2005; Annaert and De Strooper, 2010; Jinwal et al., 2011; Karran et al., 2011; Benilova et al., 2012; Choi et al., 2015). Tau accumulation and aggregation is facilitated by Hsp90 (Blair et al., 2013), and consequently efforts are directed toward developing Hsp90 inhibitors that promote Tau degradation (Dickey et al., 2007; Luo et al., 2007; Blair et al., 2014). The novel DHP-compounds, LA1011 and LA1044, were previously shown to act as co-inducers of Hsp70 and Hsp27 in the SH-SY5Y human neuroblastoma cell line (Kasza et al., 2016). Indeed, LA1011 and LA1044 increase heat shock protein expression in cells that are stressed or diseased. In that study the best co-inducer, LA1011, could protect neurons in the hippocampus, could decrease Tau-accumulation and was able to preserve dendritic spines in the APPxPS1 transgenic mouse model of AD.
Molecular chaperones together with the ubiquitin-proteasome system represent a means by which misfolded proteins in AD could be targeted for clearance (Sulistio and Heese, 2016) and some heat shock proteins show neuroprotective action in vivo, (Liu et al., 2009; Manaenko et al., 2010; West et al., 2012; Doyle et al., 2013; Bobkova et al., 2014; Eroglu et al., 2014; Cohen et al., 2015). While unregulated HSP induction could lead to the instability of the HSR (Lamech and Haynes, 2015), heat shock protein co-inducers, like LA1011, that specifically target diseased cells would offer a potentially very useful strategy against neurodegenerative diseases such as AD. Our investigations herein suggest that the molecular mechanism by which LA1011 acts is through the modulation of Hsp90 activity by binding predominantly to its C-terminal domains. It appears that binding to Hsp90 activates its ATPase activity and that these compounds appear to bind Hsp90 to both the open (Sti1 bound) and closed conformation of Hsp90 (Hsp90-AMPPNP). Furthermore, LA1011 binding to the closed Hsp90-AMPPNP-Sba1 complex was only slightly affected.
Our results show that the Kd for LA1011 binding to Hsp90 is in the region of 3.8–13.5 μM, which correlates with the 5–10 μM levels of LA1011 that had an effect in mouse models (Kasza et al., 2016).
However, effects on the in vitro ATPase activity of Hsp90 were much higher. 10 μM levels of LA1011 had marginal effects on the ATPase activity of Hsp90 and levels as high as 240 μM were required to stimulate the ATPase by 2-fold. The reason for this is that nucleotide (ADP and AMPPNP) influences the binding of LA1011 reducing its Kd from 13 to 66 μM for yeast Hsp90. While this appears to be a minor change, the fractional occupancy of the LA1011 binding site would require 0.5 mM LA1011 to achieve 88% occupancy in the presence of ATP, but only 100 μM in its absence. These levels of LA1011 correlated well with the levels used in the citrate synthase aggregation- and luciferase-assay experiments. However, the difference between in vivo levels (5–10 μM) that affect cells and those required (up to 1 mM LA1011) to affect the in vitro folding of luciferase remains around 100 times different. Such a difference in the levels of potency of small molecules that target Hsp90, between in vivo and in vitro experiments, has previously been reported and differences of up to 100-fold were seen (Kamal et al., 2003). It appears that in diseased cells Hsp90's sensitivity toward small molecules that target Hsp90 is much greater, but the exact reasons for this remains enigmatic. Finally, while it is easy to reconcile the effect of inhibition of Hsp90 by compounds such as geldanamycin, which halt chaperone cycle, and thus prevent protein folding. It is, however, not so obvious how small molecules that accelerate the chaperone cycle would have the same effect. Clearly the dwell time of the cycle must influence the productive folding of a client protein and, in fact, this has been previously reported (Zierer et al., 2016).
The mechanism by which the activation of the ATPase activity of Hsp90 occurs is likely to be similar to rhamnoside, which is thought to modulate the conformation of Hsp90 by lowering the transition state that favors N-terminal dimerization and therefore ATPase activity (Sattin et al., 2015). LA1011 is likely to work by a similar mechanism and changes in the chaperones rate might interfere with the mechanism by which client proteins are folded. For example, it has been seen that downregulating the ATPase activity of Hsp90, by displacing Aha1 from Hsp90 complex, can rescue misfolding of the delta F508 mutant CFTR that causes cystic fibrosis. Presumably the mutant CFTR requires longer to fold than wild type CFTR and benefits from a slower chaperone cycle or dwell time (Wang et al., 2006). A model by which proteostasis is maintained by Aha1 regulating the dwell time of Hsp90 with client has been proposed (Koulov et al., 2010). The model suggests that Aha1 activity is able to synchronize chaperone activity with the energetics of client folding by modulating the chaperones ATPase activity. Consequently, LA1011 is likely to be affecting the synchronization of Hsp90 activity with client protein folding. The degree of effect on client folding exerted by an increase in the ATPase activity of Hsp90 might, however, depend on the exact client protein in question. Some clients may be able to tolerate higher rates of Hsp90 activity before their folding is affected, while others may be more sensitive.
Using molecular dynamics simulations and a variety of mutant Hsp90 constructs, we were able to confirm the binding of LA1011 into a pocket formed by a variety of loops from the C-terminal and middle-domains of Hsp90. The mobility of such loops could provide a flexible platform for the binding of a variety of similar compounds that have been shown to bind the C-terminal domain of Hsp90 (Marcu et al., 2000; Donnelly and Blagg, 2008; Lee et al., 2011; Kusuma et al., 2012; Sattin et al., 2015; Vettoretti et al., 2016; Goode et al., 2017), but also the flexibility to provide binding in both the open and closed states of Hsp90. It is also noteworthy that the C-terminal domain of Hsp90 has been implicated as a client protein binding site (Genest et al., 2013) and client protein is known to activate the ATPase activity of Hsp90 (McLaughlin et al., 2002). Consequently, LA1011 may be mimicking the binding of a client protein. The flexibility of this site also raises the possibility that different client protein sequences might be accommodated. However, the structure of Hsp90 in complex with Tau has been determined and it doesn't appear that Tau, at least, interacts with the C-terminal domain of Hsp90 (Karagöz et al., 2014).
Ultimately, whether through competition for client protein binding or through modulation of Hsp90 activity, there is upregulation of the HSR that most likely results from the release of HSF1 from Hsp90 and or because of the higher demand for Hsp90 in diseased or cell stressed states. This, subsequently could be beneficial in AD because of increased levels of Hsp70, Hsp40, and Hsp27 (Heimberger et al., 2013; Blair et al., 2014; Pratt et al., 2015). Furthermore, the hyperphosphorylation of Tau, a known Hsp90 dependent process (Jinwal et al., 2011), may also be disrupted by LA1011, either through direct competition with Tau for binding to Hsp90 or because the Hsp90 cycle is dysregulated leading either to increased E3 ubiquitin ligase dependent degradation or reduced levels of hyperphosphorylation of Tau. However, whichever mechanism is responsible for the disruption of Tau hyperphosphorylation, this in turn translates to a decrease in the accumulation of neurofibrillary tangles and neurotoxicity (Drewes, 2004). Thus, both elevated chaperone levels, which may aid clearance of Aβ plaques, and decreased levels of hyperphosphorylated Tau could be changes in response to LA1011 that are likely to be beneficial to the prognosis of AD.
The C-terminal domain of Hsp90 has been shown to interact with nucleotides and it has been proposed that there is a C-terminal ATP binding site (Marcu et al., 2000; Garnier et al., 2002). However, structural studies have not revealed the presence of an obvious ATP binding pocket capable of hydrolyzing ATP (Ali et al., 2006). It is interesting to note that the binding of LA1011 in the presence of ADP or AMPPNP was identical suggesting that these nucleotides influence and weaken the binding of LA1011 to the same degree. The binding of another Hsp90 C-terminal inhibitor, C095-0286 was recently modeled to the yeast C-terminal binding domain (Jiang et al., 2017). This raises the possibility that the nucleotide and C-terminal LA1011 binding site are one and the same. If so, it might be that this site is an adenine nucleotide binding site acting as a sensor responding to total cellular levels of adenine nucleotide and/or it might act as a regulatory site with which either co-chaperones or client proteins are able to interact with. Further studies will be required to unravel the significance of this observation. Currently, we are trying to determine a co-crystal structure to confirm the molecular details of the LA1011 binding site.
CP, IH, and LV: conceptualization; CP, MSR, ZT, and BW: methodology; CP and BW: investigations; CP: writing—original draft; CP, IH, LV, and BW: writing—review and editing; CP: supervision.
CP and MSR collaborated with Vernalis for the discovery of N-terminal HSP90 inhibitors.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
CP was supported by the Wellcome Trust, 095605/Z11/Z. IH and LV were supported by the National Research Development and Innovation Office of Hungary (GINOP-2.3.2-15-2016-00060). The GPUs used for the Molecular Dynamics in this research were donated by the NVIDIA Corporation.
1. ^The PyMOL Molecular Graphics System, Version 2.0.
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Keywords: Alzheimer's disease, dementia, neuroprotection, heat shock proteins, dihydropyridine compounds, heat shock response, Hsp90
Citation: Roe MS, Wahab B, Török Z, Horváth I, Vigh L and Prodromou C (2018) Dihydropyridines Allosterically Modulate Hsp90 Providing a Novel Mechanism for Heat Shock Protein Co-induction and Neuroprotection. Front. Mol. Biosci. 5:51. doi: 10.3389/fmolb.2018.00051
Received: 20 February 2018; Accepted: 22 May 2018;
Published: 07 June 2018.
Carlos Henrique Ramos, Universidade Estadual de Campinas, Brazil
Jason C. Young, McGill University, Canada
Brian Blagg, University of Notre Dame, United States
Copyright © 2018 Roe, Wahab, Török, Horváth, Vigh and Prodromou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Chrisostomos Prodromou, chris.prodromou@sussex.ac.uk
†Co first author (conducting most experimentation).


text:
BARBARA LISTER-SINK, internationally acclaimed pianist and teacher, is a graduate
of Smith College and holds the coveted Prix d'Excellence from the Utrecht Conservatory, and an Ed.D. from Columbia University. A former member of the Artist Faculty of the Eastman School of Music, she has collaborated with some of the world's most distinguished performers, composers, and chamber ensembles. She has performed with the Harvard Chamber Players, the Rochester Society for Chamber Music, and at the New Hampshire, Skaneateles, Brevard and Chautauqua summer music festivals. Formerly keyboardist for the Royal Concertgebouw Orchestra of Amsterdam, she has also taught on the piano faculties of the Amsterdam Muziek Lyceum, Duke University, and the Brevard Music Center and is Director of the School of Music and the Music Graduate Program at Salem College, Winston-Salem, NC. Her piano teachers include Margaret Mueller, Clemens Sandresky, Edith Lateiner-Grosz, John Duke, Harold Rubens, and Guido Agosti.
As a teacher, Dr. Lister-Sink is acknowledged as a global leader in injury-preventive keyboard technique. Her video/DVD Freeing the Caged Bird
– Developing Well-Coordinated, Injury-Preventive Piano Technique won the distinguished 2002 Music Teachers National Association-Frances Clark Keyboard Pedagogy Award and was praised as "A monumental work!" by Vladimir Ashkenazy. A frequent writer for peer-reviewed music journals, she has served on the Editorial Committee of The American Music Teacher, is a contributing author of A Symposium for Pianists & Teachers, and is on the National Wellness Committee of the National Conference on Keyboard Pedagogy (NCKP)/Frances Clark Center for Piano Pedagogy. Dr. Lister-Sink has been featured artist or clinician in over 20 Music Teachers National Association state conventions. Her frequent national and international appearances as performer, researcher or clinician include numerous Music Teachers National Association (MTNA) and NCKP conventions; over 20 state MTNA conferences; 2010 Piano Texas/Cliburn Institute International Piano Academy; 2013 New South Wales Piano Teachers' Festival in Sydney, Australia; Performing Arts Medicine Association 2015 Annual Conference; 2015 and 2017 National Keyboard Pedagogy Conference; and the 2017 International Society for Music Education in Glasgow, Scotland.
Through her DVD, research, intensive training workshops, academic programs, and numerous appearances at universities, conservatories, and festivals, Dr. Lister-Sink has dedicated her life to helping thousands of keyboardists worldwide remove technical obstacles and enhance musicality. Her own playing is an eloquent demonstration of the marriage of technical freedom and musical artistry.

redpajama_set_name: RedPajamaCommonCrawl


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Armani Hotel Milano is a place that embodies Giorgio Armani's design and lifestyle philosophy, and promises a "home-away-from-home" experience, delivering luxurious comfort and a unique experience.
While Armani Hotel Milano means many things to many people, gorgeous getaway, gourmet heaven or health and wellness oasis, the only constant is our hospitality whose every facet is designed around you - our guest - to ensure you get the most out of your stay and your visit to Milan.
From the moment of your reservation, the Lifestyle Manager is there at every step before, throughout and even after your stay, from facilitating your arrival experience, to personalizing your journey with customized service.
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Melancholia Lars von Trier ( 2011 Den; Swe)
14th October 2011 Adrin NeatrourFilm Review
Melancholia Lars von Trier ( 2011 Den; Swe) Kirsten Dunst; Charlotte Gainsbourg
Viewed: 6 Oct 2011 Tyneside Cinema Ticket price: £7.99
What planet is Lars von Trier on?
When I tried to think what Melancholia was about, I arrived at the idea that it was about nothing. From its filmic form to its expressive content there is vacuity, a stylised vacuity, but still an emptiness that characterises this movie.
Melancholia (M) points to the metamorphosis of Lars von Trier (LvT) from film director into brand.
M has the look and feel of a photo installation. It's a film that makes its statement through stylistic expression rather than content. A film that has apocalypse as a theme but is not about dieing; it's about the look of dieing. Or perhaps just 'the look'. A film for people for whom the important thing is always to look cool. A film for the sort of people who are always an advert for themselves, dieing or being, level or bipolar.
M, in particular it's opening pretitle sequence, reminded me of the work of Bill Viola. The selling point is that hyper realistic images lock the viewer into the immanence of presence, a presence in which context and other determined social criteria are excess baggage. I think that this works because the hyper real rendition of image, in particular the face, determines that the focus of cognitive attention as an empathic imperative. As when we gaze at ourselves in the mirror our attention is transfixed to the surface. Some think that surface is all there is. That's OK.
LvT's M is a filmic installation structured on the idea of stylised juxtapositions divided up by title cards named after the two female roles: Justine: individual state of mind and set piece social ritual; Clair: cosmic disaster and family. The characteristic feature of the expressive elements is their overwhelming visual hyper presence and the abandonment of history and context. All that matters is the now and the watching of the performers go through the motions of projecting an image of the now.
The set piece reception is put through its formulaic paces: the bad behaviour of a dysfunctional family, the intimation of the corrupt business underlying the event, linked to a highly visible falling out between two of the occasion's central parties, and a less visible falling out of the newly weds. Justine's behaviour is erratic cool, alternatively accommodating and disruptive.. . of course whatever she does she continues to look drop-dead gorgeous. And the event continues to look sumptuous. Nothing really happens. Everything is cool. Perhaps this is LvT's point. With everything anchored in the hyperreal we gaze on image. The beautiful people remain the beautiful people. Dead and unchanging. Beautiful people like it this way.
What's in it for the audience ? Unless you're one of the 'cool set', once the eye ceases to be bewitched by image, there is little else to attend to. Even the handheld camera work, which initially lends a stylised cinematic life to the sequences, becomes tedious. It offers only repetitive movements, with its sound led splicing overlaying too many shots composed in the same way: a whip pan off action onto a talking head. Again and again and again.
Without context to anchor events, M's reliance on associative juxtaposition is similar in to TV advert for an anti-wrinkle cream. There's a before and after structure (in M's case inverse to the ad structure as the mood movement is from confidence to depression); and LvT's cosmic physics is as wonky and suspect as the science behind anti wrinkle products.
A key element of the second section of M, intertitled Clair, is the house where the action takes place. Characteristically it has form without history, it's is located nowhere without space time or social referents. It is (I think) the same house where the reception was held. It looks like a house in a hotel ad or a real estate brochure. A house in the middle of nowhere, occupied by people in the middle of nowhere; a situation in the middle of nowhere. And then! A planet from nowhere!
Justine's precognition of the final catastrophe is very detached and melds imperceptibly with her utterances – the earth is evil (wow!) – and behaviour which indicate she is a very cool person in particular when she and LvT indulge in a little 'Melancholic light' naked bathing. She looks so good and ravishing, really cool thing to do that!
Of course in the past where societies were attuned to a religious cosmology and demonology there was typically a collective response to catastrophe: mass flagellation, mass gatherings. On planet Earth today in the West there is no collective belief to sustain life. Only denial or mute acceptance. Apocalypse now will be experienced as a family occasion like an advert for a family holiday in Florida. To this extent LvT makes a point, but it seems a secondary afterthought to his primal concern, the image.
LvT ensconced in the Zentropa may feel like his little pretend household at the end of the movie. They seek out the sanctuary of the child from cosmic disaster in a little birch frame wigwam. Is Zentropa LvT's little magic cave? A bubble world from which he can look out and comment on the world. Insulated from the world remote from its concerns, he has little to say. It worked for him with Antichrist because of the psychic forces he set in motion were intertwined from a ruthlessly internalised re-mything of Freud. In Melancholia it is mere arrogant indulgence.
Vertigo Alfred Hitchcock (USA 1958)
6th October 2011 Adrin NeatrourFilm Review
Vertigo Alfred Hitchcock (USA 1958) James Steward; Kim Novak
Viewed: 24 09 2011 Star and Shadow Cinema Newcastle; Ticket: £5.00
retrocrit: sex and the cripple…
Vertigo's plot, its settings and locations are of course all typical macguffins. They are the elaborate devices and visual mechanics that only serve to frame Alfred Hitchcock's (AH) core obsession in this movie: the ritual of the fetish. The slow build up of the film, its ramshackle flaky story line with its cod parapsychological exploitation of the idea of possession and its ring a roses visual rotation of San Francisco tourist attractions, delivers the pay off in its finale: the sexual completion of the cripple. This final section in which Scottie rebuilds Madeleine ( interesting choice of character name; it is the Madeleine cake that energies Proust's erotic memory journey through time) is a pure erotic act of film. The audience (this male one anyway) shares the complicity of Scottie's psychic fixation, feels the tumescent race of blood fill out his limp erogenous tissues. Of course we see nothing; we don't have to. We are seeing with Scottie.
AH seems to have understood the consequences that the development of corporate man had for the psycho sexual functioning of the male. Both Rear Window and Vertigo explore aspects of this schizoid detached objectifying state.
The opening sequence offers a banal explanatory back story for Scottie, who at the starting point of the film's narrative is immediately presented as a 'crippled man' (symbolised by acrophobia) racked by guilt, inadequacy compounded by a sense of personal and career failure. It is during the opening section at Midge's apartment that AH lays out his key concerns. In a phallocentric culture, Scottie is a castrated impotent, incapable of sexual functioning and all too aware of it. The scenario presents Scottie and Midge (Barbara Del Geddes) as just 'friends'. Reading the interaction between them I think it's evident that this presentation of their relationship is an assured scripted feint, the old conjurer displaying his mastery of misdirection to distract the audience from what is actually happening. The couple are man and wife and they have problems with their sexual relationship. As a good wife Midge (small blood feeding mosquito = wife?) tries to help him. But he cannot be aroused by her; she doesn't pump his blood, she sucks his blood; she has no power over him. He is a castrate in a phallic corporate regime which dictates the rules of the game for both of them. He denies her both physical intimacy, of which coitus is a most powerful sign, and fertility.
This opening sequence has I think another necessary input besides the establishment of the cripple: it also brokers the appearance of the fetish.
The sexual functioning (phallic arousal), of corporate man has been conditioned to respond not to the stimulus of the body; but to the stimulus of the object or body part as object. Of course this starts with his own cock as object. But the process of objectification in particular pervades most aspects of sexuality where shoes, lingerie, breasts, legs, arse etc are projected as potent signs of legitimate sexual arousal. In linking the idea of sexual functioning to objects such as, cigarettes (at least until recently in the West) cars, interiors, fragrance, leisure, commercial interests through advertising and other channels, detach sexuality from the body onto the object fetish. In the first exchange between Midge and Scottie, AH effects the extraordinary appearance of a cantilevered brassiere (pronounced by Midge as brazeer) mounted on a stand. In response to Scotties puzzled question about it, Midge explains this new invention; its purpose is to aggressively push the breasts up and forward, accentuating their prominence making them into object signs of sexual potency. Of course for Hollywood in the '50's women's breasts were the most powerfully exploited object sign of the female stars, Monroe, Russell, Dors etc. (the maternal function of the breasts was not advertised). With the cantilever bra (possibly invented by Hughes) breasts become fetish, detached from the body and released into an independent existence available for development exploitation and celebration by Hollywood and the fashion industry. But Scotty is not fixated on the breast fetish; Midge is unable to arouse him with her cantilevered brazeer. Scottie the cripple needs another sort of object sign to enable him to find his erection.
Who better than AH with his intimate knowledge of himself and Hollywood to understand this? Hollywood as a parallel world where sex is welded to and defined by image. Image mediates sexual desire through body shape, hair style and colour, breasts, facial make up. Hollywood with its consistently ambivalent contradictory message of the sexual fetish: a magical thing available to the gaze yet simultaneously distant and inaccessible. As sex detaches from body and attaches to image, sexual functioning undergoes a significant re-conditioning process.
Fetish in contemporary English has dual (and sort of related) meanings. Firstly it points to an object believed to have magical power regarded with superstitious reverence; secondly a fetish is an object or body part that is psychologically necessary to achieve sexual gratification: no fetish no sexual arousal.
In Vertigo AH combines both meanings of the word. As a fetish it is her assemblage as sex goddess that causes Scottie to worship Madeleine. He is like a man wanting his wife to dress and look like Marilyn Monroe. From the first time he sees her with her peroxide blond hair, her demure beige clothing and shoes, she is a vision, a fount of magical power. Madeleine is constituted as pure image. Much of the film is composed of sequences of shots in which Scottie watches her from a distance. Even when he rescues her from the sea, there is little he can say to his object of worship, who at the same time becomes his object of desire, a fetish image without whom he cannot connect his mind to his sexual awakening.
On losing Madeleine , Scottie changes from cripple to dead man. He is as one dead, and only restored to life by his recreation of the fetish as the psycho sexual salve. It is an astonishing filmic realisation as Scottie item by item detail by detail recreates Madeleine. Watched by the complicit eye of the audience, he moves with increasing assurance towards both the image of the goddess and the arousal of his own crippled sexual functioning through the fetish of the image. The final assemblage of the image of Madeleine has an orgasmic intensity.
Interestingly AH was not happy with either of his stars' performances. Both players seem detached and uncomfortable with their roles. It may be that the implicit obsessions of AH in making Vertigo acted as an inhibitionary factor because the explicit message was so close to the film's surface that both Stewart and Novak were uncomfortable with the forbidden areas of psycho sexual functioning that lay at the heart of the film and of which they were the key expressive realisators.


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Lennox Scott, Chairman and CEO of John L Scott Real Estate,shares how "Walking in the Solution" and "Living Life as a Contribution®" has powered his company to $14.4B in annual sales.
Hard work and determination, the work ethics instilled in him by his grandfather who hired him to paint signs when he was ten, helped Lennox Scott grow the company from a small mom and pop business to 1,400 broker associates in 2007. Yet, those old-fashioned work ethics were not enough to sustain the company when put to the test by the Great Recession in 2007. When the housing market collapsed that year, Lennox was looking at the disheartening possibility of having to make huge cuts to his staff or even close his doors. Serendipitously, he then met a woman who introduced him to a progressive, innovative perspective she calls "Walking in the Solution" that ultimately turned his business and his life around. (That woman, Deanna Scott, is now his wife.) Annually, John L. Scott now engages over 3,000 broker associates in 110 offices across four states, closes more than 37,000 transactions totaling $14.4B in sales volume, and is now listed as the 12thlargest Real Estate Power Broker in the US. Most impressively, in 2017 the company's foundation helped contribute over $15M to children's hospitals and healthcare in the Pacific Northwest.


Fortnum & Mason has admitted that thousands of customers have had their personal data stolen in a cyber attack on the upmarket grocer.
The retailer said that around 23,000 of its shoppers had seen their names, email addresses and home addresses, as well as social media handles, compromised by hackers.
The breach occurred through Typeform, a survey company Fortnum & Mason used for its recent food and drink awards.


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"I'm deeply concerned about how the United States treats egg donors"
By Alison Motluk, HeyReprotech! | 08. 27. 2019
Support groups are meant to provide support. But sometimes you learn too much.
Reactions to Jail Sentences for "CRISPR Babies" Scientists, and What Comes Next
CGS Statement on Prison Term for "CRISPR Babies" Scientist He Jiankui
By CGS Staff | 12.30.2019
CGS Year In Review: The Fight for Genetic Justice in 2019
https://heyreprotech.substack.com/p/im-deeply-concerned-about-how-the
Ten years ago, if you were a woman who had donated eggs, there was a good chance you'd never met or spoken with anyone else who had done it. Sure, it felt exotic, but it could also feel isolating.
In the years after she donated eggs at a Toronto clinic in 2004, Claire Burns began to feel a need to connect with other egg donors. She contacted an infertility support group and suggested they start a chapter for egg donors. "Why would you need a support group?" she remembers being asked.
Why? Because there were a lot of thoughts and feelings to untangle, and ordinary people didn't always get it. In our first phone conversation, Burns told me she had been led to believe that she was just giving away something meaningless — like a piece of skin — but that now she was re-examining that. "It's my biological material. I am part of that family, even though I have to pretend I'm not," she said. "It's something I once thought I'd be able to dismiss. But it's haunting me."... see more
Embryo Research To Reduce Need For In Vitro Fertilization Raises Ethical Concerns
By Rob Stein, NPR | 01.15.2020
Researchers have conducted a controversial study that involved paying dozens of young women at a hospital near Puerto Vallarta, Mexico, to get artificially inseminated so their embryos could be flushed out of their bodies and analyzed for research purposes.
Should We Alter the Human Genome? Let Democracy Decide
By Krishanu Saha, J. Benjamin Hurlbut & Sheila Jasanoff , Scientific American | 01.15.2020
Senators Dianne Feinstein (D–Calif.), Marco Rubio (R–Fla.) and Jack Reed (D–R.I.) have introduced a resolution affirming the importance of heritage genome editing. The resolution is a valuable first step, and we urge the Senate to take it—and then to give this issue the democratic attention it deserves.
Rewriting Our Genes Is Easier Than Ever. That Doesn't Mean We Should Do It.
By Sarah Ruth Bates, WBUR Boston | 01.03.2020
Gene-editing technologies have huge potential to alleviate human suffering. But, like all very powerful technologies, they also carry enormous risks if used improperly.
What Happened to All Those Frozen Eggs?
By Sarah Elizabeth Richards, The New York Times | 12.21.2019
Egg freezing was supposed to be as revolutionary as birth control. It hasn't lived up to the hype — but it has still changed women's lives.


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La agresión es un ataque provocado producto de la práctica o del hábito de ser agresivo. Es una conducta hostil o destructiva cuya finalidad es provocar un daño a otro.

Dollard afirma que es cualquier conducta cuyo objetivo es causar un daño cierto a la persona a la que se dirige. Existen unas polémicas sobre conductas agresivas: Archer y Browne (1989) establecen tres características del caso prototípico de agresión:

 Intención de causar daño.
 Provocar daño real.
 La existencia de alteración del estado emocional.

Geen hace una tajante distinción entre agresión colérica y agresión instrumental. La primera tiene un estado emocional negativo de cólera como reacción a alguna provocación previa y persigue causar daño que, mediante la agresión, pretende anular a la persona.

Clases de agresiones
Dentro de las agresiones se pueden distinguir dos variantes.

Agresión verbal o psicológica
Este tipo de agresión se da cuando una persona es agredida mediante malos tratos o insultos de cualquier índole y los cuales pueden llegar a causar lesiones de índice psicológico.

Agresión física
La agresión física es producida por la acción de golpes de manera directa o mediante cualquier medio físico que causen lesiones directamente a la otra persona o animal.

Etología (ciencias naturales)

Los etólogos estudian la agresión en lo que respecta a la interacción y evolución de los animales en ambientes naturales. En dicho entorno la agresión puede comprender el contacto corporal mediante mordidas, golpes o empujones, pero la mayoría de los conflictos son resueltos mediante las amenazas y avances intimidatorios que no producen daño físico. Esta forma de agresión puede incluir el exponer el tamaño del cuerpo, cornamentas, garras o dientes; señales estereotipadas incluidas expresiones faciales; vocalizaciones tales como el canto de un ave; la liberación de químicos; y cambios en la coloración. La expresión comportamiento agonístico a veces es utilizada para referirse a estas formas de conducta.

La mayoría de los etólogos creen que la agresión provee ventajas biológicas. La agresión puede ayudar a que un animal controle un  territorio, incluidos recursos tales como alimentos y agua. La agresión entre machos a menudo tiene lugar para asegurar oportunidades de apareamiento, y resulta en la selección natural del animal más sano o más vigoroso. La agresión también puede producirse para autoprotegerse o para proteger a la cría. La agresión entre grupos de animales también puede resultar en ventajas; por ejemplo, un comportamiento hostil puede forzar a una población de animales a penetrar en un territorio nuevo, donde la necesidad de adaptarse a un nuevo ambiente puede dar lugar a una mayor flexibilidad genética.

Sociedad y cultura
Los seres humanos comparten aspectos relacionados con la agresión con los animales no humanos, y tienen aspectos específicos y complejos relacionados con factores asociados a la genética, etapas tempranas del desarrollo, aprendizaje social y flexibilidad, cultura y moral.
Konrad Lorenz expresó en su obra Sobre la agresión, (1963) que el comportamiento del ser humano se encuentra modelado por cuatro imperativos animales de lucha por la subsistencia o supervivencia. Considerados en forma conjunta, estos factores impulsores: hambre, miedo, reproducción y agresión, resultan en la selección natural. E. O. Wilson precisó en su obra Sobre la naturaleza humana que la agresión es un medio para tomar control sobre recursos. Por lo cual la agresión, se encuentra agravada en épocas que densidades de población elevadas devienen en escasez de recursos. Según Richard Leakey, la agresión en los humanos ha ido en aumento al acentuarse el principio de propiedad y su defensa. Sin embargo la UNESCO adoptó el Manifiesto de Sevilla sobre la Violencia en 1989 que refuta los pronunciamientos de los científicos evolucionistas, en el sentido que en la información genética se encuentra la única causa de la agresión.

Los aspectos sociales y culturales pueden interferir de manera importante con las diferentes expresiones de la agresividad. por ejemplo, una densidad de población elevada, cuando acontece junto con una disminución de los recursos disponibles, puede ser una variable significativa en el desencadenamiento de actos violentos.

Género

El género es un factor que influye tanto en la agresión humana como en la animal. Históricamente se cree que los varones suelen ser más agresivos físicamente que las mujeres desde una edad temprana, y los hombres cometen la gran mayoría de los asesinatos  Esta es una de las diferencias de comportamiento por sexo más sólidas y fiables, y se ha encontrado en muchos grupos de edad y culturas diferentes. Sin embargo, algunos estudios empíricos han encontrado que la discrepancia en la agresión masculina y femenina es más pronunciada en la infancia y que la diferencia de género en los adultos es modesta cuando se estudia en un contexto experimental.  Aun así, hay pruebas de que los hombres son más rápidos en la agresión (Frey et al. 2003) y más propensos que las mujeres a expresar su agresión físicamente. Cuando se consideran las formas indirectas de agresión no violenta, como la agresión relacional y el rechazo social, algunos científicos sostienen que las mujeres pueden ser bastante agresivas, aunque la agresión femenina rara vez se expresa físicamente. Una excepción es la violencia de pareja que ocurre entre las parejas que están comprometidas, casadas o en alguna otra forma de relación íntima.

Aunque es menos probable que las mujeres inicien la violencia física que los hombres, pueden expresar la agresión utilizando una variedad de medios no físicos. El método exacto que utilizan las mujeres para expresar la agresión es algo que varía de una cultura a otra. En la isla de Bellona, una cultura basada en la dominación masculina y la violencia física, las mujeres tienden a entrar en conflicto con otras mujeres con más frecuencia que con los hombres. Cuando entran en conflicto con los hombres, en lugar de utilizar medios físicos, inventan canciones que se burlan del hombre, que se difunden por toda la isla y lo humillan. Si una mujer quiere matar a un hombre, convence a sus parientes masculinos para que lo maten o contrata a un asesino. Aunque estos dos métodos implican violencia física, ambos son formas de agresión indirecta, ya que la propia agresora evita implicarse directamente o ponerse en peligro físico inmediato.

Véanse también los apartados sobre la testosterona y consideraciones sobre las explicaciones evolutivas de las diferencias de género.

Véase también
Agresor sexual

Referencias

Enlaces externos

 When Family Life Hurts: Family experience of aggression in children – Parentline plus, 31 de octubre de 2010
 Aggression and Violent Behavior, a Review Journal
 International Society for Research on Aggression (ISRA)
  por Johan van der Dennen, originalmente publicado en 1980
 Agresión y asimetría cerebral

Etología
Violencia
Psicología social
Problemas de comportamiento

redpajama_set_name: RedPajamaWikipedia


'Natural History Redux' from Coral Morphologic on Vimeo.
23 natural history films by Coral Morphologic, remixed/ remastered and compiled into a half-hour odyssey of the sea.
Original films created 2009-2011 Soundtrack by Coral Morphologic & Geologist ('Man O War') Additional editing by Lucas Leyva Additional sound design by Michael-John Hancock & Jorge Graupera Post production support by Borscht Corp. Produced by Coral Morphologic & Borscht Corp.


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Good fortune, dire poverty, and inequality in Baltimore: An American story
Alan Berube and Brad McDearman Monday, May 11, 2015
The unrest in Baltimore has fostered nationwide discussion about the root causes of the tensions in the city's poor neighborhoods that led to an outbreak of riots and mass protests. While criminal justice policy and police-community relationships are arguably at the core of the present debate, the economic and social context in which those actions took shape matters greatly too. Yet media coverage has obscured a few key facts about economic and social conditions in Baltimore and other major American cities. The charts below situate the distress affecting neighborhoods like Freddie Gray's Sandtown-Winchester against the backdrop of wider dynamics in Baltimore City and its metropolitan area, and in comparison to other cities and regions around the country.
Baltimore is no longer a 'Rust Belt' region
Homeownership, racial segregation, and policy solutions to racial wealth equity
Rashawn Ray, Andre M. Perry, David Harshbarger, Samantha Elizondo, and Alexandra Gibbons
Life in Baltimore, from the people who call it home: A video
Jennifer S. Vey
A modern case for regional collaboration
Amy Liu and Nathan Arnosti
Far from a struggling post-industrial region primarily characterized by stagnant or falling incomes, the Baltimore metro area today is relatively affluent. Per capita incomes in the Baltimore region rose faster than in any other major metro area from 2000 to 2013. Regionwide, African American households share in that prosperity. And the city of Baltimore itself boasts large numbers of high-paying jobs in industries such as education, health care, and professional services, particularly in its downtown core along the Inner Harbor and adjoining neighborhoods.
Yet the city's prosperity is not shared across neighborhood and racial lines
Redefining Urban and Suburban America
Edited by Alan Berube, Bruce Katz, and Robert E. Lang
The New Localism
By Bruce Katz and Jeremy Nowak
Edited by Bruce Katz and Robert E. Lang
Alan Berube
Interim Vice President and Director - Brookings Metro
Twitter berubea1
Brad McDearman
Nonresident Senior Fellow - Brookings Metro
Within the city of Baltimore, deep divisions exist by race and place. Neighborhoods just to the west and east of downtown Baltimore, including Sandtown-Winchester and extending out into suburban Baltimore County, exhibit very high rates of poverty. Those neighborhoods are predominantly black, reflecting a long history of explicit and implicit policies in the region that yielded high levels of racial and economic segregation. This racial isolation and poverty concentration help account for stark differences between Baltimore's black and white populations in key economic outcomes like education, employment, and child poverty.
These same issues affect many other major U.S. cities
In these respects, Baltimore is a typical American city. Its level of concentrated poverty is average among cities in major metropolitan areas. While Baltimore has a higher overall poverty rate than most other cities, its level of income inequality mirrors that for U.S. cities generally. And Baltimore's depressing outcomes for black residents on poverty and employment are actually better than those in a majority of other cities with large black populations.
In sum, Baltimore is a region on the upswing economically, but one in which stability and prosperity are distributed highly unequally across racial and community lines. That is more or less the norm in metropolitan America today, and a stark challenge to economic growth patterns that have left too many behind for too long.
Income Inequality & Social Mobility
U.S. Metro Areas
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Home » 2016-17 Season » Oboes on the Ohio, Dwight Parry, Oboe
Oboes on the Ohio, Dwight Parry, Oboe
November 5, 2016 at 7:30 p.m.
Dwight Parry is just rounding out his first decade as the principal oboist of the Cincinnati Symphony Orchestra. Previously, he held the same position with the San Diego Symphony and was a fellow with the New World Symphony. He has performed as guest principal oboist with groups including the New York Philharmonic, the Chicago Symphony, the San Francisco Symphony, the Atlanta Symphony, the Los Angeles Opera, and the Deutsche Symphonie of Berlin. Originally from coastal Southern California, he found his passion for music studying piano, voice, and jazz saxophone. It was not until late in high school, however, that he began playing the oboe and truly found his calling. He studied at the Cleveland Institute of Music with John Mack and at the University of Southern California with Allan Vogel and David Weiss, a gentleman who also taught him to surf.
Mr. Parry is sought after for concertos, recitals, and chamber music around the country and abroad. Past appearances have featured the works of Mozart, Goossens, Haydn, Bach, Strauss, Vivaldi, Albinoni, Barber, and Marcello. He was recently the featured soloist in Mexico City for Vaughn-Williams' wonderful concerto for oboe. In July 2016, he will be the soloist for the Strauss Oboe Concerto at the Shippensburg Music Festival. Mr. Parry will again perform the Strauss Oboe Concerto in February 2017 for his third subscription solo appearance with his own orchestra, the Cincinnati Symphony. Mr. Parry is an Adjunct Faculty member at both the Cincinnati College-Conservatory of Music and at Northern Kentucky University. He also teaches privately and gives masterclasses at schools and festivals throughout the year.
Sérénade Françaix
Oblivion Piazzolla
L'Horloge de Flore Françaix
Vocalise Rachmaninoff
Ma Mere l'Oye Ravel
Libertango Piazzolla


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By C. Sky
Sally stared up at the ceiling, arms flung wide across her bed as she contemplated her folly. She had met a nice man named Joe and they had hit it off but in her excitement to impress, she had promised to cook dinner for the pair. There was only one problem. Sally couldn't cook. Not even a little. And the reality only set in after promises where made and assurances where given.
What now? She thought. What now?
It came to her on her morning commute. An ad played on her favorite podcast that she would normally skip over but this time it held her attention.
"Try Blue Apron," the host explained in a chipper voice. "You receive fresh ingredients that are perfectly portioned to the recipe's needs."
Sally scratched her chin as she contemplated the service.
"Anyone can cook with Blue Apron!"
Anyone? Sally wondered. She planned to put that declaration to the test.
Sally received the box in time for the big night. She took a deep breath and went to work. Shrimp and fresh gnocchi was on the menu and she was determined to not screw it up. The cooking instructions came with pictures and she followed each step diligently, scrutinizing her work against the pictures.
After 30 minutes, she plated the dish. Before her was a mound of shrimp and gnocchi with bright yellow cherry tomatoes garnished with basil. Her mouth watered at the aroma and the meal matched the pictures. She nodded her head approvingly and went to clean up.
Sally watched him, probably a bit more intently than she should have, as he took the first bite. When he finished chewing, Sally realized she was holding her breath.
"Wow," Joe commented. Sally's eyes widened. "This is really good."
"Really?" Sally replied hopefully.
Joe smiled. "Yeah, this must have taken a lot of work."
Sally straightened her posture. "It was no problem at all, I'm glad you like it."
"I'm going to have to return the favor," Joe winked. "I'll cook something for you."
Sally blushed and she smiled brightly. Her mission was a success.
Joe stared at the ceiling with existential terror. Why had he promised he would cook something? Sally's food was so good, there was no way he could top it but that didn't stop him from running his mouth. Now, he had to deliver.
He opened his phone and looked through his contacts. He picked his best friend and called. He explained the situation, his friend mostly listening.
"So man, any ideas?" Joe said, finishing his tale of woe. Joe listened to his friend's reply and received a promising suggestion… Blue Apron!
Try Blue Apron for yourself by clicking on the FREE offer below:
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C. Sky
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