Celastrol Analogues as Inducers of the Heat Shock Response. Design and Synthesis of Affinity Probes for the Identification of Protein Targets

The natural product celastrol (<b>1</b>) possesses numerous beneficial therapeutic properties and affects numerous cellular pathways. The mechanism of action and cellular target(s) of celastrol, however, remain unresolved. While a number of studies have proposed that the activity of celastrol is mediated through reaction with cysteine residues, these observations have been based on studies with specific proteins or by <i>in vitro</i> analysis of a small fraction of the proteome. In this study, we have investigated the spatial and structural requirements of celastrol for the design of suitable affinity probes to identify cellular binding partners of celastrol. Although celastrol has several potential sites for modification, some of these were not synthetically amenable or yielded unstable analogues. Conversion of the carboxylic acid functionality to amides and long-chain analogues, however, yielded bioactive compounds that induced the heat shock response (HSR) and antioxidant response and inhibited Hsp90 activity. This led to the synthesis of biotinylated celastrols (<b>23</b> and <b>24</b>) that were used as affinity reagents in extracts of human Panc-1 cells to identify Annexin II, eEF1A, and β-tubulin as potential targets of celastrol

Neuronal Reprograming of Protein Homeostasis by Calcium-Dependent Regulation of the Heat Shock Response

<div><p>Protein quality control requires constant surveillance to prevent misfolding, aggregation, and loss of cellular function. There is increasing evidence in metazoans that communication between cells has an important role to ensure organismal health and to prevent stressed cells and tissues from compromising lifespan. Here, we show in <i>C. elegans</i> that a moderate increase in physiological cholinergic signaling at the neuromuscular junction (NMJ) induces the calcium (Ca²⁺)-dependent activation of HSF-1 in post-synaptic muscle cells, resulting in suppression of protein misfolding. This protective effect on muscle cell protein homeostasis was identified in an unbiased genome-wide screening for modifiers of protein aggregation, and is triggered by downregulation of <i>gei-11</i>, a Myb-family factor and proposed regulator of the L-type acetylcholine receptor (AChR). This, in-turn, activates the voltage-gated Ca²⁺ channel, EGL-19, and the sarcoplasmic reticulum ryanodine receptor in response to acetylcholine signaling. The release of calcium into the cytoplasm of muscle cells activates Ca²⁺-dependent kinases and induces HSF-1-dependent expression of cytoplasmic chaperones, which suppress misfolding of metastable proteins and stabilize the folding environment of muscle cells. This demonstrates that the heat shock response (HSR) can be activated in muscle cells by neuronal signaling across the NMJ to protect proteome health.</p></div

Modulation of AChR and GABA_R can restore post-synaptic folding.

<p>(A) At the <i>C. elegans</i> NMJ, the functional balance between GABA_R and AChR signaling regulates post-synaptic muscle function. (B) L-AChR activation with the agonist levamisole (in water) suppressed Q35 aggregation at 5 µM, but enhanced aggregation at 50 µM. Mutant AChR <i>unc-38(e264)</i> is a control for AChR-mediated effect. (C) Reduction in GABA_R function with lindane (in 10% EtOH) suppressed Q35 aggregation at 25 µM, and enhanced aggregation at 1 mM concentration (relative to EtOH control treatment). (D) Effect on Q35 aggregation by decrease in GABA with <i>unc-49</i> or <i>unc-47</i> RNAi, and by inhibition of GABA in <i>unc-47(gk192)</i> or <i>unc-30(e191)</i> mutant backgrounds. (E) Incubation with 50–200 mM GABA (in water) suppressed Q35 aggregation. GABA at 50 mM abolished the suppressor effect of <i>gei-11</i>, by “re-balancing” the GABAergic-cholinergic signaling. (F) Real-time qPCR analysis of <i>hsp-70</i> (<i>C12C8.1, F44E5.4</i>) levels in 5 day old wt animals upon treatment with ACh, levamisole or the GABA_R antagonist Lindane, or upon decrease in GABAergic signaling by either RNAi or mutant backgrounds of <i>unc-47(gk192)</i>, <i>unc-49(e407)</i> or <i>unc-30(e191)</i>. Student t-test **<i>p</i><0.01 and ***<i>p</i><0.001; data and statistics are relative to Q35;vector control (±SD) (RNAi controls: <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003711#pgen.1003711.s008" target="_blank">Table S1</a>).</p

<i>gei-11</i> knockdown effect through regulation of cholinergic receptors at the NMJ.

<p>(A) Real-time qPCR analysis of AChR subunits <i>unc-29</i>, <i>unc-38</i>, <i>unc-63</i>, <i>lev-1</i> and <i>acr-16</i>, and GABA_R<i>unc-49</i>, in 6 day old wt animals fed with <i>gei-11</i> RNAi. Data are normalized to the levels of each gene on vector-treated wt animals (±SD). (B) Suppression of Q35 aggregation by <i>gei-11</i> RNAi was abolished by co-treatment with L-AChR (<i>unc-38, unc-63, unc-29</i>) but not with N-AChR (<i>acr-16</i>) subunits RNAi (±SD). Individual RNAi controls are shown in light grey (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003711#pgen.1003711.s008" target="_blank">Table S1</a>). (C) Cholinergic sensitivity assay: 5 day old animals treated with <i>gei-11</i> or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). L-AChR mutant animals <i>unc-38(e264)</i>, <i>unc-63(x26)</i> and <i>unc-29(e1072)</i> were used as controls. Two-way ANOVA and Bonferroni test ***<i>p</i><0.001 relative to vector control. (D) AChR antagonist dTBC (2.5 mM in water) prevented suppression of Q35 aggregation by <i>gei-11</i> RNAi (±SD). Q35;<i>unc-38(e264)</i> is a control for AChR-dependent effect. Student t-test ***<i>p</i><0.001. (E) Real-time qPCR analysis of AChR subunits <i>unc-29</i>, <i>unc-38</i> and <i>unc-63</i> upon muscle-specific <i>gei-11</i> RNAi (<i>rde-1(ne219);m</i>RDE-1, 6 days old), relative to vector control (±SD). (F) Cholinergic sensitivity assay: 5 day old wt, <i>rde-1(ne219);m</i>RDE-1 and <i>rde-1(ne219)</i> animals treated with <i>gei-11</i> or vector RNAi were scored for paralysis on 1 mM levamisole plates (±SD). Two-way ANOVA and Bonferroni test ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 relative to vector control. (G) Aggregation quantification upon <i>gei-11</i> RNAi in Q35, Q35;<i>rde-1(ne219);m</i>RDE-1 (muscle-specific RNAi) and Q35;<i>rde-1(ne219)</i> (impaired RNAi); shown as a relative % to Q35;vector (±SD). Student t-test ***<i>p</i><0.001, ns/not significant.</p

Knockdown of <i>gei-11</i> suppresses polyQ aggregation and toxicity.

<p>(A) <i>gei-11</i> RNAi suppressed Q35 aggregation in BWM cells of 6 day old animals, shown by the diffuse fluorescent pattern in II, IV and VI, in contrast to a foci-like pattern in the vector control I, III, V. Scale bar: 0.1 mm (I–IV), 0.025 mm (V–VI). Boxed areas correspond to the magnified images below. (B) FRAP analysis shows relative fluorescence intensity recovery at each time-point post-photobleaching. Control Q35 foci (in black; vector) revealed no fluorescence recovery, while <i>gei-11</i>-treated animals showed complete recovery of fluorescence (in blue), analogous to the soluble Q24 control (in red). Each curve represents an average of >12 independent measurements for <i>gei-11</i> RNAi, and >5 for the controls. (C) Motility assay for 6 day old Q35 and wt animals fed with vector, <i>gei-11</i> or <i>yfp</i> RNAi, measured in body-length-per-second and relative to wt speed in vector control (100%) (±SEM, Student t-test **<i>p</i><0.01, ***<i>p</i><0.001).</p

EGL-19- and RYR-mediated Ca²⁺ influx are components of the proteostasis rescue mechanism.

<p>Ca²⁺ relevance for <i>gei-11</i> effect on (A) Q35 aggregation (B) and <i>hsp-70</i> (<i>C12C8.1, F44E5.4</i>) upregulation, tested by employing a hypomorphic mutant <i>egl-19(n582)</i>, a weak hypermorph <i>egl-19(n582ad952)</i>, a hypermorph <i>egl-19(ad695), egl-19</i> RNAi (control RNAi in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003711#pgen.1003711.s008" target="_blank">Table S1</a>) or the specific EGL-19 antagonist Nemadipine A (0.75 µM, DMSO). Student t-test *<i>p</i><0.05, ***<i>p</i><0.001, ns/not significant; data relative to vector control or control in DMSO (±SD). (C) The RYR agonists ryanodine (50 nM, EtOH) and 4-CmC (10 µM, water) suppressed Q35 aggregation in a similar way to <i>gei-11</i> RNAi, but were less efficient in Q35<i>;egl-19(n582)</i> hypomorphic mutant animals. Treatment with the RYR antagonist DS (DMSO) together with <i>gei-11</i> RNAi prevented suppression of Q35 aggregation. Student t-test ***<i>p</i><0.001, ns/not significant; data relative to vector control in respective compound % solvent (±SD). (D) Real-time qPCR analysis of <i>hsp-70</i> (<i>C12C8.1, F44E5.4</i>) levels: RYR agonists Ryr (50 nM) and 4-CmC (10 µM) up-regulated <i>hsp-70</i> in wt animals but not in mutant <i>egl-19(n582</i>) animals. Chaperone induction by <i>gei-11</i> RNAi was prevented in the RYR mutant (<i>unc-68(kh30)</i>) and by co-treatment with DS (±SD). <i>gei-11</i> levels were 0.27±0.150 upon RNAi, relative to vector sample. (E) Model for <i>gei-11</i> modulation of proteostasis in BWM. [a] Knockdown of <i>gei-11</i> by RNAi leads to an increase in L-AChR expression at the NMJ (dashed line: proposed genetic interaction). [b] This causes a shift in the cholinergic/GABAergic signaling at the NMJ towards higher (thick arrow) excitatory signaling into the muscle. ACh binding to AChRs activates the VGCC EGL-19. [c] Depolarization, conformational changes and Ca²⁺ influx through EGL-19 triggers the opening of RYR at the SR and further release of Ca²⁺ into the cytosol [d]. Ca²⁺ activates signaling cascades to promote muscle contraction [e], HSF-1 activation [f ] and expression of cytosolic chaperones that rescue protein folding in the cytosol [g]. Dashed lines represent proposed and simplified sequence of events.</p

Ca²⁺-dependent kinases required for activation of the HSR and folding enhancement.

<p>(A) Cholinergic signaling at the NMJ activates muscle EGL-19 and Ca²⁺ flux into the cytoplasm of muscle cells, which further activates the ryanodine receptor (RYR) at the SR for muscle contraction. (B) Double knockdown of <i>gei-11</i> with calmodulin <i>cal-1</i>, <i>cal-2</i>, or <i>cal-4</i>; or Ca²⁺-dependent kinase <i>unc-43</i>, <i>pkc-1</i>, <i>pkc-3</i>, or <i>gsk-3</i>, prevented suppression of Q35 aggregation (±SD). % of foci are relative to Q35 in vector RNAi; Student t-test <i>p</i><0.001. (C) Real-time qPCR analysis of <i>hsp-70</i> levels in wt animals upon double RNAi of <i>gei-11</i> with the indicated genes (±SD). Data are relative to vector-treated wt animals. <i>gei-11</i> levels were 0.23±0.101 upon RNAi, relative to vector sample.</p

Spreading of a Prion Domain from Cell-to-Cell by Vesicular Transport in <i>Caenorhabditis elegans</i>

<div><p>Prion proteins can adopt self-propagating alternative conformations that account for the infectious nature of transmissible spongiform encephalopathies (TSEs) and the epigenetic inheritance of certain traits in yeast. Recent evidence suggests a similar propagation of misfolded proteins in the spreading of pathology of neurodegenerative diseases including Alzheimer's or Parkinson's disease. Currently there is only a limited number of animal model systems available to study the mechanisms that underlie the cell-to-cell transmission of aggregation-prone proteins. Here, we have established a new metazoan model in <i>Caenorhabditis elegans</i> expressing the prion domain NM of the cytosolic yeast prion protein Sup35, in which aggregation and toxicity are dependent upon the length of oligopeptide repeats in the glutamine/asparagine (Q/N)-rich N-terminus. NM forms multiple classes of highly toxic aggregate species and co-localizes to autophagy-related vesicles that transport the prion domain from the site of expression to adjacent tissues. This is associated with a profound cell autonomous and cell non-autonomous disruption of mitochondrial integrity, embryonic and larval arrest, developmental delay, widespread tissue defects, and loss of organismal proteostasis. Our results reveal that the Sup35 prion domain exhibits prion-like properties when expressed in the multicellular organism <i>C. elegans</i> and adapts to different requirements for propagation that involve the autophagy-lysosome pathway to transmit cytosolic aggregation-prone proteins between tissues.</p> </div

Positive Regulators of the HSR.

<p>Positive Regulators of the HSR.</p

The prion domain forms biophysically and morphologically distinct aggregate types.

<p>(A) FRAP analysis of the indicated transgenic animals revealed mobile and immobile aggregate types that were grouped into two categories (see text). Aggregate mobility in animals expressing NMm::YFP and R2E2m::YFP correlated with a certain aggregate morphology. Roman numbers next to the FRAP graphs refer to representative foci (or diffuse staining pattern in case of RΔ2-5) that are shown to the right of each graph. Arrows indicate bleached ROI. The YFP only control is shown in yellow. RFI = relative fluorescence intensity in [%]. (B, C) Collapsed confocal z-stack images of R2E2m::YFP expressing transgenic <i>C. elegans</i>. (B) Aggregate shapes differed between muscle cells within one animal. (C) Aggregate types differed between the same cells of different animals. Arrows highlight fibril-like aggregates. Scale bars: 10 µm.</p