1. Volume 24, Issue 5, Pages 614-623.e6 (May 2017)
Mitochondrial Chaperonin HSP60 Is the Apoptosis-Related Target for Myrtucommulone 
Katja Wiechmann, Hans Müller, Stefanie König, Natalie Wielsch, Aleš Svatoš, Johann Jauch, Oliver Werz 
Cell Chemical Biology 
Volume 24, Issue 5, Pages e6 (May 2017)
DOI: /j.chembiol
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2. Cell Chemical Biology 2017 24, 614-623. e6DOI: (10. 1016/j. chembiol
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3. Figure 1
Myrtucommulone Induces Cytochrome c Release in Isolated Mitochondria
Mitochondria were incubated with compounds or vehicle (0.3% DMSO) for 1 hr at 37°C. Pellet (A) and supernatant (B) fractions were analyzed for cytochrome c (Cyt c) by western blot and quantified by densitometric analysis. Means ± SEM; n = 4; **p < 0.01, ***p < versus vehicle control, ANOVA plus Bonferroni. Statistics applied to logarithmized data.
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4. Figure 2 Myrtucommulone Derivatives and the Synthesis Thereof
(A) MC and derivatives as well as their corresponding pentacyclic structures used for cellular and biochemical studies. Immobilized derivatives were coupled to EAH Sepharose 4B and used for target fishing. Gray sphere represents Sepharose beads.
(B and C) Synthesis of immobilized MC (9). Reagents and conditions: (a) EtOH, H2SO4 cat., cyclohexane, reflux, 96 hr, 58%; (b) SOCl2, CHCl3, reflux, 14 hr, quant.; (c) AlCl3, CH2Cl2, CH3NO2, reflux, 10 min, 58%; (d) NaOH, H2O, isoPrOH, reflux, 3 hr, 50%; (e) CH2Cl2, isobutyl aldehyde, piperidine, room temperature, 5 min, transfer to step (f) without further purification; (f) HCl, NH4Cl, room temperature, 15 min, transfer to step (g) without further purification; (g) NaH, THF, room temperature, 2 hr, 80%; (h) EAH Sepharose 4B, EDCI, dioxane/H2O (1:1, v/v), room temperature, 96 hr, 90%–100%.
See also Figure S1.
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5. Figure 3
Impact of MC and Derivatives on Cell Viability of HL-60 Cells and Cytochrome c Release from Isolated Mitochondria
(A) HL-60 cells were treated with test compounds or vehicle (0.3% DMSO) for 24 hr at 37°C, 5% CO2. MTT was added and incubation was continued until blue staining of the vehicle-treated control. Afterward, cell lysis absorbance was measured at 570 nm. Staurosporine served as positive control leading to complete cell death (1.5% ± 0.8% cell viability, data not shown). Means ± SEM; n = 3. +p < 0.05, +++p < 0.001, ###p <
(B) Mitochondria were incubated with test compounds or vehicle (0.3% DMSO) for 1 hr at 37°C, and supernatant (upper panel) and pellet (lower panel) fractions were analyzed by western blot (representative blots out of two to three experiments are shown) and quantified by densitometric analysis. means ± SEM; n = 2–3. *p < 0.05, **p < 0.01, ***p < versus vehicle control, ANOVA plus Bonferroni. Statistics applied to logarithmized data.
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6. Figure 4 Target Identification Using Immobilized MC Derivatives
(A) Mitochondrial lysates derived from HL-60 cells were incubated with the fishing constructs (see Figure 2A) overnight at 4°C. After washing, bound proteins were eluted by SDS lysis (5 min, 95°C) and analyzed by SDS-PAGE followed by Coomassie staining. Protein bands of interest were cut out, digested, and analyzed by nanoLC-MS/MS. The Coomassie-stained gel shown is representative of at least three independent experiments; proteins (bands framed by dashed rectangles) were quantified by densitometric analysis. Means ± SEM; n = 3; ***p < versus MC-penta-immob., ANOVA plus Bonferroni. Statistics applied to logarithmized data.
(B) Fishing samples (see A) were analyzed by western blot for HSP60 and quantified by densitometric analysis; result shown is representative of three independent experiments. Means ± SEM; n = 3; ***p < versus MC-penta-immob., ANOVA plus Bonferroni. Statistics applied to logarithmized data.
(C) Beads were blocked with milk powder and then incubated with human recombinant HSP60 overnight at 4°C. Beads were washed and proteins eluted by SDS lysis (5 min, 95°C). Protein samples were analyzed by western blot; milk powder served as loading control. Densitometric analysis; result shown is representative of three independent experiments. Means ± SEM; n = 3; **p < 0.01 versus MC-penta-immob., ANOVA plus Bonferroni.
See also Table S1.
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7. Figure 5 Myrtucommulone Inhibits HSP60 Chaperone Activity
HSP60/HSP10 were preincubated with compounds or vehicle (1% DMSO) for 15 hr at 4°C. MDH was denatured with 10 mM HCl for 2 hr at 25°C. MDH refolding was initiated by 2 mM ATP for 30 min at 27°C. The reaction was terminated by addition of glucose/hexokinase, NADH and oxaloacetate were added, and kinetics was monitored at 360 nm at 30°C. (A) MC inhibits HSP60 chaperone activity. (B) Effect of MC on native MDH. Means ± SEM; n = 3; *p < 0.05, **p < 0.01, ***p < versus vehicle control, ANOVA plus Bonferroni.
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8. Figure 6 Myrtucommulone Induces Mitochondrial Protein Aggregation
Mitochondria were incubated with compounds or vehicle (0.3% DMSO) for 20 min at the indicated temperatures. Samples were centrifuged and pelleted mitochondria were lysed. After centrifugation at 125,000 × g, the supernatants were analyzed by SDS-PAGE and Coomassie staining.
(A) Coomassie-stained polyacrylamide gel. Results are representative of three independent experiments.
(B) Proteins showing different amounts compared with the control samples (lane a and b, bands framed by dashed rectangles) were cut out and identified by LC-MS/MS.
(C) Effect of MC and derivatives on mitochondrial protein aggregation. Samples were analyzed by western blot for HSP60, LRP130, or LONP and quantified by densitometric analysis.
(D) MC triggers mitochondrial protein aggregation in a concentration-dependent manner. Samples were analyzed as described above. Means ± SEM; n = 3.
(E) Mitochondrial lysates derived from HL-60 cells were incubated with the fishing constructs (see Figure 2A) overnight at 4°C. After washing, bound proteins were eluted by SDS lysis (5 min, 95°C) and analyzed by western blot for LONP and LRP130. Loading control: unspecific protein band at approximately 50 kDa. Densitometric analysis; means ± SEM; n = 3.
#,∗p < 0.05, ##,∗∗p < 0.01, ###,∗∗∗p < versus vehicle control, Student’s t test. Statistics applied to logarithmized data. See also Table S2.
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