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Volume 24, Issue 11, Pages 1416-1427.e5 (November 2017)
Multiplexed Thiol Reactivity Profiling for Target Discovery of Electrophilic Natural Products Caiping Tian, Rui Sun, Keke Liu, Ling Fu, Xiaoyu Liu, Wanqi Zhou, Yong Yang, Jing Yang Cell Chemical Biology Volume 24, Issue 11, Pages e5 (November 2017) DOI: /j.chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions
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Cell Chemical Biology 2017 24, 1416-1427. e5DOI: (10. 1016/j. chembiol
Copyright © 2017 Elsevier Ltd Terms and Conditions
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Figure 1 Method Development and Validation of MTRP
(A) Schematic workflow of MTRP. (B) Representative MS/MS spectrum of THUMPD1 C31 peptide labeled by both IPM and iTRAQ used for sequence assignment and reporter ion quantification. HeLa proteomes were labeled with the alkyne-tagged thiol-reactive reagent, IPM, and digested into tryptic peptides. The digest mixture was split into eight identical aliquots. Each was then labeled with one of the eight isobaric iTRAQ reagents, and the derivatized digests combined in a predefined ratio (1:1:2:2:5:5:10:10). After click chemistry and affinity enrichment, the alkylated peptides were photoreleased and analyzed by LC-MS/MS. The iTRAQ-tagged N terminus in the peptide sequence is labeled with red asterisk. (C) Intensity of iTRAQ reporter ions for IPM-modified peptides were measured as (B). Ratios were calculated relative to the mean peak intensity at and The distributions of these ratios demonstrate the accuracy of MTRP. Data are displayed using a log 2 scale on the x axis. Cell Chemical Biology , e5DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions
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Figure 2 Chemical Structures of Electrophilic Natural Products Used in this Study The major pharmacore (α,β-unsaturated ketone) of GA is shown in blue color, while the α-methylene-γ-butyrolactone motifs are depicted in red color. Cell Chemical Biology , e5DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions
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Figure 3 Identification and Functional Validation of XPO2 as a Major Cellular Target of GA (A) Boxplot showing the distribution of measured ratios for the cysteines quantified by MTRP in each concentration group. (B) IC50 curves derived from MTRP experiment showing the potency of GA on C842 and C939 in XPO2. (C) Representative MS/MS spectra of XPO2 C842 (upper) and C939 (lower) peptide labeled by both IPM and iTRAQ tag. The iTRAQ-tagged N terminus and/or lysine in the peptide sequence are labeled with red asterisks. (D–E) Western blot derived from iso-thermo dose-response experiment (D) and thermo shift assay (E) confirming the stabilization of XPO2 by GA in U2OS cell lysates. (F) Densitometric analysis of XPO2 bands in the U2OS cell lysates treated with or without GA shown in (E). Data are presented as mean values ± SD, n = 3. (G) Binding of XPO2 in U2OS cells to different dose of GA-biotin were captured by streptavidin beads and detected by western blotting. (H) Protein binding to GA-biotin (5 μM) with or without GA competition (2.5 μM) was subjected to streptavidin enrichment and western blotting. (I) GA disrupts XPO2-mediated nuclear transportation. U2OS cells were treated with 5 μM GA for 1 hr. The distribution of XPO2 (red) and its cargo protein KPNA2 (green) were detected by immunofluorescence as described in the STAR Methods. Scale bars represent 10 μm. Cell Chemical Biology , e5DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions
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Figure 4 Working Model: GA Binds to XPO2 and Interrupts Its Function
Cell Chemical Biology , e5DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions
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Figure 5 Analysis of Interactions between Electrophilic Lactones and Thiol Proteome (A) Comparison of the proteomic reactivities of lactones screened at 30 versus 300 μM concentration in cell lysates. (B) Heatmap showing R values for all cysteines that can be targeted by at least one γ-lactone examined at a 30 μM concentration (R≤0.5). (C) Representative MS/MS spectra of EEF2 C41 peptide labeled by both IPM and iTRAQ tag from low (upper) and high (lower) concentration treatment experiments. The iTRAQ-tagged N terminus and/or lysine in the peptide sequence are labeled with red asterisks. (D) Principal coordinates analysis showing disparate target profiles of seven lactones at 30 and 300 μM concentrations in cell lysates. Cell Chemical Biology , e5DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions
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Figure 6 Confirmation and Functional Analysis of ACE-Cysteine Interactions in HSP60 (A) Crystal structure of human HSP60 (PDB: 4PJ1). The structures were visualized using Discovery Studio v2.5. The annotated cysteine residues were shown in stick style. (B) Heatmap showing R values for the reactivity of three cysteines of HSP60 toward seven examined γ-lactones. (C) Representative MS/MS spectrum of HSP60 C442 peptide labeled by both IPM and iTRAQ tag (113, DMSO; 114, COS; 115, YEJ; 116, EUA; 117, ACE; 118, ISO; 119, DEH; 121, EUB). The iTRAQ-tagged N terminus in the peptide sequence is labeled with red asterisks. (D) ACE preferentially blocked BIAM labeling of human recombinant HSP60. Human recombinant HSP60 (1 μM) was pre-incubated with the indicated lactones (3 μM) for 2 hr at 37°C and then a labeled 2 μM BIAM probe for 1 hr at room temperature. The BIAM-labeled proteins were detected with western blotting using HRP-streptavidin. (E) Representative MS/MS spectrum of HSP60 C442 peptide covalently modified with ACE. (F) ACE inhibited HSP60 chaperone activity in a concentration-dependent manner. Denatured MDH was incubated with pre-formed HSP60-HSP10 complex for 5 min at 27°C. HSP60 was treated with or without ACE. The refolding reaction was initialed by 2 mM ATP for 30 min at 27°C, and terminated by addition of glucose/hexohinase, NADH, and oxaloacetate. Absorbance was monitored at 360 nm at 30°C. YEJ was used as a negative control. (G) ACE exhibited no inhibitory effect on native MDH activity. For (F) and (G), data are presented as mean values ± SD, n = 3. Cell Chemical Biology , e5DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions
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Figure 7 Liganded Cysteines Targeted by Electrophilic Lactones
(A) Heatmap showing R values for all γ-lactone-liganded cysteines. (B) Percentage of proteins with γ-lactone-liganded cysteines found in DrugBank. (C) Functional classification of DrugBank and non-DrugBank proteins with liganded cysteines. Cell Chemical Biology , e5DOI: ( /j.chembiol ) Copyright © 2017 Elsevier Ltd Terms and Conditions
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