Covalent inhibition of endoplasmic reticulum chaperone GRP78 disconnects the transduction of ER stress signals to inflammation and lipid accumulation in diet-induced obese mice
- School of Chinese Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, China
- Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, China
- Colleage of Chemistry and Pharmacy, College of Chemistry & Pharmacy, Northwest A&F University, China
Figures
Figure 1
Celastrol ameliorates adipose hypertrophy and lipid metabolism.
(A, B) Hematoxylin and eosin (H&E) staining of mouse hepatic tissues and epididymal fat pads. After 21-day treatment with vehicle or celastrol, the livers and epididymal fat pads were recovered from …
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Figure 1—source data 1
Data for Figure 1A–D.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig1-data1-v1.zip
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Figure 1—source data 2
Data for Figure 1E, F.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig1-data2-v1.xlsx
Figure 2
Celastrol restores the expression of the biomarkers in lipid metabolism in livers and epididymal adipose tissues.
(A) One-week effects of celastrol on the biomarkers of lipid metabolism in liver tissues. After 7-day treatment, total RNAs were extracted from livers and analyzed by quantitative real-time PCR …
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Figure 2—source data 1
Data for Figure 2A–C.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig2-data1-v1.xlsx
Figure 3
Celastrol suppresses endoplasmic reticulum (ER) stress signaling in livers and epididymal adipose tissues.
(A) Immunostaining of GRP78 in livers. After 21-day treatment, mouse livers were recovered and stained with antibodies against GRP78 and CD68, whereas 2-(4-Amidinophenyl)-6-indolecarbamidine …
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Figure 3—source data 1
Data for Figure 3A.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig3-data1-v1.zip
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Figure 3—source data 2
Data for Figure 3B.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig3-data2-v1.zip
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Figure 3—source data 3
Data for Figure 3C, D.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig3-data3-v1.zip
Figure 4
RNA-seq transcriptome profiling of celastrol-regulated genes.
(A) Hierarchical cluster analysis of celastrol-regulated differentially expressed genes (DEGs) in high-fat diet (HFD)-induced obese mice. Expression values are determined by next-generation RNA-seq …
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Figure 4—source data 1
Data for Figure 4A–C.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig4-data1-v1.zip
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Figure 4—source data 2
Differentially expressed genes list for comparison.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig4-data2-v1.zip
Figure 5
Celastrol attenuates palmitate-induced endoplasmic reticulum (ER) stress in RAW264.7 macrophages.
(A) Western blot analysis for the effects of celastrol on the ER stress biomarkers in palmitate-challenged RAW264.7 cells. RAW264.7 cells were treated with celastrol for 8 hr and subsequently with …
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Figure 5—source data 1
Original immunoblots.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig5-data1-v1.zip
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Figure 5—source data 2
Data for Figure 5B.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig5-data2-v1.xlsx
Figure 6
Celastrol forms covalent conjugate with endoplasmic reticulum (ER) chaperone GRP78.
(A) Click chemistry biotinylation and affinity isolation of celastrol-bound proteins. Following 24-hr treatment with celastrol-PEG4-alkyne, the cellular proteins were isolated from RAW264.7 cells …
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Figure 6—source data 1
Original immunoblots and figure with the uncropped blots with the relevant bands labeled.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig6-data1-v1.zip
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Figure 6—source data 2
Data for Figure 6C.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig6-data2-v1.xlsx
Figure 7
Covalent binding of celastrol attenuated the chaperone activity of GRP78 toward peptide.
(A) Validation of peptide binding to GRP78. After the immobilization of biotinylated GRP78 to the streptavidin-coated Sensor Chip SA, GRP78-targeting peptide (WDLAWMFRLPVG) was pumped into the …
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Figure 7—source data 1
Data for Figure 7A, B.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig7-data1-v1.xlsx
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Figure 7—source data 2
Original immunoblots and figure with the uncropped blots with the relevant bands labelled.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig7-data2-v1.zip
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Figure 7—source data 3
Data for the structures, HRMS spectrum, 1H NMR spectrum, and 13C NMR spectrum of celastrol analog.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig7-data3-v1.pptx
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Figure 7—source data 4
Synthesis route of celastrol analog and data for the structures, HRMS spectrum, 1H NMR spectrum, and 13C NMR spectrum of intermediates.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig7-data4-v1.pptx
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Figure 7—source data 5
Data for the structures, HRMS spectrum, 1H NMR spectrum, and 13C NMR spectrum of celastrol-PEG3-biotin.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig7-data5-v1.pptx
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Figure 7—source data 6
Data for the structures, HRMS spectrum, 1H NMR spectrum, and 13C NMR spectrum of analog-PEG3-biotin.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig7-data6-v1.pptx
Figure 8
Intracellular colocalization of celastrol labeling and GRP78.
Following the challenge with palmitate, RAW264.7 cells were treated with celastrol or celastrol-PEG4-alkyne, washed, fixed, reacted with fluorescence dye AFDye555-picolyl azide. The cells were …
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Figure 8—source data 1
Original immunofluorescence staining result of RAW264.7 cells treated with celastrol-PEG4-alkyne.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig8-data1-v1.zip
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Figure 8—source data 2
Original immunofluorescence staining result of RAW264.7 cells treated with celastrol.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig8-data2-v1.zip
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Figure 8—source data 3
Data for the structures, HRMS spectrum, 1H NMR spectrum, and 13C NMR spectrum of celastrol-PEG4-alkyne.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig8-data3-v1.pptx
Figure 9
Quinone methide is essential to induce weight loss, reduce fat mass, and restore glucose tolerance and insulin tolerance in diet-induced obese C57BL/6N mice.
(A) Images of experimental mice. Diet-induced obese mice were treated with celastrol or analog at the dose of 5.0 mg kg−1 d−1 for 21 days, whereas control mice were fed with normal diet. …
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Figure 9—source data 1
Data for Figure 9B–F.
- https://cdn.elifesciences.org/articles/72182/elife-72182-fig9-data1-v1.xlsx
Figure 10
Potential mechanisms underlying the activities of celastrol against endoplasmic reticulum (ER) stress, inflammation, and lipid accumulations.
The in vitro and in vivo results from the present study suggest that celastrol covalently inhibits the chaperone activity of GRP78 and disconnects the transduction of ER stress signals to downstream …
Author response image 1
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Author response image 3
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-GRP78 (rabbit polyclonal) | Abcam | Cat. #: ab21685 | WB (1:1000)IF (1:500) |
| Antibody | Anti-IRE1α (rabbit monoclonal) | Cell Signaling Technology | Cat. #: 3294 | WB (1:1000) |
| Antibody | Anti-XBP1 (rabbit polyclonal) | Abcam | Cat. #: ab37152 | WB (1:1000) |
| Antibody | Anti-Calnexin (goat polyclonal) | Abcam | Cat. #: ab192439 | IF (1:500) |
| Antibody | Anti-ATF-6 (rabbit monoclonal) | Cell Signaling Technology | Cat. #: 65,880 | WB (1:1000) |
| Antibody | Anti-Phospho-eIF2α (rabbit monoclonal) | Cell Signaling Technology | Cat. #: 3398 | WB (1:1000) |
| Antibody | Anti-eIF2α (rabbit monoclonal) | Cell Signaling Technology | Cat. #: 5324 | WB (1:1000) |
| Antibody | Anti-GAPDH (rabbit monoclonal) | Cell Signaling Technology | Cat. #: 2118 | WB (1:1000) |
| Antibody | Anti-β-actin (rabbit polyclonal) | Thermo Fisher Scientific | Cat. #: PA1-183 | WB (1:1000) |
| Antibody | Anti-GRP78 (mouse monoclonal) | Abcam | Cat. #: ab212054 | IF (1:500) |
| Antibody | Anti-CD68 (rabbit polyclonal) | Abcam | Cat. #: ab125212 | IF (1:500) |
| Antibody | Anti-rabbit IgG (H + L) Alexa Fluor 594 (goat polyclonal) | Thermo Fisher Scientific | Cat. #: A-11012 | IF (1:500) |
| Antibody | Anti-mouse IgG (H + L) Alexa Fluor 488 (goat polyclonal) | Thermo Fisher Scientific | Cat. #: A-11001 | IF (1:500) |
| Antibody | Anti-goat IgG H&L Alexa Fluor 647 (donkey polyclonal) | Abcam | Cat. #: ab150131 | IF (1:500) |
| Antibody | Anti-rabbit HRP-conjugated IgG secondary antibody (goat polyclonal) | Sigma-Aldrich | Cat. #: A0545 | WB (1:10,000) |
| Chemical compound, drug | Celastrol | Nanjing Spring and Autumn Biological Engineering Co., Ltd | Purity ＞98% (HPLC) | |
| Chemical compound, drug | Lipopolysaccharides from Escherichia coli O111:B4 | Sigma-Aldrich | Cat #: L4391 | LPS |
| Chemical compound, drug | Sodium palmitate | Sigma-Aldrich | Cat #: P9767 | |
| Chemical compound, drug | Biotin-PEG3-amine | Hunan Huateng Pharmaceutical Company | Cat#: 11,025 | |
| Chemical compound, drug | Alkyne-PEG4-amine | Hunan Huateng Pharmaceutical Company | Cat#: 10,749 | |
| Cell line (Mus musculus) | RAW264.7 cells | ATCC | TIB-71 | Authenticated (STR profiling) and mycoplasma test (absent) (Genetic Testing Biotechnology Corporation, Suzhou, China) |
| Commercial assay or kit | TRIzol Reagent | Thermo Fisher Scientific | Cat. #: 15596026 | |
| Commercial assay or kit | RevertAid first-strand cDNA synthesis kit | Thermo Fisher Scientific | Cat. #: K1622 | |
| Commercial assay or kit | SYBR Green mix | QIAGEN | Cat. #: 204,056 | |
| Sequence-based reagent | Mm_Cfd_1_SG | QIAGEN | Cat. #: QT00250495 | Cfd |
| Sequence-based reagent | Mm_Fabp4_1_SG | QIAGEN | Cat. #: QT00091532 | Fabp4 |
| Sequence-based reagent | Mm_Cebpa_1_SG | QIAGEN | Cat. #: QT00311731 | Cebpa |
| Sequence-based reagent | Mm_Cpt1a_1_SG | QIAGEN | Cat. #: QT00106820 | Cpt1 |
| Sequence-based reagent | Mm_Lep_1_SG | QIAGEN | Cat. #: QT00164360 | Lepa |
| Sequence-based reagent | Mm_Pparg_1_SG | QIAGEN | Cat. #: QT00100296 | Pparg |
| Sequence-based reagent | Mm_Retn_1_SG | QIAGEN | Cat. #: QT00093450 | Retn |
| Sequence-based reagent | Mm_Ucp2_1_SG | QIAGEN | Cat. #: QT00138943 | Ucp2 |
| Sequence-based reagent | Mm_Crat_1_SG | QIAGEN | Cat. #: QT00111405 | Cat |
| Sequence-based reagent | Mm_Acox1_1_SG | QIAGEN | Cat. #:QT00174342 | Aco |
| Sequence-based reagent | Mm_Gapdh_3_SG | QIAGEN | Cat. #: QT01658692 | Gapdh |
| Strain, strain background (Escherichia coli) | BL21(DE3) | Thermo Fisher Scientific | Cat. #: EC0114 | Competent cells |
| Recombinant DNA reagent | pET-28a | Sigma-Aldrich | Cat. #: 69,864 | |
| Recombinant DNA reagent | pET28a-GRP78 plasmid | This manuscript | See Materials and methods | |
| Software, algorithm | Prism | GraphPad | https://www.graphpad.com/scientific-software/prism/ | |
| Software, algorithm | NIH Image J | NIH | http://imagej.net/ImageJ2 |
