Urolithin A Protects Hepatocytes from Palmitic Acid-Induced ER Stress by Regulating Calcium Homeostasis in the MAM
<p>Effects of UA on cellular response, ER stress, and lipid accumulation in PA-treated hepatocytes. (<b>A</b>) The MTT assay was performed on HepG2 cells treated with various concentrations of UA (1–100 μM) for 24, 48, and 72 h to evaluate cell proliferation. (<b>B</b>) The MTT assay was used to measure the cell proliferation of HepG2 cells treated with PA (0.5 mM) and UA (20, 40 μM) for 24 h. (<b>C</b>) The Trypan blue exclusion assay was performed to evaluate the cell viability of HepG2 cells treated with PA (0.5 mM) and UA (20, 40 μM) for 24 h. (<b>D</b>) mCherry-CHOP stable HEK293 cells were treated with PA (0.5 mM) and UA (20, 40 μM) for 24 h, and mCherry fluorescence signals were measured. The expression of CHOP was visualized as a fluorescence signal and observed using confocal microscopy (scale bar: 10 μM). (<b>E</b>) HepG2 cells were treated with PA (0.5 mM) and UA (20, 40 μM) for 24 h, followed by staining of intracellular lipid droplets with BODIPY and observation via confocal microscopy (scale bar: 20 μM) (* <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001).</p> "> Figure 2
<p>CETSA-LC-MS/MS for the identification of the target protein of UA. (<b>A</b>) Overview of the CETSA-LC-MS/MS method. HEK293 cells were treated with DMSO (control) or UA (20 μM) and subjected to thermal treatment (55 °C or 60 °C). Proteins were then extracted, digested with trypsin, and labeled with TMT reagents. The labeled peptides were analyzed by HPLC and LC-MS/MS. (<b>B</b>) Schematic diagram of the target selection identification criteria of the UA process. (<b>C</b>) Heatmap of target candidates of UA localized in the ER and mitochondria. Proteins are clustered by their functions, as indicated on the left side of the heatmap.</p> "> Figure 3
<p>Validation of UA binding to SERCA. (<b>A</b>) Western blot analysis to evaluate the thermal stability of SERCA in HepG2 cells treated with UA (40 μM). (<b>B</b>) Comparison of the binding affinity between UA and two calcium-regulating target proteins: SERCA and CCDC47. HepG2 cells were treated with various concentrations of UA (1–100 μM), followed by isothermal CETSA at 52 °C. (<b>C</b>) The 2D diagram represents amino acids involved in UA binding to SERCA. (<b>D</b>) Predicted binding of UA to the actuator domain of SERCA, as visualized using Discovery Studio software 2018 (CDOCKER energy: −20.57 kcal/mol). (<b>E</b>) The 3D structure of the full-length SERCA protein (PDB: 7E7S), highlighting its four domains. (<b>F</b>) HEK293 cells were transfected with FLAG-SERCA(WT), FLAG-SERCA(R198A), or FLAG-SERCA(K234A) for 48 h and then treated with UA (40 μM). After heat treatment at 52 °C for 3 min, proteins were extracted and analyzed using a Western blot. (<b>G</b>) Measurement of ATPase activity of ER proteins extracted from LX2 cells. UA (20, 40 μM) and thapsigargin (0.1 μM) were each treated for 30 min before ATPase activity was assessed.</p> "> Figure 4
<p>Intracellular calcium levels in PA-treated HepG2 cells with UA. (<b>A</b>) HepG2 cells were transfected with the ER calcium indicator ER-LAR-GECO vector for 48 h, followed by treatment with UA (40 μM), CDN1163 (10 μM), or TG (0.1 μM) for 6 h (scale bar: 20 μm). (<b>B</b>) ER calcium levels were measured in HepG2 cells treated with PA (500 μM) for 6 h, either alone or co-treated with UA (40 μM) and CDN1163 (10 μM) (scale bar: 10 μm). (<b>C</b>) Cytosolic calcium levels were assessed using the Fluo-4-AM after 6 h of treatment with PA, either alone or co-treated with UA (40 μM) and CDN1163 (10 μM) (scale bar: 20 μm). (<b>D</b>) Mitochondrial calcium levels were determined using the Rhod-2-AM after 24 h of treatment with PA, either alone or co-treated with UA (40 μM) and CDN1163 (10 μM) (scale bar: 20 μm) (* <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001).</p> "> Figure 5
<p>ER stress markers in PA-treated HepG2 cells with UA. (<b>A</b>) Western blot results showing changes in ER stress markers in HepG2 cells treated with PA. (<b>B</b>) HepG2 cells were treated with PA for 6 h in the absence or presence of UA (40 μM), CDN1163 (10 μM), and thapsigargin (0.1 μM). UA down-regulated the level of ER stress-related proteins. (<b>C</b>) HepG2 cells were treated with PA for 24 h in the absence or presence of UA (40μM), CDN1163 (10 μM), and thapsigargin (0.1 μM). UA down-regulated the level of CHOP (* <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, ns: not significant).</p> "> Figure 6
<p>Impact of <span class="html-italic">SERCA</span> knockdown on UA activity in PA-treated cells. (<b>A</b>–<b>C</b>) Following <span class="html-italic">SERCA</span> knockdown with si-SERCA for 24 h, cells were co-treated with PA and UA (40 μM) for 6 h. ER, cytosolic, and mitochondrial calcium levels were then measured using Mag-Fluo-4 AM, Fluo-4 AM, and Rhod-2 AM, respectively (scale bar: 40 μm). (<b>D</b>) <span class="html-italic">SERCA</span> knockdown using si-<span class="html-italic">SERCA</span> for 24 h confirmed the effect of UA (40 μM) on PA-induced ER stress protein levels, as shown by Western blot (* <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001, ns: not significant).</p> "> Figure 7
<p>Schematic summary of the target proteins and mechanisms of action of UA. HepG2 cells stimulated with PA release ER calcium through IP<sub>3</sub>R, leading to ER stress. UA binds to SERCA, the ER calcium pump, replenishing ER calcium levels and maintaining calcium homeostasis. This mechanism helps protect the cells from stress-induced damage.</p> ">
Abstract
:1. Introduction
2. Methods and Materials
2.1. Cell Culture and Treatment
2.2. Western Blot
2.3. CETSA
2.4. Sample Preparation for LC-MS/MS Analysis
2.5. High pH Reversed-Phase Liquid Chromatography for Peptide Fractionation
2.6. Liquid Chromatography–Tandem Mass Spectrometry
2.7. Protein Identification and Quantitation
2.8. In Silico Docking Study
2.9. ATPase Activity Assay
2.10. Lipid Droplet Staining
2.11. Calcium Analysis
2.12. Mitochondrial ROS Measurement
2.13. Transfection
2.14. Materials
2.15. Statistical Analysis
3. Results
3.1. Effects of UA on Palmitic Acid-Induced Cellular Stress
3.2. Target Identification of UA by CETSA-LC-MS/MS
3.3. Validation of Target Protein of UA
3.4. Modulation of Calcium Levels in Cellular Organelles by UA
3.5. UA-Mediated Regulation of Ca2+ Homeostasis and Its Role in ER Stress Reduction
3.6. Influence of SERCA Knockdown on UA Activity
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
CETSA | cellular thermal shift assay |
CDN | CDN1163 |
LC-MS/MS | liquid chromatography–mass spectrometry/mass spectrometry |
PA | palmitic acid |
TG | thapsigargin |
MTT | 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide |
IC50 | half maximal inhibitory concentration |
EC50 | half maximal effective concentration |
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Ryu, G.; Ko, M.; Lee, S.; Park, S.I.; Choi, J.-W.; Lee, J.Y.; Kim, J.Y.; Kwon, H.J. Urolithin A Protects Hepatocytes from Palmitic Acid-Induced ER Stress by Regulating Calcium Homeostasis in the MAM. Biomolecules 2024, 14, 1505. https://doi.org/10.3390/biom14121505
Ryu G, Ko M, Lee S, Park SI, Choi J-W, Lee JY, Kim JY, Kwon HJ. Urolithin A Protects Hepatocytes from Palmitic Acid-Induced ER Stress by Regulating Calcium Homeostasis in the MAM. Biomolecules. 2024; 14(12):1505. https://doi.org/10.3390/biom14121505
Chicago/Turabian StyleRyu, Gayoung, Minjeong Ko, Sooyeon Lee, Se In Park, Jin-Woong Choi, Ju Yeon Lee, Jin Young Kim, and Ho Jeong Kwon. 2024. "Urolithin A Protects Hepatocytes from Palmitic Acid-Induced ER Stress by Regulating Calcium Homeostasis in the MAM" Biomolecules 14, no. 12: 1505. https://doi.org/10.3390/biom14121505
APA StyleRyu, G., Ko, M., Lee, S., Park, S. I., Choi, J. -W., Lee, J. Y., Kim, J. Y., & Kwon, H. J. (2024). Urolithin A Protects Hepatocytes from Palmitic Acid-Induced ER Stress by Regulating Calcium Homeostasis in the MAM. Biomolecules, 14(12), 1505. https://doi.org/10.3390/biom14121505