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. 2022 Apr 16;11(8):1363.
doi: 10.3390/cells11081363.

Organismal and Cellular Stress Responses upon Disruption of Mitochondrial Lonp1 Protease

Affiliations

Organismal and Cellular Stress Responses upon Disruption of Mitochondrial Lonp1 Protease

Eirini Taouktsi et al. Cells. .

Abstract

Cells engage complex surveillance mechanisms to maintain mitochondrial function and protein homeostasis. LonP1 protease is a key component of mitochondrial quality control and has been implicated in human malignancies and other pathological disorders. Here, we employed two experimental systems, the worm Caenorhabditis elegans and human cancer cells, to investigate and compare the effects of LONP-1/LonP1 deficiency at the molecular, cellular, and organismal levels. Deletion of the lonp-1 gene in worms disturbed mitochondrial function, provoked reactive oxygen species accumulation, and impaired normal processes, such as growth, behavior, and lifespan. The viability of lonp-1 mutants was dependent on the activity of the ATFS-1 transcription factor, and loss of LONP-1 evoked retrograde signaling that involved both the mitochondrial and cytoplasmic unfolded protein response (UPRmt and UPRcyt) pathways and ensuing diverse organismal stress responses. Exposure of worms to triterpenoid CDDO-Me, an inhibitor of human LonP1, stimulated only UPRcyt responses. In cancer cells, CDDO-Me induced key components of the integrated stress response (ISR), the UPRmt and UPRcyt pathways, and the redox machinery. However, genetic knockdown of LonP1 revealed a genotype-specific cellular response and induced apoptosis similar to CDDO-Me treatment. Overall, the mitochondrial dysfunction ensued by disruption of LonP1 elicits adaptive cytoprotective mechanisms that can inhibit cancer cell survival but diversely modulate organismal stress response and aging.

Keywords: C. elegans; CDDO-Me; LonP1; aging; cancer; mitochondria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Disruption of C. elegans LONP-1 activity impairs normal development, fecundity, and lifespan. (A) Schematic of the operon CEOP1636, the CRISPR/Cas9-generating lonp-1(ko) null allele, and the lonp-1(tm5171) loss-of-function allele. Western blot analysis, using an antibody against human LonP1 and anti-β-ACTIN as a loading control, showed the absence of LONP-1 protein in both lonp-1 mutant strains. (B) Domain structure and functional regions of LONP-1 protein. The mitochondrial _target sequence (MTS) directs the translocation of the precursor protein across the mitochondrial membranes; the N domain is involved in substrate recognition, along with the central AAA+ module, which contains the Walker Box A and B motifs for ATP-hydrolysis and the sensor- and substrate-discrimination (SSD) domain. The C-terminal proteolytic domain (P domain) contains the serine (S) and lysine (K) catalytic dyad residues. The graphic showing the gene structure of LONP-1 was created using the Exon-Intron Graphic Maker (http://wormweb.org/exonintron, last accessed on 10 December 2021). Black boxes represent exons linked by lines corresponding to introns. The bracket points to the sequences deleted in the tm5171 allele. In the right panel, the molecular modeling of the SSD domain of human (HsLonP1), bacterial (EcLon), Arabidopsis (AtLon1), and worm (CelLonP1) proteases is presented. (C) The growth rate of lonp-1 mutants 72 h post-egg-laying at 20 °C versus the wild-type N2 animals (wt). At this time point, wt worms have reached the adult stage, while lonp-1 mutants were at the larval stage 3 or 4 (L3, L4). (D) Fertility assays of lonp-1 mutants showing the mean number of viable progenies per individual in all biological replicates. An unpaired t-test was used to assess significance (**** p value <0.0001). (E) Lifespan assays of lonp-1 mutants at 20 °C. Replicates and statistical analysis of lifespan assays are shown in Supplementary Table S3.
Figure 2
Figure 2
Disturbed mitochondrial network and increased ROS production in lonp-1 mutants. (A) Representative confocal images of vitally stained C. elegans mitochondria using fluorescent dye Mitotracker Green and Mitotracker Red CMXRos in N2 (wt) and lonp-1(ko) animals. Scale bar, 10 μm. (B) Representative confocal images of the myo-3p::GFPmt transgenic animals that express GFP localized to muscle mitochondria, stained with Mitotracker Red CMXRos. Scale bar, 10 μm. (C) Representative microscopy images and quantification of fluorescence of the myo-3p::GFPmt reporter in wt and lonp-1(ko) mutants. Scale bar, 100 μm. (D) Representative microscopy images of transgenic animals expressing a cyp-14A4p::gfp fusion gene that is induced by mitochondrial dysfunction in wt and lonp-1(ko) mutants. Scale bar, 100 μm. (E) Quantification of fluorescence in wt and lonp-1(ko) animals stained with the ROS-sensitive dye dihydroethidium (DHE). (F) Representative confocal images and quantification of the ratio of oxidized to reduced ratiometric reporter HyPer in wt and lonp-1(ko) worms expressing jrIs1[rpl-17p::HyPer]. Scale bar, 100 μm. All experiments were performed on 1-day adults, and an unpaired t-test was used to assess significance (**** p value < 0.0001).
Figure 3
Figure 3
Deletion of lonp-1 induces UPRmt in C. elegans. (A) Quantification of the relative mRNA levels of endogenous hsp-6 and hsp-60 genes in wt and lonp-1 worms on the first day of adulthood. The normalized mean fold-change of all biological replicates relative to control strain is shown, and significance was assessed by paired t-test. (B) Representative microscopy images and GFP fluorescence quantification of the UPRmt reporters hsp-6p::gfp and hsp-60p::gfp in wt and lonp-1 adults. In both graphs, an unpaired t-test was used to assess significance (p value). (C) Representative microscopy images and GFP quantification of the UPRmt reporter hsp-6p::gfp in 1-day adult wt and lonp-1 mutants, subjected throughout their life to RNAi against atfs-1, dve-1, or ubl-5, compared with animals fed the empty vector. Two-way ANOVA followed by post hoc Tukey’s test was used to assess the significance of treatment in each strain (showed as asterisks in each graph) and the interaction between genotype and RNAi treatment (p < 0.0001 for atfs-1(RNAi) in both lonp-1 mutants, p = 0.2510 for dve-1(RNAi) and p = 0.4728 for ubl-5(RNAi) in lonp-1(ko) worms). In all panels, asterisks denote statistical significance: * p = 0.01–0.05, ** p = 0.001–0.01, *** p = 0.0001–0.001, **** p < 0.0001.
Figure 4
Figure 4
Activation of antioxidant and stress response mechanisms in lonp-1 mutants. (A) Quantification of the relative mRNA levels of endogenous skn-1 and its _target genes, gst-4 and gst-13, in 1-day adult wt and lonp-1(ko) worms grown at 20 °C. The normalized mean fold-change of all biological replicates relative to control strain is shown, and significance was assessed by paired t-test. (B) Representative microscopy images of gst-4p::gfp reporter in wt and lonp-1(ko) 1-day adults, under normal conditions and GFP fluorescence quantification in these animals as well as following exposure to ROS-generator H2O2 (10 mM for 30 min, followed by 30 min recovery before visualization). To assess significance, an unpaired t-test was used for worms under normal conditions, while two-way ANOVA followed by post hoc Tukey’s test was used to assess the significance of H2O2 treatment in each strain (showed as asterisks in the graph) and the interaction between genotype and treatment (p < 0.0001). (C) GFP quantification of the gst-4p::gfp reporter in wt and lonp-1(ko) 1-day adults subjected from eggs to RNAi against atfs-1 or skn-1. Two-way ANOVA followed by post hoc Tukey’s test was used to assess the significance of treatment in each strain (showed as asterisks in graphs) and the interaction between genotype and each RNAi (p < 0.0001 for atfs-1(RNAi) and p = 0.4774 for skn-1(RNAi) treatment). (D) Representative microscopy images and quantification of fluorescent nuclei in wt and lonp-1(ko) young adults expressing the muIs71[daf-16ap::gfp::daf-16a(bKO)] translational reporter, under normal or mild heat-shock conditions (HS for 15 or 30 min at 35 °C), with white arrows to indicate localization of the reporter in intestinal and epidermal nuclei. In the right graph, quantification of the relative mRNA levels of endogenous DAF-16 _target genes in 1-day adult wt and lonp-1(ko) worms grown at 20 °C is shown. The normalized mean fold-change of all biological replicates is shown, and significance was assessed by paired t-test. In all panels, asterisks denote statistical significance: * p = 0.01–0.05, ** p = 0.001–0.01, *** p = 0.0001–0.001, **** p < 0.0001.
Figure 5
Figure 5
Loss of lonp-1 enhances heat shock response (HSR) and induces diverse organismal responses to various stresses. (A) Quantification of the relative mRNA levels of endogenous HSR genes, and UPRER-related genes, in lonp-1 worms under normal growth temperature (20 °C). The normalized mean fold-change of all biological replicates relative to control strain is shown, and significance was assessed by paired t-test. (B) Representative microscopy images and GFP fluorescence quantification of the hsp-16.2p::gfp reporter in wt and lonp-1(ko) worms subjected to heat shock (HS at 35 °C for 90 min). Two-way ANOVA followed by post hoc Tukey’s test was used to assess the significance of treatment in each strain (showed as asterisks in the graph) and the interaction between genotype and HS (p < 0.0001). Scale bar, 100 μm. Resistance to (C) HS (35 °C for 6 h), (D) osmotic stress (500 mM NaCl for 24 h), (E) paraquat (30 mM for 48 h) or antimycin A (40 μm for 24 h) and (F) H2O2 (10 mM for 30 min) or tBHP (10 mM), of lonp-1 mutants at day 1 or 2 of adulthood, at the indicated time periods. (G) On the contrary, lonp-1 mutants exhibit increased sensitivity to oxidants sodium arsenite (7.5 mM), sodium azide (1.5 mM), and rotenone (10 μm) after 24 h exposure on day 1 of adulthood. The percentage survival for all biological replicates was plotted, and an unpaired t-test was used to assess significance (p value). In all panels, asterisks denote statistical significance: * p = 0.01–0.05, ** p = 0.001–0.01, *** p = 0.0001–0.001, **** p < 0.0001.
Figure 6
Figure 6
CCDO-Me induces specific stress-responsive genes in wt worms. (A) Quantification of the relative mRNA levels of endogenous HSR genes in wt 1-day adult worms, treated with 10 μm CDDO-Me from eggs (long-term) or at the L4 stage for 24 h (short-term), under normal growth temperature (20 °C). (B) Quantification of the relative mRNA levels of endogenous oxidative stress genes in wt 1-day adult worms, treated with 10 μm CDDO-Me from eggs (long-term), under normal growth temperature (20 °C). (C) Quantification of the relative mRNA levels of all tested stress genes in lonp-1(ko) 1-day adults, treated with 10 μm CDDO-Me from eggs (long-term), under normal growth temperature (20 °C). (D) Quantification of the relative mRNA levels of endogenous UPRmt genes in wt and lonp-1(ko) 1-day adult worms, treated with 10 μm CDDO-Me from eggs (long-term), under normal growth temperature (20 °C). In all graphs, the normalized mean fold-change of all biological replicates relative to control strain is shown, and significance was assessed by paired t-test (* p = 0.01–0.05, ** p = 0.001–0.01, *** p = 0.0001–0.001).
Figure 7
Figure 7
Effects of LonP1 silencing on stress-related gene expression in cancer cells. (A) Quantification of LonP1 relative mRNA levels and Western blot analysis presenting LonP1 versus β-ACTIN protein levels upon LonP1 silencing. Three biological replicates are represented in the blot. Quantification of relative mRNA levels of genes related to ISR (Integrated Stress Response), UPRmt (mitochondrial Unfolded Protein Response), HSR (Heat Shock Response), and antioxidant responses in (B) fibrosarcoma (HT1080) and (C) metastatic melanoma (WM266-4) cell lines upon LonP1 silencing. In all graphs, the normalized mean fold-change of three biological replicates relative to the control is shown. Statistical significance was assessed by paired t-test. In all panels, asterisks denote statistical significance: * p = 0.01–0.05, ** p = 0.001–0.01, *** p = 0.0001–0.001, **** p < 0.0001.
Figure 8
Figure 8
Effects of LonP1 pharmacological inhibition with CDDO-Me on stress-related gene expression. Quantification of relative mRNA levels after CDDO-Me treatment (1 μΜ, 24 h) of genes related to ISR (Integrated Stress Response), UPRmt (mitochondrial Unfolded Protein Response), HSR (Heat Shock Response), and antioxidant responses in (A) fibrosarcoma (HT1080) and (B) metastatic melanoma (WM266-4) cell lines. In all graphs, the normalized mean fold-change of three biological replicates relative to the control is shown. Statistical significance was assessed by paired t-test (* p = 0.01–0.05, ** p = 0.001–0.01, *** p = 0.0001–0.001, **** p < 0.0001). (C) Western blot analysis of LonP1, HSP70, ATF4, and β-ACTIN from total protein extracts, in WM266-4 and HT1080 cancer cell lines, upon 1 μM CDDO-Me treatment for 24 h. Experiments were repeated three times, while here, one representative blot is shown.
Figure 8
Figure 8
Effects of LonP1 pharmacological inhibition with CDDO-Me on stress-related gene expression. Quantification of relative mRNA levels after CDDO-Me treatment (1 μΜ, 24 h) of genes related to ISR (Integrated Stress Response), UPRmt (mitochondrial Unfolded Protein Response), HSR (Heat Shock Response), and antioxidant responses in (A) fibrosarcoma (HT1080) and (B) metastatic melanoma (WM266-4) cell lines. In all graphs, the normalized mean fold-change of three biological replicates relative to the control is shown. Statistical significance was assessed by paired t-test (* p = 0.01–0.05, ** p = 0.001–0.01, *** p = 0.0001–0.001, **** p < 0.0001). (C) Western blot analysis of LonP1, HSP70, ATF4, and β-ACTIN from total protein extracts, in WM266-4 and HT1080 cancer cell lines, upon 1 μM CDDO-Me treatment for 24 h. Experiments were repeated three times, while here, one representative blot is shown.
Figure 9
Figure 9
Effects of LonP1 disruption on mitochondrial function and motility in cancer cells. (A) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed in fibrosarcoma (HT1080) and metastatic melanoma (WM266-4) cell lines after LonP1 silencing (left) or 1 μΜ CDDO-Me treatment for 24 h (right). MTT assays were carried out at least three times, whereas significance was assessed by paired t-test (**** p < 0.0001). (B) Western blotting of ATP6 (25 kDa) upon LonP1 silencing (above) or 1 μM CDDO-Me treatment for 24 h (below) in WM266-4 and HT1080 cancer cells. β-ACTIN was used as a protein of reference. (C) Scratch-wound assays were carried out for 24 h using HT1080 and WM266-4 cancer cells under control conditions versus LonP1 silencing or treatment with 500 nM of CDDO-Me. Observations were made under an inverted microscope, and pictures were taken at 4× magnification. Experiments were repeated three times, while here, one representative experiment is shown.
Figure 9
Figure 9
Effects of LonP1 disruption on mitochondrial function and motility in cancer cells. (A) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed in fibrosarcoma (HT1080) and metastatic melanoma (WM266-4) cell lines after LonP1 silencing (left) or 1 μΜ CDDO-Me treatment for 24 h (right). MTT assays were carried out at least three times, whereas significance was assessed by paired t-test (**** p < 0.0001). (B) Western blotting of ATP6 (25 kDa) upon LonP1 silencing (above) or 1 μM CDDO-Me treatment for 24 h (below) in WM266-4 and HT1080 cancer cells. β-ACTIN was used as a protein of reference. (C) Scratch-wound assays were carried out for 24 h using HT1080 and WM266-4 cancer cells under control conditions versus LonP1 silencing or treatment with 500 nM of CDDO-Me. Observations were made under an inverted microscope, and pictures were taken at 4× magnification. Experiments were repeated three times, while here, one representative experiment is shown.

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References

    1. Rigas S., Daras G., Tsitsekian D., Hatzopoulos P. The multifaceted role of Lon proteolysis in seedling establishment and maintenance of plant organelle function: Living from protein destruction. Physiol. Plant. 2012;145:215–223. doi: 10.1111/j.1399-3054.2011.01537.x. - DOI - PubMed
    1. Tsitsekian D., Daras G., Alatzas A., Templalexis D., Hatzopoulos P., Rigas S. Comprehensive analysis of Lon proteases in plants highlights independent gene duplication events. J. Exp. Bot. 2019;70:2185–2197. doi: 10.1093/jxb/ery440. - DOI - PMC - PubMed
    1. Venkatesh S., Lee J., Singh K., Lee I., Suzuki C.K. Multitasking in the mitochondrion by the ATP-dependent Lon protease. Biochim. Biophys. Acta. 2012;1823:56–66. doi: 10.1016/j.bbamcr.2011.11.003. - DOI - PMC - PubMed
    1. Yang J., Chen W., Zhang B., Tian F., Zhou Z., Liao X., Li C., Zhang Y., Han Y., Wang Y., et al. Lon in maintaining mitochondrial and endoplasmic reticulum homeostasis. Arch. Toxicol. 2018;92:1913–1923. doi: 10.1007/s00204-018-2210-3. - DOI - PubMed
    1. Suzuki C.K., Suda K., Wang N., Schatz G. Requirement for the yeast gene LON in intramitochondrial proteolysis and maintenance of respiration. Science. 1994;264:273–276. doi: 10.1126/science.8146662. - DOI - PubMed

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