Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Dec;174(23):4409-4429.
doi: 10.1111/bph.14045. Epub 2017 Nov 7.

Lon Protease: A Novel Mitochondrial Matrix Protein in the Interconnection Between Drug-Induced Mitochondrial Dysfunction and Endoplasmic Reticulum Stress

Affiliations
Free PMC article

Lon Protease: A Novel Mitochondrial Matrix Protein in the Interconnection Between Drug-Induced Mitochondrial Dysfunction and Endoplasmic Reticulum Stress

Miriam Polo et al. Br J Pharmacol. .
Free PMC article

Abstract

Background and purpose: Mitochondria-associated membranes (MAMs) are specific endoplasmic reticulum (ER) domains that enable it to interact directly with mitochondria and mediate metabolic flow and Ca2+ transfer. A growing list of proteins have been identified as MAMs components, but how they are recruited and function during complex cell stress situations is still not understood, while the participation of mitochondrial matrix proteins is largely unrecognized.

Experimental approach: This work compares mitochondrial/ER contact during combined ER stress/mitochondrial dysfunction using a model of human hepatoma cells (Hep3B cell line) treated for 24 h with classic pharmacological inducers of ER stress (thapsigargin), mitochondrial dysfunction (carbonyl cyanide m-chlorophenyl hydrazone or rotenone) or both (the antiretroviral drug efavirenz used at clinically relevant concentrations).

Key results: Markers of mitochondrial dynamics (dynamin-related protein 1, optic atrophy 1 and mitofusin 2) were expressed differently with these stimuli, pointing to a specificity of combined ER/mitochondrial stress. Lon, a matrix protease involved in protein and mtDNA quality control, was up-regulated at mRNA and protein levels under all conditions. However, only efavirenz decreased the mitochondrial content of Lon while increasing its extramitochondrial presence and its localization to MAMs. This latter effect resulted in an enhanced mitochondria/ER interaction, as shown by co-immunoprecipitation experiments of MAMs protein partners and confocal microscopy imaging.

Conclusion and implications: A specific dual drug-induced mitochondria-ER effect enhances the MAMs content of Lon and its extramitochondrial expression. This is the first report of this phenomenon and suggests a novel MAMs-linked function of Lon protease.

Figures

Figure 1
Figure 1
Expression of the main molecular mediators of mitochondrial dynamics. Cells were treated for 24 h with increasing concentrations of efavirenz (EFV), vehicles (MeOH or DMSO), thapsigargin (TG) 2 μM, rotenone (Rot) 25 μM or CCCP 10 μM. (A) Immunoblot analysis showing representative Western blotting image and histograms expressing quantification of the main regulators of mitochondrial fusion and fission. Data (mean ± SEM, n = 6 for OPA1; n = 5 for Drp1 y p‐Drp1; n = 8 for Mfn2) are expressed as % of control, the negative control (untreated cells) being considered 100%. (B) Gene expression analysed by quantitative RT‐PCR. Data (mean ± SEM, n = 5) are expressed as mRNA content in relation to that of control (untreated cells, considered 100%) after normalization with the expression of the housekeeping gene ACTB (β‐actin). Statistical analysis was performed by one‐way ANOVA (* P < 0.05 for efavirenz vs. MeOH and # P < 0.05 for thapsigargin, rotenone or CCCP vs. DMSO).
Figure 2
Figure 2
Subcellular protein expression p‐Drp1 and Mfn2. (A) Analysis of the purity of mitochondria‐enriched and cytosolic protein extracts. Representative Western blotting image and histograms expressing quantification of several mitochondrial (TOM20, CIV‐II and porin) and cytosolic (LDH, tubulin and β‐actin) proteins after 24 h of treatment. Data (mean ± SEM, n = 6) are expressed as % of control in the mitochondrial fraction, untreated cells considered 100%. (B) Western blotting analysis of p‐Drp1 and Mfn2 in mitochondrial and cytosolic extracts in cells treated for 24 h with increasing concentrations of efavirenz (EFV), vehicles (MeOH and DMSO), thapsigargin (TG) 2 μM, rotenone (Rot) 25 μM or CCCP 10 μM. A representative image and histograms of the quantified data are shown. Data (mean ± SEM, n = 6) are expressed as % of control, untreated cells considered 100%. (C) Translocation of p‐Drp1 to mitochondria. Cells were treated for 24 h with increasing concentrations of efavirenz, vehicle, thapsigargin 2 μM, rotenone 25 μM or CCCP 10 μM. Representative confocal microscopic images (63×) of cells labelled with Hoechst 33342 (nuclei), anti‐p‐Drp1 (Ser616) and anti‐TOM20 (mitochondria). Histogram showing the index of correlation between p‐Drp1 and mitochondria (mean ± SEM, n = 5). Statistical analysis was performed by one‐way ANOVA (# P < 0.05 for rotenone or CCCP vs. DMSO).
Figure 3
Figure 3
Study of MAMs. Cells were treated for 24 h with increasing concentrations of efavirenz (EFV), vehicles (MeOH or DMSO), thapsigargin (TG) 2 μM, rotenone (Rot) 25 μM or CCCP 10 μM. (A, B) Analysis of contact between specific MAM protein partners by co‐immunoprecipitation using protein A sepharose beads. Representative Western blotting images and histograms expressing quantification of (A) VAP B/C after immunoprecipitation of PTPIP51 and (B) Grp75 after immunoprecipitation of porin. A negative control (without primary antibody) was used as a control for the immunoprecipitation. (C) Western blotting analysis of PTPIP51 expression in whole‐cell extracts. Data (mean ± SEM, n = 5) are expressed as % of control (untreated cells, considered 100%). Statistical analysis was performed by one‐way ANOVA (*P < 0.05 for efavirenz vs. MeOH).
Figure 4
Figure 4
Analysis of the expression of Lon. Cells were treated for 24 h with increasing concentrations of efavirenz (EFV), vehicles (MeOH or DMSO), thapsigargin (TG) 2 μM, rotenone (Rot) 25 μM or CCCP 10 μM except for (C). (A) Representative Western blotting images and histograms expressing quantification of Grp75 and Lon in mitochondria‐enriched and cytosolic protein extracts (mean ± SEM, n = 6). (B) Western blotting analysis of Lon expression in whole‐cell protein extracts (mean ± SEM, n = 6). (C) Western blotting analysis of Lon expression in whole‐cell protein extracts obtained from Hep3B cells after 24 h treatment with rilpivirine (RPV; mean ± SEM, n = 5). (D) Quantitative RT‐PCR analysis (mean ± SEM) of LONP1 in Hep3B (n = 6 except for MeOH, rotenone and thapsigargin n = 7), U‐251MG (n = 6 except for efavirenz 10 n = 5, efavirenz 25 and thapsigargin n = 7) and HUVEC (n = 6). (E) Analysis of the recruitment of NF‐κB to the promoter of LONP1. Representative image of semiquantitative PCR after ChIP with anti‐NF‐κB; a non‐related antibody anti‐IgG and a sample of the input chromatin were used as controls. Aside from the aforementioned stimuli, cells were also treated with a pro‐inflammatory stimulus a cocktail of LPS, IFN‐γ and TNF‐α (C.LPS), a known activator of NF‐κB, which was employed as an additional control condition. (F) Study of ClpX expression by immunoblot in total cell extracts showing a representative image and a histogram of the quantified data (mean ± SEM, n = 6). Results are expressed as % of control (untreated cells considered 100%). Statistical analysis was performed by one‐way ANOVA (*P < 0.05 for efavirenz vs. MeOH and # P < 0.05 for thapsigargin, rotenone or CCCP vs. DMSO).
Figure 5
Figure 5
Analysis of the regulation of the expression of Lon. Cells were pretreated with the pPERK‐inhibitor GSK2656157 or the catalase/SOD mimetic MnTMPyP for 1 h and then treated for 24 h with increasing concentrations of efavirenz (EFV), vehicle, thapsigargin (TG) 2 μM, rotenone (Rot) 25 μM or CCCP 10 μM. (A) and (B) Western blotting analysis of PERK phosphorylation and Lon expression in whole‐cell extracts. Representative immunoblots and histograms of the quantified data are shown. (C) Quantitative RT‐PCR analysis of LONP1. Results (mean ± SEM) are expressed as % of control (untreated cells considered 100%); (A) left panel n = 7 for cells without pretreatment and n = 5 for pretreated cells; (B) n = 5 and (C) n = 9 for cells without pretreatment and n = 5 for pretreated cells. Statistical analysis was performed by one‐way ANOVA (*P < 0.05 for efavirenz vs. MeOH and # P < 0.05 for thapsigargin or rotenone vs. DMSO and & P < 0.05 for cells with vs. those without pretreatment).
Figure 6
Figure 6
Analysis of the participation of Lon in the effect of efavirenz. Cells were transfected with siRNA Control or siLONP1 and treated for 24 h with efavirenz (EFV) or vehicle (MeOH). (A) Representative Western blotting image and histogram expressing quantification of Lon expression. (B) Histograms showing mean MitoSox (mitochondrial superoxide production), TMRM (mitochondrial membrane potential) and NAO fluorescence (mitochondrial mass – upper panel) and number of nuclei (visualized with Hoechst fluorescence) – lower panel. Data [mean ± SEM, n = 5 in (A) and in (B)‐upper panel, and n = 9 in (B)‐lower panel] are expressed as % of control (considered 100%). Statistical analysis was performed by one‐way ANOVA (*P < 0.05 for efavirenz vs. MeOH).
Figure 7
Figure 7
Analysis of the presence of Lon in the ER and mitochondria by confocal microscopy. Cells were treated for 24 h with increasing concentrations of efavirenz EFV), vehicle, thapsigargin (TG) 2 μM, rotenone (Rot) 25 μM or CCCP 10 μM and stained with (A) Hoechst 33342 (nuclei), anti‐calnexin (ER) antibody and anti‐Lon antibody or (B) Hoechst 33342 (nuclei), anti‐Lon antibody and Mitoview (MM fluorescent marker). Representative confocal microscopy images of 63× maginification with 3× optical zoom (A) or 3× optical + 1.5× digital zoom (B), and a histogram showing the index of correlation between Lon and ER (A) and Lon and mitochondria (B). White arrows show mitochondria that do not overlap with Lon. Data are shown as mean ± SEM, n = 5. Statistical analysis was performed by one‐way ANOVA (*P < 0.05 for efavirenz vs. MeOH and # P < 0.05 for thapsigargin, rotenone or CCCP vs. DMSO).
Figure 8
Figure 8
Analysis of the extramitochondrial expression of Lon. (A) Analysis of the purity of different cell fractions (mitochondrial, ER, cytosolic and MAMs) in untreated cells. Representative Western blotting image showing the expression of several mitochondrial (porin, cytochrome c, TOM20, Lon), ER (IP3R3), cytosolic (tubulin) and MAMs (FACL4, Grp75) proteins in basal conditions. (B) Western blotting analysis of Lon, Grp75 and PTPIP51 in cytosolic, ER, mitochondrial and MAM fraction obtained in cells treated for 24 h with efavirenz (EFV), thapsigargin (TG) 2 μM or CCCP 10 μM. A representative experiment is shown, quantified data are expressed as % of control (with the expression in the untreated cells in each fraction considered 100%). Ponceau staining of proteins was used as a loading control to normalize the results – equal relative amounts of protein were loaded per lane for each extract.

Comment in

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

MeSH terms

Feedback