SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma

Abstract

Mitochondrial dysfunction and abnormal energy metabolism are major features of cancer. However, the mechanisms underlying mitochondrial dysfunction during cancer progression are far from being clarified. Here, it is demonstrated that the expression level of succinyl-coenzyme A (CoA) synthetase GDP-forming subunit β (SUCLG2) can affect the overall succinylation of lung adenocarcinoma (LUAD) cells. Succinylome analysis shows that the deletion of SUCLG2 can upregulate the succinylation level of mitochondrial proteins and inhibits the function of key metabolic enzymes by reducing either enzymatic activity or protein stability, thus dampening mitochondrial function in LUAD cells. Interestingly, SUCLG2 itself is also succinylated on Lys93, and this succinylation enhances its protein stability, leading to the upregulation of SUCLG2 and promoting the proliferation and tumorigenesis of LUAD cells. Sirtuin 5 (SIRT5) desuccinylates SUCLG2 on Lys93, followed by tripartite motif-containing protein 21 (TRIM21)-mediated ubiquitination through K63-linkage and degradation in the lysosome. The findings reveal a new role for SUCLG2 in mitochondrial dysfunction and clarify the mechanism of the succinylation-mediated protein homeostasis of SUCLG2 in LUAD, thus providing a theoretical basis for developing anti-cancer drugs targeting SUCLG2.

Keywords: SIRT5; SUCLG2; TRIM21; mitochondrial dysfunction; succinylation.

Figure 1

SUCLG2 is essential for the proliferation of LUAD cells and closely related to the poor survival of LUAD patients. A) The protein levels of SUCLG2 and SUCLA2 were detected by western blotting in LUAD cell lines and human bronchial epithelial cell line BEAS‐2B. B) The protein levels of SUCLG2 and SUCLA2 in paired LUAD tissues and adjacent normal tissues were detected by western blotting (left‐hand panels). N: adjacent normal tissue, T: tumor tissue. Protein quantification was performed using ImageJ software (right‐hand panel), and the results represent the average of 16 independent experiments (mean ± SD). ns > 0.05, ***p < 0.001. C and D) A549 cells and H1299 cells with or without SUCLG2 were seeded in 24‐well plates. At the indicated times, the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The dye was extracted with 10% acetic acid, and the relative proliferation was assessed based on the increase in absorbance at 595 nm. The data represent the average of three independent experiments (mean ± SD). ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. E–G) A549 cells (1 × 10⁷) with CTL‐KO or SUCLG2 depletion (SUCLG2‐KO) were subcutaneously injected into the flanks of nude mice (six mice per group). Twenty‐eight days later, the tumors were dissected out and photographed (E). The weight (F) and volume (G) of the xenograft tumors were measured. The P value was calculated by paired t‐test: *p < 0.05, **p < 0.01. H) Representative IHC images of xenograft tumors with anti‐Ki67 (left), anti‐TTF1 (middle), and SUCLG2 (right) antibodies (upper panels). Quantification of the IHC staining was performed using ImageJ software (bottom panel), and the results represent the average of six independent experiments (mean ± SD). **p < 0.01, ***p < 0.001. I) Microscopic evaluation of the IHC staining of a LUAD tissue microarray with an anti‐SUCLG2 antibody. N: adjacent normal tissue, T: tumor tissue. J) Quantification of the IHC staining shown in Figure 1I. K) Kaplan–Meier survival curve of 90 patients in the LUAD tissue microarray. Patient tissues were divided into two groups based on the average staining density of SUCLG2 in the tumor tissues of the tissue array (high expression: n = 45, low expression: n = 45; a log‐rank [Mantel–Cox] test was used for the statistical analysis).

Figure 2

SUCLG2 knockout induces mitochondrial dysfunction by affecting the metabolism of LUAD cells. A and B) A549‐SUCLG2‐KO and A549‐WT cells were used for untargeted negative ion mode metabolomics analysis (A). The negative ion mode metabolomics relative pathway analysis is shown (B). n = 6 per group. C) Mitochondrial morphology was observed by TEM. Scale bars: 2 µm (upper panels) and 1 µm (bottom panels). D) qPCR analysis of relative mtDNA content. Data represent the average of three independent experiments (mean ± SD). **p < 0.01, ***p < 0.001. E) ROS generation was assessed with 5 µM H2DCFDA added to A549 and H1299 cells or SUCLG2 knockout stable cell lines, followed by confocal microscopy. Data represent the average of three independent experiments (mean ± SD). ***p < 0.001. F) A549 and H1299 cells were transfected with individual siRNAs targeting SUCLG2. The intracellular ATP concentration was determined based on luminescence values and normalized to the protein content in each sample. Data represent the average of three independent experiments (mean ± SD). **p < 0.01, ***p < 0.001. G) The MMP of A549 and H1299 cells and SUCLG2 knockout stable cell lines was determined by adding JC‐1 and measuring immunofluorescence based on the absorbance at 490 nm/530 nm. Data represent the average of three independent experiments (mean ± SD). ***p < 0.001.

Figure 3

SUCLG2 knockout induces a global increase in protein succinylation. A) Succinate levels in A549 and H1299 cells and SUCLG2 knockout stable cell lines. Data represent the average of three independent experiments (mean ± SD). ***p < 0.001. B) The succinylation of proteins was detected in A549‐SUCLG2‐KO and A549‐WT cells using western blotting. C and D) The succinylation of proteins in A549‐SUCLG2‐KO and A549‐WT cells was analyzed with a 4D mass spectrometer. Intracellular distribution of succinylated proteins (C). Biological process analysis showing that upregulated succinylated proteins were significantly enriched in processes related to mitochondrial dysfunction (D). n = 3 per group. E) Schematic depicting the key enzymes related to mitochondrial function by knock outing SUCLG2. F–J) The succinylation levels of GAPDH, ME2, IDH2, MDH2, and ACOT9 in A549‐SUCLG2‐KO and A549‐WT cells measured by mass spectrometry. Data represent the average of three independent experiments (mean ± SD). *p < 0.05, **p < 0.01.

Figure 4

SUCLG2 affects protein stability or enzymatic activity by regulating succinylation. A–E) Western blotting was used to detect the succinylation of the GAPDH, ME2, IDH2, MDH2, and ACOT9 proteins in A549‐WT and A549‐SUCLG2‐KO cells. WCL: whole cell lysate. F–J) Western blotting was used to detect the succinylation of the GAPDH, ME2, IDH2, MDH2, and ACOT9 proteins in cells with or without SUCLG2 overexpression. K) The expression levels of GAPDH, ME2, IDH2, MDH2, and ACOT9 proteins in wild‐type or SUCLG2 knockout stable cell lines were detected by western blotting. L) The cells were treated with 20 µg mL⁻¹ CHX and collected at the indicated time. The degradation rate of the ME2 and ACOT9 proteins was detected by western blotting. M–P) The activity of GAPDH, ME2, IDH2, and MDH2 was detected using a specific enzyme activity detection kit. Data represent the average of three independent experiments (mean ± SD). ***p < 0.001.

Figure 5

TRIM21 induces SUCLG2 degradation through the K63‐linkage ubiquitin lysosomal pathway. A) BEAS‐2B, A549, HCC827, and H1299 cells were treated with 20 µg mL⁻¹ CHX and collected at the indicated time. The degradation rate of SUCLG2 was tested by western blotting (left‐hand panels). Relative SUCLG2 expression was analyzed using ImageJ (right‐hand panel). Data represent the average of three independent experiments (mean ± SD). ***p < 0.001. B) CHX (20 µg mL⁻¹) was added to A549 cells for 24 h, and CQ (20 µm) or MG132 (20 µm) was added at the same time. The expression of SUCLG2 was detected using western blotting. C) pcDNA3.1‐His‐SUCLG2 was overexpressed in A549 cells treated with MG132 (20 µm) or CQ (20 µm) for 24 h. Co‐IP and western blotting were used to detect the ubiquitination level of SUCLG2. D) The indicated plasmids were transfected into A549 cells treated with CQ (20 µm) for 24 h. Co‐IP was used to detect the K63‐linkage ubiquitination level of SUCLG2. E) The E3 ligase for SUCLG2 identified by mass spectrometry is shown. F) The interaction between SUCLG2 and TRIM21 was detected using co‐IP in A549 cells transfected with pcDNA3.1‐His‐SUCLG2 and pCMV‐HA‐TRIM21. G) A549 cells were transfected with pCMV‐HA‐TRIM21, and western blotting was used to detect the expression of the indicated proteins. H) A549 cells transfected with or without pCMV‐HA‐TRIM21 were treated with CHX (20 µg mL⁻¹) for the indicated times. The protein stability of SUCLG2 was detected by western blotting. I) The protein levels of SUCLG2 and TRIM21 were detected by western blotting in LUAD cell lines and human bronchial epithelial cell line BEAS‐2B. J) The protein levels of SUCLG2, TRIM21, and SIRT5 in 8 LUAD tissues were detected by western blotting (upper panels). T: tumor tissue. Protein correlation analysis was performed using ImageJ software and Prism 8 (bottom panels). K) The indicated plasmids were transfected into A549 cells. Co‐IP and western blotting were used to detect the K63 ubiquitination level of SUCLG2. L) The ubiquitination sites of SUCLG2 predicted by PhosphoSitePlus. M) A549 cells were transfected with the indicated plasmids, and the ubiquitination of SUCLG2 was detected by co‐IP and western blotting. N) A549 cells were transfected with the indicated plasmids, then treated with CHX (20 µg mL⁻¹) for 0, 3, 6, 9, 12, and 24 h. The degradation rate of SUCLG2 was detected by western blotting.

Figure 6

The K93‐succinylation of SUCLG2 affects the TRIM21‐mediated degradation of SUCLG2. A) pcDNA‐3.1‐His‐SUCLG2 was transfected into A549 cells, and co‐IP and western blotting were used to detect the succinylation level of SUCLG2. B) The succinylation sites of SUCLG2 predicted by PhosphoSitePlus. C) A549 cells overexpressing the SUCLG2‐WT or SUCLG2 lysine residue‐mutated plasmids were prepared, and co‐IP and western blotting were used to detect the succinylation of protein. D) A549 cells were transfected with the pcDNA‐His‐SUCLG2 or pcDNA‐His‐SUCLG2^K93R plasmids. The protein degradation rate of SUCLG2 was detected by western blotting. E) Differences in ubiquitination levels between SUCLG2‐WT and SUCLG2^K93R were assessed. Co‐IP and western blotting were used to detect the ubiquitination level of SUCLG2. F) A549 cells were transfected with the indicated plasmids, and co‐IP and western blotting were used to detect the interaction between SUCLG2 and TRIM21. G) A549 cells were transfected with the indicated plasmids. The ubiquitination of the SUCLG2 protein was detected by co‐IP and western blotting. H) A549 cells were transfected with pcDNA‐His‐SUCLG2 or pcDNA‐His‐SUCLG2^K93R plasmids, and co‐IP and western blotting were used to detect the interaction between SUCLG2 and p62.

Figure 7

SIRT5 is the desuccinylase for SUCLG2 and affects the degradation of the SUCLG2 protein. A and B) In A549 cells, SIRT5 was overexpressed (A) or knocked down (B) and the succinylation of SUCLG2 was detected by co‐IP and western blotting. C and D) In A549 cells, SUCLG2 (C) or SIRT5 (D) was overexpressed, and the interaction between SIRT5 and SUCLG2 was assessed using co‐IP and western blotting. E and F) The co‐localization of SUCLG2 and SIRT5 was detected by immunofluorescence (E) and mitochondrial isolation, followed by western blotting (F). G and H) In A549 cells, SIRT5 was overexpressed (G) or knocked down (H) and the SUCLG2 protein was detected by western blotting. I) SIRT5 was overexpressed in A549 cells. The degradation rate of the SUCLG2 protein was detected by western blotting. J and K) The indicated plasmids or SIRT5 siRNAs were transfected into A549 cells. Co‐IP and western blotting were used to detect the ubiquitination level of SUCLG2. L) The indicated plasmids were transfected into A549 cells. Co‐IP and western blotting were used to detect the ubiquitination level of SUCLG2. M) Co‐IP was performed in A549 cells transfected with the indicated plasmids before subjecting them to western blotting. N) Co‐IP was performed in A549 cells transfected with SUCLG2 and SIRT5 before subjecting them to western blotting with an anti‐p62 antibody. O) Co‐IP was performed in A549 cells transfected with SUCLG2^K93R and SIRT5 before subjecting them to western blotting with a succinylation antibody.

Figure 8

Inhibition of succinylation at the SUCLG2 K93 site leads to the mitochondrial dysfunction and reduced tumorigenesis of LUAD cells. A and B) LUAD cell lines A549 (A) and H1299 (B) were transfected with His‐SUCLG2 and HA‐TRIM21 plasmids. Twenty‐four hours later, cells were seeded in 24‐well plates. At the indicated times, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The dye was extracted with 10% acetic acid, and the relative proliferation was assessed based on the increase in absorbance at 595 nm (upper panels). Western blotting was used to determine the overexpression efficiency (bottom panels). The data represent the average of three independent experiments (mean ± SD). **p < 0.01, ***p < 0.001. C and D) LUAD cell lines A549 (C) and H1299 (D) were transfected with His‐SUCLG2 and Flag‐SIRT5 plasmids. Cell growth assays were performed. (upper panels). Western blotting was used to determine the overexpression efficiency (bottom panels). The data represent the average of three independent experiments (mean ± SD). ***p < 0.001. E) A549‐SUCLG2^WT and A549‐SUCLG2^K93R cell lines were constructed by CRISPR‐Cas9. The expression of the SUCLG2 protein was detected by western blotting. F) Mitochondrial morphology was observed using TEM. Scale bars: 2 µm (upper panels) and 1 µm (bottom panels). G) qPCR analysis of mtDNA content in A549‐SUCLG2^WT and A549‐SUCLG2^K93R cells. Data represent the average of three independent experiments (mean ± SD). **p < 0.01. H) ROS generation was assessed with 5 µm H2DCFDA added to A549‐SUCLG2^WT and A549‐SUCLG2^K93R cells, followed by confocal microscopy. The data represent the average of three independent experiments (mean ± SD). ***p < 0.001. I) The intracellular ATP concentration was determined in A549‐SUCLG2^WT and A549‐SUCLG2^K93R using luminescence values and normalized to the protein content in each sample. The data represent the average of three independent experiments (mean ± SD). **p < 0.01. J) A549‐SUCLG2^WT and A549‐SUCLG2^K93R cells (5 × 10³) were seeded in 24‐well plates. At the indicated times, the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The dye was extracted with 10% acetic acid, and the relative proliferation was assessed based on the increase in absorbance at 595 nm. The data represent the average of three independent experiments (mean ± SD). **p < 0.01. K–M) A549‐SUCLG2WT and A549‐SUCLG2K93R cells were subcutaneously injected into the flanks of nude mice (six mice per group). Twenty‐eight days later, the tumors were dissected out and photographed (K). The weight (L) and volume (M) of xenograft tumors were measured. The data represent the average of six independent experiments (mean ± SD). **p < 0.01, *p < 0.05. N) Representative IHC images of xenograft tumors with anti‐Ki67 and anti‐TTF1 (left‐hand panels). Quantification of the IHC staining was performed using ImageJ software (right‐hand panel), and the results represent the average of four independent experiments (mean ± SD). **p < 0.01, ***p < 0.001.