Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

Abstract

Background: Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. Apart from Parkin, little is known about additional Ub (ubiquitin) ligases that mediate mitochondrial ubiquitination and turnover, particularly in highly metabolically active organs such as the heart.

Methods: In this study, we have combined in silico analysis and biochemical assay to identify CRL (cullin-RING ligase) 5 as a mitochondrial Ub ligase. We generated cardiomyocytes and mice lacking RBX2 (RING-box protein 2; also known as SAG [sensitive to apoptosis gene]), a catalytic subunit of CRL5, to understand the effects of RBX2 depletion on mitochondrial ubiquitination, mitophagy, and cardiac function. We also performed proteomics analysis and RNA-sequencing analysis to define the impact of loss of RBX2 on the proteome and transcriptome.

Results: RBX2 and CUL (cullin) 5, 2 core components of CRL5, localize to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, increased cardiomyocyte cell death, and has a global impact on the mitochondrial proteome. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to the rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. The action of RBX2 in mitochondria is not dependent on Parkin, and Parkin gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 (PTEN-induced kinase 1) in mitochondria.

Conclusions: These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that regulates mitophagy and cardiac homeostasis in a Parkin-independent, PINK1-dependent manner.

Keywords: heart failure; mitochondria; mitophagy; protein kinases; ubiquitination.

Figure 1.. RBX2 and Cul5 localize to the mitochondria.

A, Schematic of APEX2-catalyzed biotinylation at the mitochondrial outer membrane (MOM) via fusion to a mitochondrial targeted peptide derived from MAVS (mitochondrial antiviral-signaling protein). Overlap of APEX2-MOM data with the ESI (E3-substrate interaction) network reveals the association of CRLs with mitochondria. B, Representative Western blot of biotinylated proteins in neonatal rat ventricular cardiomyocytes (NRVCs) with adenoviral (Ad) expression of APEX2-MOM. NRVCs were treated with CCCP (10 μM) before H₂O₂ activation. The resultant biotinylated proteins were enriched by streptavidin beads. C, Representative Western blot of cytosol and mitochondrial fractions from NRVCs treated with CCCP (10 μM) for the indicated times. Arrowhead, neddylated CUL5. Tubulin and VDAC serve as cytosol and mitochondrial markers, respectively. D, Immuno-gold electron microscopic images showing the localization of RBX2 on mitochondrial membranes (arrowheads) in NRVCs expressing HA-RBX2. NRVCs infected with Ad-GFP and Ad-HA-OMP25 (MOM protein) serve as negative and positive controls, respectively. Scale bars, 0.5 μm. E, Representative Western blot of mitochondria with or without proteinase K (PK) treatment for 30 min on ice after isolation from NRVCs. F, Confocal images showing the colocalization (arrowhead) of RBX2 with mitochondria in CCCP-treated cardiomyocytes. NRVCs with adenoviral expression of HA-RBX2 were treated with CCCP for 3 hours and stained or immunostained as indicated. HA, green. Mitotracker, red. TOMM20, blue. Line scan co-localization analysis was done for all channels. Scale bars, 50 μm.

Figure 2.. RBX2 mediates mitochondrial ubiquitination and mitophagy.

A, Western blot of total cell lysates. NRVCs were transfected with indicated siRNAs, followed by CCCP (10 μM) treatment for the indicated times. B, Western blot of mitochondrial (mito) and cytosol (cyto) extracts. Cells were treated as described in A. C, Representative confocal images of live NRVCs stained with Mtphagy dye (red) and LysoTracker (blue) showing mitophagic vesicles. D, Quantification of mitophagic puncta per cell. E, Western blot of mitochondrial extracts. NRVCs were transfected with indicated siRNAs and treated with lysosomal protease inhibitors, E64d (E, 10 μg/ml) and pepstatin A (P, 10 μg/ml) for 6 hours before harvest. Fold-change of LC3-II and p62 levels between cells treated with and without lysosome inhibitors, indicative of mitophagy flux, is quantified on the right. F, Oxygen consumption rate (OCR) assessed by Seahorse analysis. Basal (Bas.) and maximal (Max.) respiration (resp.) are quantified on the right. siLuci: n=6, siRBX2: n=5 biological replicates. Data are representative of 3 repeats. G, Representative confocal images of NRVC stained with TMRM (1 μM, red) and MitoTracker (1 μM, green). The mean fluorescent intensity per view from 7–8 views (over 100 cells) per group is quantified on the right. H, Representative confocal images of NRVC stained with MitoSOX (red). The mean fluorescent intensity per view from 10 views (over 100 cells) per group is quantified on the right. Two-way ANOVA with the Tukey multiple comparisons test was used in D, the Mann-Whitney test in E-F, and unpaired t test in G-H.

Figure 3.. Deletion of Rbx2 in adult heart leads to heart failure and lethality.

A, Schematics of creation of tamoxifen-inducible, cardiac-specific RBX2 knockout (iCKO) mice. B, Western blot of indicated proteins in mouse hearts at 12 days after tamoxifen injection. C, Quantification of B. D, Survival curve. E, Gross morphology of mouse heart (top) and hematoxylin and eosin staining of myocardium section (bottom) at 12 days after tamoxifen injection. F, Heart weight to tibial length ratio and lung weight to tibial length ratio. F/F: n=13, MCM: n=5, iCKO: n=18 mice. G, Representative B-mode images. H, Quantification of echocardiographic parameters before (F/F: n=17, MCM: n=6, iCKO: n=23 mice) and after (F/F: n=12, MCM: n=6, iCKO: n=12 mice) tamoxifen treatment. I, Wheat germ agglutinin (WGA) staining (left) of myocardium sections and quantification of cardiomyocyte (CM) cross-sectional area (right). More than 100 cells/heart and 3 and 12 hearts from F/F and iCKO mice, respectively, were quantified. J, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of myocardium sections (left) and quantification (right). Three fields per heart, and 3 hearts per group, were quantified. K, qPCR analysis of the indicated genes. F/F: n=4, iCKO: n=4 mice. The Kruskal-Wallis test with the Dunn multiple comparisons test was used in C, F and H, Log-rank (Mantel-Cox) test in D, the Mann-Whitney U test in I-K.

Figure 4.. RBX2-regulated mitochondrial proteome.

A, Scheme of procedures for identification Rbx2-regulated proteome in NRVCs. NRVCs were transfected with indicated siRNAs, followed by CCCP (10 μM) treatment for 6 hours. Cell lysates were collected for trypsin digestion. The resultant peptides were labeled by TMT before mass spectrometry analysis. B, Principal component analysis (PCA) of normalized protein expression in whole cell proteome. CTL, siLuci. KD, siRBX2. C, Analysis of the percentage of differential expression of proteins (DEPs) in each group. D, Volcano plot of differentially expressed proteins (Sig., blue and red) in CCCP-treated RBX2-deficient CMs (KD) compared with CCCP-treated CTL. E, Venn diagram showing overlap of RBX2-regulated proteome and mitochondrial proteins annotated in MitoCarta 3.0. F, Venn diagram showing the identification of RBX2-regulated MOMs. G, Heatmap showing the relative expression of MOM proteins amongst the groups of cells. H, Western blot of MOM proteins in control and RBX2-deficient cardiomyocytes. N=4 biological replicates per group. Multiple unpaired t test with the Holm-Šídák correction was used. I, Western blot of WT and RBX2KO Hela cells treated with cycloheximide (CHX, 100 nM) for indicated times. RBX2 was deleted in Hela cells via CRISPR/Cas9-mediated gene editing. J, WT and RBX2KO Hela cells were transfected with plasmids expressing HA-Ub (pCDNA3-HA-Ub), treated with a proteasome inhibitor Bortezomib (BZM, 100 nM) for 6 hours, and subjected to immunoprecipitation followed by Western blot. The Mann-Whitney U test was used in I.

Figure 5.. Impaired mitochondrial ubiquitination and mitophagy in RBX2-deficient hearts.

Adult MCM and RBX2^iCKO (iCKO) mice were administered with tamoxifen (50 mg/kg/d for 5 days). Tissues were collected for indicated analyses (A-H) at 12 days after tamoxifen injections. A, Representative confocal images (left) of MCM and RBX2^iCKO myocardium sections immunostained with pUb (green), HSP60 (red, mitochondrial marker) and DAPI. Scale bars, 10 μm. B, Quantification of pUb+ foci normalized by mitochondria (HSP60+) area. A total of 16 views from two hearts per group were quantified. C-D, Western blot (C) and quantification (D) of mitochondrial (mito) and cytosolic (cyto) P62 in mouse hearts. E-F, Western blot (E) and quantification (F) of mitochondrial (mito) and cytosolic (cyto) LC3-II in mouse hearts. Mice at 12 days after tamoxifen administration were intraperitoneally injected with bafilomyocin A1 (BFA, 3 μmol/kg) for 3 hours before tissue harvest. 4 hearts per group were quantified. G, Representative confocal images of mt-Keima at 488 nm and 568 nm, respectively, in epicardial cardiomyocytes and the derived heatmaps. Neonatal MCM and RBX2^iCKO mice were transduced with AAV9-mt-Keima (1X10¹¹ GC/pup). At 10 weeks of age, mice were treated with tamoxifen and intact mouse hearts were excised 12 days later and scanned for mt-Keima signals in epicardial cardiomyocytes in situ with confocal microscope. Scale bars, 20 μm. H, Quantification of relative 568/488 ratio. 20–40 views per heart, 3 hearts per group were quantified. I, Western blot of indicated proteins in adult cardiomyocytes isolated from 2-month-old CTL (RBX2^F/F) or RBX2^CKO (CKO) mouse hearts. Results from two different batches of cells are shown. The Mann-Whitney test was used in B, D and F, and nested unpaired t test in H.

Figure 6.. Alterations in metabolic pathways and mitochondrial homeostasis in RBX2-deficient hearts.

A-B, Bulk RNA sequencing of mouse hearts at 12 days after tamoxifen (50 mg/kg/day for 5 days) treatment. A, KEGG pathways enriched in downregulated (top) and upregulated (bottom) genes in iCKO hearts. B, Chord plot showing downregulated genes involved in the indicated metabolic processes. C, Transmission electron microscopic images showing myofibril lysis (*), degenerating mitochondria (yellow arrowheads) surrounding by lysosomes (arrows), and abundant mitophagic vesicles (red arrowheads) in mutant cardiomyocytes. The number of mitophagic vesicles was quantified. Over 50 cardiomyocytes per group were quantified. D, Representative confocal images of myocardium sections immunostained with LAMP1 (green), TOMM20 (red) and DAPI (blue). Quantification of LAMP1+ puncta in mitochondria is shown. 5–6 views per heart, 2 hearts per group were quantified. E, Seahorse analysis of mitochondria isolated from mice at 8 days after tamoxifen injections. n=4 technical replicates/heart, 3 hearts/group were analyzed. Basal and maximal oxygen consumption rates are shown. F, Western blot of mitochondrial proteins in mouse hearts. *, cleaved form. Mann-Whitney test was used in C and D, and nested t test in E.

Figure 7.. The role of Parkin in RBX2-regulated mitochondrial ubiquitination and cardiac homeostasis.

A, Western blot of Parkin in NRVCs. Cells were infected with Ad-Parkin and transfected with indicated siRNAs. B, Representative Western blots of three independent repeats. Neonatal mouse ventricular CMs (NMVCs) were isolated from WT or Parkin^−/− mouse hearts, transfected with siRNA, and treated with CCCP (10 μM) for 12 hours. C, Western blots of cell lysates from NRVCs transfected with siRNAs and treated with CCCP (10 μM). D, Western blots of cell lysates from NRVCs infected with Ad-Parkin, transfected with siRNAs, and treated with CCCP (10 μM). E, Schematics of generation of RBX2 and Parkin double knockout (RBX2^CKO/Parkin^−/−) mice. F, Ejection fraction and fractional shortening at 5 (Parkin^−/−: n=5, RBX2^Het/Parkin^+/−: n=8, RBX2^CKO: n=12. RBX2^CKO/Parkin^−/−, n=8 mice) and 8 (Parkin^−/−: n=7, RBX2^Het/Parkin^+/−: n=7, RBX2^CKO: n=11. RBX2^CKO/Parkin^−/−, n=8 mice) months of age. G, Survival curves of indicated mice. Mann-Whitney test was used in A, one-way ANOVA followed by post hoc Tukey test in F, and Log-rank (Mantel-Cox) test in G.

Figure 8.. RBX2 stabilizes PINK1.

A, Western blots of indicated proteins in NRVCs. Cells were infected with Ad-PINK1, transfected with indicated siRNAs and treated with or without CCCP (10 μM) for 12 hours. B, Western blots. RBX2 was deleted in Hela cells via CRISPR/Cas9 using a single guided RNA against RBX2 (gRBX2). WT and RBX2KO cells with treated with CCCP (10 μM) for indicated times before harvest. C, Analysis of Pink1 transcript levels in NRVCs transfected with indicated siRNAs by qPCR. D, Western blots. NRVCs were infected with Ad-PINK1, transfected with siRNAs, and treated with Bortezomib (BZM, 100 nM) for 6 hours. E, Cycloheximide-based pulse chase assay. NRVCs were transfected with siRNAs, treated with CCCP (10 μM) for 3 hours, followed by removal of CCCP, and then chased for the indicated time in the presence of cycloheximide (CHX, 100 nM). F, Immunoprecipitation of PINK1, followed by Western blots. NRVCs were transfected with indicated siRNAs and treated with or without Bortezomib (BZM, 100 nM) for 6 hours. G, Western blots of cell lysates from NRVCs transfected with indicated siRNAs and treated with CCCP (10 μM) for 3 hours. H, A proposed model showing the role of RBX2-CRL5 in regulation of physiological mitophagy and cardiac homeostasis. Mann-Whitney test was used in C.