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. 2016 Feb;15(2):445-61.
doi: 10.1074/mcp.M115.051516. Epub 2015 Oct 8.

Succination Is Increased on Select Proteins in the Brainstem of the NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome

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Succination Is Increased on Select Proteins in the Brainstem of the NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome

Gerardo G Piroli et al. Mol Cell Proteomics. .
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Abstract

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys(77) and Cys(48) were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.

Figures

Fig. 1.
Fig. 1.
Protein succination is increased in the brainstem (BS) of Ndufs4 KO mice. A, Total cell lysates (30 μg protein) from BS of early (3 weeks old), middle (Mid, 6 weeks old) and late (9 weeks old) WT and Ndufs4 KO mice were separated by SDS/PAGE, and succinated proteins were detected using a polyclonal anti-2SC antibody (2SC panel), as described under “Experimental Procedures.” Protein succination in the BS of KO mice increased in association with the age of the mice and progression of disease versus WT controls. Succination was detected on a range of proteins and is especially prominent for proteins of ∼70–80 and ∼25–35 kDa (2SC panel). Note that intense tubulin (T) succination is present in all the lanes, and seems to increase with age in the BS of Ndufs4 KO mice. The bracket denotes the area circled in Fig. 3B. B, Succination of proteins is unchanged in skeletal muscle of Ndufs4 KO mice. Note that even when actin (A) is much more abundant (Coomassie panel), tubulin (T) is the primary succinated protein, confirming the selectivity of the modification. The identity of the tubulin band was confirmed in the BS by LC-MS/MS. C, Protein modification by the reactive lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and (D) glutathione (GSH) is unchanged in the BS of Ndufs4 KO mice. E, The total 2SC content of BS and muscle was determined by GC-MS/MS after acid hydrolysis of the protein. Note that 2SC content (expressed as mmol/mol lysine) is much higher in BS than in muscle. Results are presented as mean ± S.E. No significant differences between ages and genotypes were observed. In all the panels, triplicate samples were used for each age and genotype. In A, B, C, and D molecular masses of marker proteins are indicated on the left-hand side.
Fig. 2.
Fig. 2.
Endogenous succination does not alter tubulin polymerization in the brainstem of WT and Ndufs4 KO mice. A, BS samples were homogenized and centrifuged to obtain a cleared supernatant (Sup) as described under “Experimental Procedures.” When Sup was subjected to polymerization in the presence of taxol and high speed centrifugation, succinated tubulin (2SC panels) appeared in the microtubule pellet (Mt) but not in the remaining supernatant (SMt). Tubulin subunits (α-tubulin and β-tubulin panels) followed exactly the same pattern of distribution, indicating that endogenous succination of tubulin did not impair polymerization. Duplicate samples are shown for each genotype in this experiment; the same results were obtained in another experiment using a different set of samples. B, Total 2SC content (in mmol/mol Lys) was determined in Mt and SMt fractions after tubulin polymerization of BS samples by GC-MS/MS as described under “Experimental Procedures.” The distribution of succinated proteins favors the Mt fractions, especially in the BS of WT mice, whereas the BS of KO mice show a trend to increased total succination in the SMt fractions. Results are shown as the average of duplicate samples. C, Protein succination in the ∼50 kDa region of BS extracts from WT and Ndufs4 KO mice. For 30 μg protein samples succination increases in the KO group (30 μg, 2SC panel), but a slightly lower MW band is present in all samples (30 μg, Coomassie panel). With a reduced protein load of 5 μg, succination increases for the lower MW band in the BS of KO mice, with no changes in tubulin succination (5 μg, 2SC panel). β-tubulin and Coomassie staining are included to show even loading of the lanes (5 μg, β-tubulin and Coomassie panels). D–E, Cys376α is a prominent site of succination in the BS of the mouse. Mt fractions were resolved by SDS/PAGE and the tubulin bands at ∼50 kDa were excised and digested with trypsin prior to LC-MS/MS analysis as detailed under “Experimental Procedures.” D, MS/MS sequencing showing succination of Cys376α (C2SC) in the peptide AVCMLSNTTAIAEAWAR; the pyridylethylated version of Cys376α (CPE) is shown in (E).
Fig. 3.
Fig. 3.
VDAC1 and 2 are succinated in the brainstem of Ndufs4 KO mice. Protein (200 μg) from late stage (∼9 week old) WT and Ndufs4 KO BS was isoelectrically focused across a 4–7 pH range, followed by 2D electrophoresis and immunoblotting as described under “Experimental Procedures.” A–B, Increased succination was observed for a large number of proteins in the KO (panel B) versus WT (panel A) BS. The series of 2SC spots in the ∼25–35 kDa region (dashed oval in B) corresponds to the region in the bracket in Fig. 1A, where a significant increase in succination was observed in the KO BS. This series of spots was analyzed by mass spectrometry to identify succinated proteins (see E and F). The abundance of 2SC-tubulin is labeled (arrows). C–D, After stripping the blots shown in A and B, probing was performed with a DJ-1 antibody. Some overlapping with 2SC immunoreactivity was observed, suggesting DJ-1 succination (solid ovals). E–G, Duplicate 2D gels from WT and KO BS were stained with Coomassie blue, or transferred to PVDF for immunoblotting with our 2SC antibody (G, upper panel) and after stripping with an antibody to VDAC2 (G, lower panel). Spot picking from stained gels in the ∼25–35 kDa region using the 2SC blot as a guide, followed by in-gel digestion and MS/MS analysis allowed for the identification of VDAC1 and VDAC2 as prominent succinated proteins (see supplemental Table S2 for identified sequences). The MS/MS spectra for two specific peptides are shown in E, VTQSNFAVGYK for murine VDAC1 (MH+ = 1213.62480); and F, YQLDPTASISAK for murine VDAC2 (MH+ = 1293.67204). There is precise overlap between several 2SC-modified spots and those identified by anti-VDAC2 (dashed ovals, G). In A, B, C, D, and G, molecular masses of marker proteins are indicated on the left-hand side.
Fig. 4.
Fig. 4.
Protein succination is increased in brainstem and cerebellar nuclei of Ndufs4 KO mice. A, Micropunches obtained from the vestibular nuclei (VN) and inferior olive/gigantocellular reticular nucleus (IO/Gi) of the BS, and the fastigial nucleus (FN) and crus 1 ansiform lobule (Crus 1) of the CB were homogenized, and proteins separated by SDS-PAGE. Probing with the anti-2SC antibody revealed increased protein succination in all the regions in the Ndufs4 KO mice, particularly in the BS nuclei (2SC panel). VDAC1 immunoreactivity (VDAC1 panel) shows overlap with a band at ∼32 kDa that is more succinated in the BS nuclei of Ndufs4 KO mice. α-tubulin was used as a loading control (α-tubulin panel). B, C, Protein (150 μg) from late stage WT and Ndufs4 KO VN was isoelectrically focused across a 4–7 pH range, followed by 2D electrophoresis and immunoblotting as described under “Experimental Procedures.” 2D blots show a considerable increase in protein succination in the VN of Ndufs4 KO mice (C, 2SC panel), particularly in the ∼25–35 kDa region. Co-localization of VDAC1 immunoreactivity with protein succination occurs in both WT (B, VDAC1 and 2SC panels) and Ndufs4 KO (C, VDAC1 and 2SC panels), but the degree of succination is increased in VN of KO mice. The profile is similar for VDAC2, with a third spot of colocalization in Ndufs4 KO VN (C, VDAC2 and 2SC panels). α-tubulin (highlighted with a black oval) shows a similar pattern of succination (B and C, 2SC and α-tubulin panels). Molecular masses of marker proteins are indicated on the left-hand side.
Fig. 5.
Fig. 5.
Identification of succination sites in VDAC2. A, B, Mitochondrial-enriched fractions from Ndufs4 KO mouse brainstem were resolved by SDS-PAGE, gels were stained with Coomassie Brilliant Blue, bands at ∼30–35 kDa were excised, destained and digested with trypsin as described under “Experimental Procedures.” MS/MS spectra showing that Cys77 in the peptide YKWC2SCEYGLTFTEK of VDAC2 is endogenously succinated in vivo (A); the unmodified Cys77 in the peptide WCPEEYGLTFTEK was also identified after alkylation with 4-vinylpyridine (B). C, D, MS/MS spectra showing that Cys48 in the peptide SC2SCSGVEFSTSGSSNTDTGK of VDAC2 is endogenously succinated in vivo (C); the unmodified Cys48 in the peptide SCPESGVEFSTSGSSNTDTGK was also identified after alkylation with 4-vinylpyridine (D).

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