Journal: The Journal of Biological Chemistry

Article Title: Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

doi: 10.1016/j.jbc.2022.101723

Figure Lengend Snippet: Characterization of the SIRT5 crKO cell line. A , schematic of the CRISPR-Cas9 system used to generate the SIRT5 crKO cell line. B , schematic of the human SIRT5 gene showing the sites of crRNA targets within Exon5/4 the four known SIRT5 coding transcripts in humans. C , immunoblot for SIRT5 protein from HEK293T crWT ( circles ) clone and crKO ( blank ) clone (using CRISPR guide E, Table S1 ). D – F , immunoblots of SIRT5 crWT and crKO whole-cell lysates blotted using antibodies against glutaryl-lysine ( D ), glutaryl-succinyl-lysine ( E ), and malonyl-lysine ( F ). G – I , relative quantification of acylated proteins normalized to β-actin levels: glutarylation ( G ), succinylation ( H ), and malonylation ( I ) (mean ± SD, n = 3, ∗ p -value ≤ 0.05). crRNA, crispr RNA; PAM, protospacer adjacent motif; SIRT5, sirtuin 5; tracrRNA, trans-activating crispr RNA.

Article Snippet: For immunoprecipitation of endogenous SIRT5 (Millipore-Sigma #HPA022002), GCDH (Millipore-Sigma # HPA048492; Thermo # PA5-60294), glutaryl-lysine- (Cell Signaling Technologies, non-commercial #14943MF; PTMScan #26101), and succinyl-lysine- (Cell Signaling Technologies, noncommercial #13599) proteins, 1 to 10 mg of precleared lysate/500 μl of lysis buffer was incubated with 2 to 10 μg of IP antibody overnight at 4 °C.

Techniques: CRISPR, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

doi: 10.1016/j.jbc.2022.101723

Figure Lengend Snippet: Glutarylation-state of GCDH is regulated by Sirt5. A , immunoblot for immunopurified GCDH-FLAG by Flag-M2 resin or glutaryl-proteins by anti-glutaryl-lysine antibody (Glut-K) from HEK293T cells grown in complete media. Blots representative of at least three independent experiments. B , immunoblot for immunopurified GCDH-FLAG by Flag-M2 resin from SIRT5 crWT or crKO cells grown in complete media or acylation media (DMEM without glucose, glutamine, pyruvate, 10% FBS). Blots are representative of at least three independent experiments and quantitative values of glutaryl-lysine intensity normalized to FLAG intensity expressed as ratios relative to control (overexpressed GCDH-FLAG in SIRT5 crWT cells grown in complete media, lane 2). C , immunoblot of immunopurified GCDH-FLAG by Flag-M2 resin from HEK293T cells grown in complete media with or without coexpressed SIRT5-HA. Blots representative of at least three independent experiments and quantitative values of glutaryl-lysine intensity normalized to FLAG intensity expressed as ratios relative to control (overexpressed GCDH-FLAG, lane 2). D , immunoblot for immunopurified GCDH-FLAG by Flag-M2 resin from SIRT5 crWT or crKO cells grown in complete media or glutarylation media (EBSS containing 5 mM glucose, +50 mM Hepes, +0.8 mM lysine) with or without coexpressed SIRT5-HA. Blots are representative of at least three independent experiments and quantitative values of glutaryl-lysine intensity normalized to FLAG intensity expressed as ratios relative to control (overexpressed GCDH-FLAG in SIRT5 crWT cells grown in complete media, lane 1). E , immunoblot for pan-glutaryl lysine immunopurified proteins by endogenous GCDH and glutaryl-lysine from 3-month-old wild-type (SIRT5WT) and Sirt5 −/− (SIRT5KO) mice liver tissue that were refed or starved for 1 h following a 24 h starvation. Red arrow indicates the GCDH band within the glutaryl blot. Blots representative of at least three mice per group (n = 3 per group). F , relative quantification of the immunoblots in ( E ). Data representative of a single experiment from six SIRT5WT and six SIRT5KO mouse livers starved for 24 h from which half were refed (3 SIRT5WT and 3 SIRT5KO) normal diet for 1 h, while the remaining half (three SIRT5WT and three SIRT5KO) continued starvation for additional 1 h (mean ± SD, n = 3 per group). WT and Gcdh −/− (GCDHKO) mice (n = 1) were used as positive and negative controls, respectively, for identifying the correct GCDH band. One-way ANOVA followed by Tukey’s post hoc test was performed for multiple comparisons across groups where ∗ p -value = 0.0173, ∗∗ p -value = 0.0018, ∗∗∗ p -value = 0.0005, ∗∗∗∗ p -value < 0.0001. DMEM, Dulbecco’s modified Eagle’s medium; EBSS, Earle's Balanced Salt Solution; endo-SIRT5, endogenous SIRT5; FBS, fetal bovine serum; GCDH, glutaryl-CoA dehydrogenase.

Article Snippet: For immunoprecipitation of endogenous SIRT5 (Millipore-Sigma #HPA022002), GCDH (Millipore-Sigma # HPA048492; Thermo # PA5-60294), glutaryl-lysine- (Cell Signaling Technologies, non-commercial #14943MF; PTMScan #26101), and succinyl-lysine- (Cell Signaling Technologies, noncommercial #13599) proteins, 1 to 10 mg of precleared lysate/500 μl of lysis buffer was incubated with 2 to 10 μg of IP antibody overnight at 4 °C.

Techniques: Western Blot, Modification

Journal: The Journal of Biological Chemistry

Article Title: Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

doi: 10.1016/j.jbc.2022.101723

Figure Lengend Snippet: Glutarylation impairs GCDH activity and SIRT5 partially restores it. A , immunoblot of chemically modified GCDH (hGCDH-6xHIS) using glutaryl-CoA with or without incubation with recombinant SIRT5 (mSIRT5-6xHIS). Quantitative values of glutaryl-lysine intensity normalized to total protein are expressed as ratios relative to control (glutaryl-modified recombinant GCDH, lane 2). B , enzymatic activity of unmodified ( black ), glutarylated ( dark purple ), and deglutarylated ( light purple ) GCDH determined by using PMS/DCPIP as artificial electron acceptors and glutaryl-CoA as electron donor (mean ± SD, n = 6, 6, and 5 for unmodified, modified, and deacylated GCDH respectively; ∗ p -value ≤ 0.05). C , enzymatic activity of unmodified ( black ), glutarylated ( dark purple ), and de-glutarylated ( light purple ) GCDH determined by using ETF/DCPIP as an electron acceptor and glutaryl-CoA as electron donor (mean ± SD, n = 4, 4, and 5 for unmodified, modified, and deacylated GCDH respectively, ∗ p -value ≤ 0.05). D and E , glutaryl-lysine sites identified by label-free quantitative LC-MS/MS on recombinant GCDH mapped to full-length human GCDH protein schematic with domain and residues important for cofactor, substrate, or tetramer interactions. Panel D shows glutaryl-lysine sites in chemically glutarylated GCDH sample expressed as a ratio over unmodified GCDH (glutarylation: glutarylated GCDH/unmodified). Panel E shows glutaryl-lysine sites on chemically glutarylated GCDH altered by SIRT5 incubation expressed as a ratio over glutarylated GCDH (deglutarylation: (glutarylated GCDH + SIRT5)/glutarylated GCDH). Plots representing Log 2 fold change (y-axis) and p -value (larger circle size = more significant p -value). F and G , homotetrameric structure of human GCDH with each subunit colored separately ( purple , blue , red , and green ). Yellow sticks indicate modified lysine residues. The FAD cofactor and CoA substrate are black and light gray spheres , respectively. H , far-UV CD spectra for glutarylated GCDH ( purple ) and unmodified GCDH ( black ) corrected for protein concentration (5 μM). I , thermal stability profiles for glutarylated GCDH ( open circles , purple curve ) and unmodified GCDH ( closed squares , dark curve ). The solid lines represent two-state sigmoid curves from which the apparent midpoint temperatures were determined. J , immunoblot of native-PAGE separated GCDH tetramer and SDS-PAGE for denatured GCDH isolated from liver mitochondria of 24 h starved SIRT5WT and SIRT5KO mice. K , quantitation of GCDH tetramer intensity normalized to ETF complex intensity. Data representative of two independent experiments (mean ± SD, n = 3 for both SIRT5WT and SIRT5KO). ETF, electron transfer flavoprotein; GCDH, glutaryl-CoA dehydrogenase; SIRT5, sirtuin 5.

Article Snippet: For immunoprecipitation of endogenous SIRT5 (Millipore-Sigma #HPA022002), GCDH (Millipore-Sigma # HPA048492; Thermo # PA5-60294), glutaryl-lysine- (Cell Signaling Technologies, non-commercial #14943MF; PTMScan #26101), and succinyl-lysine- (Cell Signaling Technologies, noncommercial #13599) proteins, 1 to 10 mg of precleared lysate/500 μl of lysis buffer was incubated with 2 to 10 μg of IP antibody overnight at 4 °C.

Techniques: Activity Assay, Western Blot, Modification, Incubation, Recombinant, Liquid Chromatography with Mass Spectroscopy, Protein Concentration, Clear Native PAGE, SDS Page, Isolation, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

doi: 10.1016/j.jbc.2022.101723

Figure Lengend Snippet: SIRT5 ablation impairs lysine oxidation and glutaryl-CoA oxidation in cells and mice. A , schematic of lysine oxidation enzymes with steps that release carbon dioxide (CO 2 ). B , oxidation of [U- 14 C]-lysine to [ 14 C]-CO 2 in 293T SIRT5 crWT and crKO cells. Data representative of three independent experiments (mean ± SD, n = 6; ∗ p -value ≤ 0.05). C , oxidation of [1, 5- 14 C]-glutaryl-CoA to [ 14 C]-CO 2 in mitochondria isolated from 3 h starved 293T SIRT5 crWT and crKO cells. Data representative of single experiment (mean ± SD, n = 4; ∗ p -value ≤ 0.05). D , oxidation of [1, 5- 14 C]-glutaryl-CoA to [ 14 C]-CO 2 in mitochondria isolated from SIRT5WT and SIRT5KO mouse liver. Data representative of single experiment (mean ± SD, n = 6, 8 for SIRT5WT and SIRT5KO respectively; ∗ p -value ≤ 0.05). SIRT5, sirtuin 5.

Article Snippet: For immunoprecipitation of endogenous SIRT5 (Millipore-Sigma #HPA022002), GCDH (Millipore-Sigma # HPA048492; Thermo # PA5-60294), glutaryl-lysine- (Cell Signaling Technologies, non-commercial #14943MF; PTMScan #26101), and succinyl-lysine- (Cell Signaling Technologies, noncommercial #13599) proteins, 1 to 10 mg of precleared lysate/500 μl of lysis buffer was incubated with 2 to 10 μg of IP antibody overnight at 4 °C.

Techniques: Isolation

Journal: The Journal of Biological Chemistry

Article Title: Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

doi: 10.1016/j.jbc.2022.101723

Figure Lengend Snippet: Characterization of the SIRT5 crKO cell line. A , schematic of the CRISPR-Cas9 system used to generate the SIRT5 crKO cell line. B , schematic of the human SIRT5 gene showing the sites of crRNA targets within Exon5/4 the four known SIRT5 coding transcripts in humans. C , immunoblot for SIRT5 protein from HEK293T crWT ( circles ) clone and crKO ( blank ) clone (using CRISPR guide E, Table S1 ). D – F , immunoblots of SIRT5 crWT and crKO whole-cell lysates blotted using antibodies against glutaryl-lysine ( D ), glutaryl-succinyl-lysine ( E ), and malonyl-lysine ( F ). G – I , relative quantification of acylated proteins normalized to β-actin levels: glutarylation ( G ), succinylation ( H ), and malonylation ( I ) (mean ± SD, n = 3, ∗ p -value ≤ 0.05). crRNA, crispr RNA; PAM, protospacer adjacent motif; SIRT5, sirtuin 5; tracrRNA, trans-activating crispr RNA.

Article Snippet: We immunopurified glutarylated proteins using the PTMScan pan-glutaryl-lysine affinity resin (Cell Signaling Technologies) and immunoblotted for endogenous GCDH.

Techniques: CRISPR, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

doi: 10.1016/j.jbc.2022.101723

Figure Lengend Snippet: Glutarylation-state of GCDH is regulated by Sirt5. A , immunoblot for immunopurified GCDH-FLAG by Flag-M2 resin or glutaryl-proteins by anti-glutaryl-lysine antibody (Glut-K) from HEK293T cells grown in complete media. Blots representative of at least three independent experiments. B , immunoblot for immunopurified GCDH-FLAG by Flag-M2 resin from SIRT5 crWT or crKO cells grown in complete media or acylation media (DMEM without glucose, glutamine, pyruvate, 10% FBS). Blots are representative of at least three independent experiments and quantitative values of glutaryl-lysine intensity normalized to FLAG intensity expressed as ratios relative to control (overexpressed GCDH-FLAG in SIRT5 crWT cells grown in complete media, lane 2). C , immunoblot of immunopurified GCDH-FLAG by Flag-M2 resin from HEK293T cells grown in complete media with or without coexpressed SIRT5-HA. Blots representative of at least three independent experiments and quantitative values of glutaryl-lysine intensity normalized to FLAG intensity expressed as ratios relative to control (overexpressed GCDH-FLAG, lane 2). D , immunoblot for immunopurified GCDH-FLAG by Flag-M2 resin from SIRT5 crWT or crKO cells grown in complete media or glutarylation media (EBSS containing 5 mM glucose, +50 mM Hepes, +0.8 mM lysine) with or without coexpressed SIRT5-HA. Blots are representative of at least three independent experiments and quantitative values of glutaryl-lysine intensity normalized to FLAG intensity expressed as ratios relative to control (overexpressed GCDH-FLAG in SIRT5 crWT cells grown in complete media, lane 1). E , immunoblot for pan-glutaryl lysine immunopurified proteins by endogenous GCDH and glutaryl-lysine from 3-month-old wild-type (SIRT5WT) and Sirt5 −/− (SIRT5KO) mice liver tissue that were refed or starved for 1 h following a 24 h starvation. Red arrow indicates the GCDH band within the glutaryl blot. Blots representative of at least three mice per group (n = 3 per group). F , relative quantification of the immunoblots in ( E ). Data representative of a single experiment from six SIRT5WT and six SIRT5KO mouse livers starved for 24 h from which half were refed (3 SIRT5WT and 3 SIRT5KO) normal diet for 1 h, while the remaining half (three SIRT5WT and three SIRT5KO) continued starvation for additional 1 h (mean ± SD, n = 3 per group). WT and Gcdh −/− (GCDHKO) mice (n = 1) were used as positive and negative controls, respectively, for identifying the correct GCDH band. One-way ANOVA followed by Tukey’s post hoc test was performed for multiple comparisons across groups where ∗ p -value = 0.0173, ∗∗ p -value = 0.0018, ∗∗∗ p -value = 0.0005, ∗∗∗∗ p -value < 0.0001. DMEM, Dulbecco’s modified Eagle’s medium; EBSS, Earle's Balanced Salt Solution; endo-SIRT5, endogenous SIRT5; FBS, fetal bovine serum; GCDH, glutaryl-CoA dehydrogenase.

Article Snippet: We immunopurified glutarylated proteins using the PTMScan pan-glutaryl-lysine affinity resin (Cell Signaling Technologies) and immunoblotted for endogenous GCDH.

Techniques: Western Blot, Modification

Journal: The Journal of Biological Chemistry

Article Title: Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

doi: 10.1016/j.jbc.2022.101723

Figure Lengend Snippet: Glutarylation impairs GCDH activity and SIRT5 partially restores it. A , immunoblot of chemically modified GCDH (hGCDH-6xHIS) using glutaryl-CoA with or without incubation with recombinant SIRT5 (mSIRT5-6xHIS). Quantitative values of glutaryl-lysine intensity normalized to total protein are expressed as ratios relative to control (glutaryl-modified recombinant GCDH, lane 2). B , enzymatic activity of unmodified ( black ), glutarylated ( dark purple ), and deglutarylated ( light purple ) GCDH determined by using PMS/DCPIP as artificial electron acceptors and glutaryl-CoA as electron donor (mean ± SD, n = 6, 6, and 5 for unmodified, modified, and deacylated GCDH respectively; ∗ p -value ≤ 0.05). C , enzymatic activity of unmodified ( black ), glutarylated ( dark purple ), and de-glutarylated ( light purple ) GCDH determined by using ETF/DCPIP as an electron acceptor and glutaryl-CoA as electron donor (mean ± SD, n = 4, 4, and 5 for unmodified, modified, and deacylated GCDH respectively, ∗ p -value ≤ 0.05). D and E , glutaryl-lysine sites identified by label-free quantitative LC-MS/MS on recombinant GCDH mapped to full-length human GCDH protein schematic with domain and residues important for cofactor, substrate, or tetramer interactions. Panel D shows glutaryl-lysine sites in chemically glutarylated GCDH sample expressed as a ratio over unmodified GCDH (glutarylation: glutarylated GCDH/unmodified). Panel E shows glutaryl-lysine sites on chemically glutarylated GCDH altered by SIRT5 incubation expressed as a ratio over glutarylated GCDH (deglutarylation: (glutarylated GCDH + SIRT5)/glutarylated GCDH). Plots representing Log 2 fold change (y-axis) and p -value (larger circle size = more significant p -value). F and G , homotetrameric structure of human GCDH with each subunit colored separately ( purple , blue , red , and green ). Yellow sticks indicate modified lysine residues. The FAD cofactor and CoA substrate are black and light gray spheres , respectively. H , far-UV CD spectra for glutarylated GCDH ( purple ) and unmodified GCDH ( black ) corrected for protein concentration (5 μM). I , thermal stability profiles for glutarylated GCDH ( open circles , purple curve ) and unmodified GCDH ( closed squares , dark curve ). The solid lines represent two-state sigmoid curves from which the apparent midpoint temperatures were determined. J , immunoblot of native-PAGE separated GCDH tetramer and SDS-PAGE for denatured GCDH isolated from liver mitochondria of 24 h starved SIRT5WT and SIRT5KO mice. K , quantitation of GCDH tetramer intensity normalized to ETF complex intensity. Data representative of two independent experiments (mean ± SD, n = 3 for both SIRT5WT and SIRT5KO). ETF, electron transfer flavoprotein; GCDH, glutaryl-CoA dehydrogenase; SIRT5, sirtuin 5.

Article Snippet: We immunopurified glutarylated proteins using the PTMScan pan-glutaryl-lysine affinity resin (Cell Signaling Technologies) and immunoblotted for endogenous GCDH.

Techniques: Activity Assay, Western Blot, Modification, Incubation, Recombinant, Liquid Chromatography with Mass Spectroscopy, Protein Concentration, Clear Native PAGE, SDS Page, Isolation, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

doi: 10.1016/j.jbc.2022.101723

Figure Lengend Snippet: SIRT5 ablation impairs lysine oxidation and glutaryl-CoA oxidation in cells and mice. A , schematic of lysine oxidation enzymes with steps that release carbon dioxide (CO 2 ). B , oxidation of [U- 14 C]-lysine to [ 14 C]-CO 2 in 293T SIRT5 crWT and crKO cells. Data representative of three independent experiments (mean ± SD, n = 6; ∗ p -value ≤ 0.05). C , oxidation of [1, 5- 14 C]-glutaryl-CoA to [ 14 C]-CO 2 in mitochondria isolated from 3 h starved 293T SIRT5 crWT and crKO cells. Data representative of single experiment (mean ± SD, n = 4; ∗ p -value ≤ 0.05). D , oxidation of [1, 5- 14 C]-glutaryl-CoA to [ 14 C]-CO 2 in mitochondria isolated from SIRT5WT and SIRT5KO mouse liver. Data representative of single experiment (mean ± SD, n = 6, 8 for SIRT5WT and SIRT5KO respectively; ∗ p -value ≤ 0.05). SIRT5, sirtuin 5.

Article Snippet: We immunopurified glutarylated proteins using the PTMScan pan-glutaryl-lysine affinity resin (Cell Signaling Technologies) and immunoblotted for endogenous GCDH.

Techniques: Isolation

Journal: The Journal of Biological Chemistry

Article Title: Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

doi: 10.1016/j.jbc.2022.101723

Figure Lengend Snippet: Characterization of the SIRT5 crKO cell line. A , schematic of the CRISPR-Cas9 system used to generate the SIRT5 crKO cell line. B , schematic of the human SIRT5 gene showing the sites of crRNA targets within Exon5/4 the four known SIRT5 coding transcripts in humans. C , immunoblot for SIRT5 protein from HEK293T crWT ( circles ) clone and crKO ( blank ) clone (using CRISPR guide E, Table S1 ). D – F , immunoblots of SIRT5 crWT and crKO whole-cell lysates blotted using antibodies against glutaryl-lysine ( D ), glutaryl-succinyl-lysine ( E ), and malonyl-lysine ( F ). G – I , relative quantification of acylated proteins normalized to β-actin levels: glutarylation ( G ), succinylation ( H ), and malonylation ( I ) (mean ± SD, n = 3, ∗ p -value ≤ 0.05). crRNA, crispr RNA; PAM, protospacer adjacent motif; SIRT5, sirtuin 5; tracrRNA, trans-activating crispr RNA.

Article Snippet: Antibodies: anti-MG-lysine (Millipore-Sigma # ABS2120-25UG), anti-SIRT5 (Millipore-Sigma #HPA022002 [for human SIRT5] and Cell Signaling Technologies #8782 [for mouse SIRT5]), anti-GCDH (Millipore-Sigma # HPA048492), mouse anti-β-actin (Cell Signaling Technologies, #3700), rabbit anti-β-actin (Cell Signaling Technologies #8457), anti-glutaryl-lysine (Cell Signaling Technologies, noncommercial #14943MF and PTM-Biolabs #1151), anti-succinyl-lysine (Cell Signaling Technologies, noncommercial # 13599 and PTM-Biolabs #401), anti-malonyl-lysine (Cell Signaling Technologies #14942), anti-acetyl-lysine (Cell Signaling Technologies, #9441), IRDye 680RD donkey anti-mouse IgG (LI-COR #926-68072), and IRDye 800CW donkey anti-rabbit IgG (LI-COR #926-32213).

Techniques: CRISPR, Western Blot