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Acylspermidines are conserved mitochondrial sirtuin-dependent metabolites

Nature Chemical Biology (2024)Cite this article

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Abstract

Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent protein lysine deacylases regulating metabolism and stress responses; however, characterization of the removed acyl groups and their downstream metabolic fates remains incomplete. Here we employed untargeted comparative metabolomics to reinvestigate mitochondrial sirtuin biochemistry. First, we identified N-glutarylspermidines as metabolites downstream of the mitochondrial sirtuin SIR-2.3 in Caenorhabditis elegans and demonstrated that SIR-2.3 functions as a lysine deglutarylase and that N-glutarylspermidines can be derived from O-glutaryl-ADP-ribose. Subsequent targeted analysis of C. elegans, mouse and human metabolomes revealed a chemically diverse range of N-acylspermidines, and formation of N-succinylspermidines and/or N-glutarylspermidines was observed downstream of mammalian mitochondrial sirtuin SIRT5 in two cell lines, consistent with annotated functions of SIRT5. Finally, N-glutarylspermidines were found to adversely affect C. elegans lifespan and mammalian cell proliferation. Our results indicate that N-acylspermidines are conserved metabolites downstream of mitochondrial sirtuins that facilitate annotation of sirtuin enzymatic activities in vivo and may contribute to sirtuin-dependent phenotypes.

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Fig. 1: Comparative metabolomics uncovers decrease in N-glutarylspermidines in sir-2.3 mutants.
Fig. 2: Protein lysine deglutarylation is dependent on sir-2.3.
Fig. 3: OAADPR can serve as an acyl donor.
Fig. 4: N-acylspermidines are mammalian metabolites and dependent on SIRT5.
Fig. 5: N-glutarylspermidine production is linked to mitochondrial metabolism.
Fig. 6: Bioactivity of N-glutarylspermidines and model for N-acylspermidine production and function.

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Data availability

Information on newly identified metabolites has been deposited in the SMID database (https://www.smid-db.org) and the ChEBI dictionary (https://www.ebi.ac.uk/chebi/); web links and accession codes for each metabolite are listed in Supplementary Table 1. HPLC–MS data are available in the MassIVE database under accession number MSV000091755, including C18LC–MS data of WT and sir-2.3-mutant C. elegans for untargeted metabolomic analysis, HILIC–MS data showing separation of N-glutarylspermidine isomers, HILIC-targeted MS/MS data for characterization of N-acylspermidines and N-acylspermines and HILIC–MS detection of N-acylspermidines and N-acylspermines in mouse tissues. Protein sequences were retrieved from UniProt (https://www.uniprot.org), and protein 3D structure predictions were retrieved from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk) under accession codes SIRT4 (human), Q9Y6E7; SIRT4 (mouse), Q8R216; SIR-2.2 (C. elegans), Q20480; SIR-2.3 (C. elegans), Q20481. Source data are provided with this paper.

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Acknowledgements

We thank the CGC for C. elegans strains, N. Burgess-Brown for the MACROD1 plasmid, S. Abrams for AT-3 cells, J. Auwerx for Sirt5-KO mice, J. Blum for mouse Sirt5 sgRNA constructs, M. Deininger for the plasmid with SIRT5-targeted shRNA, D. Fajardo Palomino for maintaining and genotyping C. elegans strains, the Cornell Institute of Biotechnology for DNA sequencing, T. H. Won for advice on mouse metabolite extraction, H. Lin and the laboratory of F.C.S. for advice and the laboratory of F.C.S. for comments on the manuscript. This work was partly supported by the NIH (R35 GM131877 to F.C.S., R01 CA223534 to R.S.W., P40 OD010440 to the CGC, GM69702 for APBS-PDB2PQR software). I.R.F. was supported by HHMI Gilliam Fellowship GT11525, and J.M. was supported by NIH grant F30 CA250451.

Author information

Authors and Affiliations

  1. Boyce Thompson Institute, Cornell University, Ithaca, NY, USA

    Bingsen Zhang, Andreas H. Ludewig, Tyler R. Bales & Frank C. Schroeder

  2. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

    Bingsen Zhang, Irma R. Fernandez & Frank C. Schroeder

  3. Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA

    James Mullmann, Irma R. Fernandez & Robert S. Weiss

  4. Department of Molecular Medicine, Cornell University, Ithaca, NY, USA

    James Mullmann

  5. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

    Tyler R. Bales

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Contributions

R.S.W. and F.C.S. supervised the study. B.Z., R.S.W. and F.C.S. designed experiments. J.M. and I.R.F. prepared mammalian samples. A.H.L. generated the sir-2.2;sir-2.3 double mutant strain and performed C. elegans-aging assays. J.M. performed compound testing in mammalian cells. B.Z. and T.R.B. performed all other chemical and biological experiments. B.Z. and F.C.S. wrote the manuscript with input from the other authors.

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Correspondence to Frank C. Schroeder.

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F.C.S. is a cofounder and stockholder of Holoclara and Ascribe Bioscience, which develop nematode-derived small molecules for biomedical and agricultural applications. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Model for the regulation of mitochondrial protein lysine acylation by sirtuins.

a, Non-enzymatic protein lysine acylation occurs in the high-pH environment of mitochondria. Four or five-carbon (main chain) dicarboxyl-CoAs can form reactive anhydride intermediates and can be further captured by lysine residues. Sirtuin-mediated deacylation requires NAD+ as a cofactor: following formation of a complex of acylated substrate-ADP-ribose and sirtuin, a conserved histidine residue of the sirtuins deprotonates the 2′-OH of ribose that then attacks the carbonyl carbon of the alkylimidate intermediate to form a 1′,2′-cyclic intermediate, which is finally hydrolyzed to 2′/3′-O-acyl-ADP-ribose (OAADPR). b, Table summarizing the localization and in vitro deacylation activity of seven mammalian sirtuins, with their C. elegans orthologs. C. elegans has two mitochondrial sirtuins, sir-2.2 and sir-2.3; both are orthologs of mammalian SIRT4 that robustly removes five-carbon (main chain) dicarboxyl modifications.

Extended Data Fig. 2 Decreased abundance of N-glutarylspermidine in sir-2.3 null mutants.

a, Untargeted comparative metabolomics of the exo-metabolome from WT and sir-2.2(n5134) mutant C. elegans. Bubble sizes reflect peak areas. n = 4. b, Extracted ion chromatograms (EICs) for m/z 302.2074 ± 5 ppm (C14H28N3O4+) showing that this feature is absent in bacterial food (OP50) and downregulated in the sir-2.3 mutant compared to WT. Representative of n = 5. c, daspid#4 (N1-glutaryl isomer) is more abundant than daspid#3 (N8-glutaryl isomer) quantified by HILIC-MS1. N2 n = 6, sir-2.3 n = 4, sir-2.2;sir-2.3 n = 2. d, Numbering scheme of spermidine. e, f, N-acetyl (e), N-succinyl (f), and N-malonyl (f) spermidines remained unchanged upon sir-2.3 deletion. n = 4. g, Major fragmentation reactions and resulting fragment ions of maspid#3 and maspid#4 in tandem mass spectrometry. Comparison of levels of maspid#3 and maspid#4 in different biological conditions were based on HILIC-PRM. EIC is ESI+ of indicated m/z ± 5 ppm. h, daspid#3 and daspid#4 are mainly secreted, and maspid#4 are mainly retained in worm bodies. daspid#3 and daspid#4: WT, n = 8; sir-2.3, n = 6; sir-2.2;sir-2.3, n = 3. maspid#3 and maspid#4: WT, n = 5; sir-2.3, n = 3; sir-2.2;sir-2.3, n = 2. i, HPLC-MS-based quantification of daspid#3 and daspid#4 in exo-metabolomes collected at different time intervals, corresponding to different developmental stages. daspid#3 and daspid#4 are mainly produced starting in young adulthood. Data were normalized to ascr#3. n = 2. j, Only trace amounts of N-glutarylspermidine derivatives are present in the metabolome of bacterial food (OP50), compared to the C. elegans metabolome. Representative of n > 3. n represents the number of independent experiments in a, e, f, and h, and the number of biologically distinct samples in c and i. P values were calculated by unpaired, two-tailed t-test with Welch correction in a. Data are mean (±s.d.) in c, e, f, and h.

Source data

Extended Data Fig. 3 MS/MS analyses of newly characterized dicarboxylic acid-derived N-acylspermidines.

a–l, Major fragmentation reactions and resulting fragment ions are indicated with MS/MS spectra of synthetic standards of the glutaryl, succinyl, or malonyl spermidine derivatives, all obtained in ESI+ mode.

Extended Data Fig. 4 O-acyl-ADP-ribose can serve as an acyl donor.

a, Scheme for comparing acyl transfer from acetyl-CoA and O-acetyl-ADPR to spermidine at neutral or slightly basic pH. Products of acyl transfer were derivatized with isobutyl chloroformate and analyzed by HPLC-MS. b, c, Estimation of the second-order rate constants for acyl transfer to form N1-acetylspermidine from AcCoA (b) or OAcADPR (c) at pH 8.0. Number of independent assays, n = 2. d, daspid#3 and daspid#4 are also produced from supplemented D4-glutaric acid. Representative of three experiments. e, Amounts of daspid#3 and daspid#4 produced from supplemented D4-glutaric acid (detected at m/z 306.2325) do not differ between WT and sir-2.3. Number of independent experiments, n = 3. f, Cartoon illustrating the production of N-glutarylspermidine from extracellular lysine and glutaric acid. g, Expression of human MacroD1 (ΔN58aa truncated) with an infusion thioredoxin and His6 tag in Rosetta(DE3) cells grown in LB induced by isopropyl β-D-1-thiogalactopyranoside. Elutions from a Ni-NTA column for recombinant protein purification are shown. h, TEV sequence was cleaved by TEV protease to release the MacroD1 for assays. Shown is the input and column-purified product. i, Steady-state kinetic analysis of OAcADPR and OGADPR hydrolysis catalyzed by human MacroD1 (ΔN58aa truncated) reveals both deacylation reactions follow saturation kinetics. The steady-state kinetic parameters Km and Vmax are determined by HPLC-MS for ADP-ribose formation to be 427 ± 74 µM and 0.17 ± 0.01 s−1 for OAcADPR, and 1020 ± 283 µM and 0.46 ± 0.08 s−1 for OGADPR. The reaction mixtures contain 0.5 µM MacroD1. Number of independent assays using the same batch of purified enzyme, n = 3. Data are mean ± s.d. in e and i. P values were calculated using unpaired, two-tailed t-tests with Welch correction in e. g and h are results from one experiment without attempts of replication.

Source data

Extended Data Fig. 5 Characterization of acylated polyamines in mouse.

a, b, Representative EICs of characteristic fragment ions of different N-acylspermidines using targeted MS/MS across different mouse tissues, demonstrating that dicarboxylic acid-derived spermidines are mammalian metabolites. Gaussian smoothing of the HPLC-MS raw data was enabled for the following metabolites: daspid#3, daspid#7, daspid#8, maspid#7 and #8 (using 5 raw data points); daspid#5, maspid#3 and #4 (using 7 data points). c, d, MS/MS analyses of N1-glutarylspermine (c) and N1-succinylspermine (d). Major fragmentation reactions and resulting fragment ions are indicated with MS/MS spectra of synthetic standards obtained in ESI+ mode. e, Representative EICs of N1-acylspermines across different mouse tissues. The dicarboxylic acid-derived spermines were below detection limit.

Extended Data Fig. 6 Relative abundances of N-acylspermidines detected in mouse liver, brain, and heart samples.

N-acylspermidine levels did not significantly differ between WT and Sirt5 KO samples, except for a decrease of maspid#3 (N8-glutaryl) and maspid#5 (N8-succinyl) in the Sirt5 KO brain samples. Data represent total peak intensity in each sample, based on detected peak area and sample volume, per tissue weight. Mice were all female. n = 5, except for WT mouse brain, n = 4. Data are mean ± s.d. and P values were calculated using unpaired, two-tailed t-tests with Welch correction (P ≥ 0.1 are not shown).

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Extended Data Fig. 7 N-acylspermidines levels in WT and SIRT5 KO cell cultures.

a-g. Comparison of N-acylspermidine levels in WT and Sirt5 KO AT-3 cell cultures, corresponding to Fig. 4b. h-o. Comparison of N-acylspermidine levels in WT and SIRT5 KO HCT116 cell cultures, corresponding to Fig. 4c. Number of biologically distinct samples in each of the three independent experiments, n = 3. Data are mean ± s.d., and P values were calculated by unpaired, two-tailed t-tests with Welch correction (P ≥ 0.15 are not shown).

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Extended Data Fig. 8 N-glutarylspermidine production is linked to mitochondrial metabolism.

a, The lysine origin of the glutaryl moiety in daspid#3 and daspid#4 can be inferred from robust incorporation of five 13C atoms in the molecular ion (a) as well as in the glutaryl-containing fragment (Fig. 5c) in isotope tracing experiments with 13C6,15N2-L-lysine. b, The ratio of labelled daspid#3 and daspid#4 closely reflected that of the corresponding unlabeled compounds in the lysine tracing experiment and was similar to the ratio of products obtained from reaction of O-glutaryl-ADP-ribose with spermidine (Fig. 3d), but differed from that of in the D4-glutaric acid labelling experiments (Fig. 3f), consistent with sir-2.3-dependent formation of daspid#3 and daspid#4 via mitochondrial lysine catabolism. Number of biologically distinct samples, n = 3. c, Representative images for delayed/aborted development of lysine-supplemented sir-2.3 mutant animals. Developmentally delayed animals are indicated by arrows. Scale bar: 1 mm. Spectra in a are representative of three independent experiments. Images in c are representative of four experiments.

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Extended Data Fig. 9 Bioactivity of N-glutarylspermidines.

a, Abundance of N-glutarylspermidines in worm bodies after supplementation of each compound at 5 µM in the growth media. The endogenous levels were first shown in Fig. 3g. Number of biologically distinct samples, n = 3. b, maspid#3 inhibited the clonogenic growth of AT-3 cells. Colony formation assay with AT-3 cells over 12 days after treatment with maspid#3 or maspid#4 in the first 3 days. See Supplementary Fig. 6 for additional images. c, maspid#3 inhibited the clonogenic growth of AT-3 cells culturing in both regular and heat-inactivated bovine serum. d, maspid#3 inhibited the clonogenic growth of MDA-MB-231 cells. +Dox, doxycycline-induced knockdown of SIRT5. e, Cells took up maspid#3 and maspid#4 to similar extents. Cells were treated with 10 µM of compound for 48 h, and the abundance of compounds in cell pellets was measured by HILIC-HRMS. Number of biologically distinct samples n = 4. f, Known mammalian oxidation reactions of polyamines and polyamine derivatives produce reactive nucleophiles (aldehydes) and hydrogen peroxide. Polyamine oxidase (PAOX) oxidizes N1-acetylated spermidine or spermine. Spermine oxidase (SMOX) oxidizes spermine. g, Hypothetical oxidation intermediates and products of N-glutarylspermidines that contain isotope labels in the glutaryl moiety. Oxidation sites are shown in colored bonds (top). One of the predicted oxidation products of maspid#3 was observed (see h for characterization) when supplementing AT-3 cells. h, Supplementation with maspid#3 resulted in production of N-glutarylputrescine, consistent with polyamine oxidase acting on maspid#3, in AT-3 cells. The glutaryl-containing fragments of N-glutarylputrescine contained the D4-label derived from N8-glutaryl(D4)-spermidine, confirming that N-glutarylputrescine originated from supplemented maspid#3. Representative of three experiments. i, Oxidase(s) in the bovine serum can also oxidize maspid#3 to form N-glutarylputrescine, but this activity can be largely inhibited by heat inactivation before the assay. The growth-inhibition effect of maspid#3 reproduced when culturing cells using heat-inactivated serum (c), indicating that oxidase activity in the culture media was not responsible for the observed effects of maspid#3 on cell proliferation. Number of biologically distinct samples, n = 3. Data are mean ± s.d. in a, e and i.

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Extended Data Fig. 10 Examination of effects of N-glutarylspermidines on SIRT5 in vitro.

a, maspid#3 and maspid#4 were not SIRT5 substrates. O-glutaryl-ADP-ribose was not produced when incubating 100 µM maspid#3 or maspid#4 with 1 µM recombinant SIRT5 and 1 mM NAD+ in Tris buffer (pH 7.5) for 2 hr at 37 °C. A peptide with succinylated lysine was used as positive control. n = 2. b, N-glutarylspermidines had no inhibitory effects on SIRT5. maspid#3 to maspid#4 ratio was 1:1. Nicotinamide was used as a positive control, IC50 = 22 µM. n = 2. n, number of assays using the same commercial enzyme sample.

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Supplementary information

Supplementary Information

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Zhang, B., Mullmann, J., Ludewig, A.H. et al. Acylspermidines are conserved mitochondrial sirtuin-dependent metabolites. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-023-01511-2

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