Metabolism is a fundamental cellular process that is coordinated with cell cycle progression. Despite this association, a mechanistic understanding of cell cycle phase-dependent metabolic pathway regulation remains elusive. Here we report the mechanism by which human de novo pyrimidine biosynthesis is allosterically regulated during the cell cycle. Combining traditional synchronization methods and metabolomics, we characterize metabolites by their accumulation pattern during cell cycle phases and identify cell cycle phase-dependent regulation of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase (CAD), the first, rate-limiting enzyme in de novo pyrimidine biosynthesis. Through systematic mutational scanning and structural modelling, we find allostery as a major regulatory mechanism that controls the activity change of CAD during the cell cycle. Specifically, we report evidence of two Animalia-specific loops in the CAD allosteric domain that involve sensing and binding of uridine 5′-triphosphate, a CAD allosteric inhibitor. Based on homology with a mitochondrial carbamoyl-phosphate synthetase homologue, we identify a critical role for a signal transmission loop in regulating the formation of a substrate channel, thereby controlling CAD activity.
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We sincerely appreciate E. Freinkman for cell cycle LC–MS metabolomics analysis, Q. Spillier, R. Bahn and M. Pacold for technical advice on biochemistry and CETSA, S. Parker for GC–MS operation and analysis, D. Jones, R. Rose, Y. Siu and A. Korman for UTP-targeted LC–MS analysis, M. Becerra Flores for ICM-Pro operation, P. Lopez and F. Joseph for FACS analysis, M. Pagano and V. Donato for advice on cell cycle analysis, P. A. Bullock, L. Wu, I. Mohr, C. Y. Park and members of the R.P. laboratory for general discussion, and V. Rubio for discussion on CPS structure, enzymology and allostery. plentiCRISPR v2 was a gift from F. Zhang. Funding was provided by National Institutes of Health grants T32CA009161 (supporting J.S.) R01CA214948 (to R.P.) and R01GM132491 (to R.P.), Pew Charitable Trusts (to R.P.), Alexander and Margaret Stewart Trust (to R.P.) and the American Cancer Society (to R.P.). The metabolomics laboratory was supported by National Institute of Health grant P30CA016087.
The authors declare no competing interests.
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Extended Data Fig. 1 Acquisition of cell populations at different phases of the cell cycle for targeted metabolomics or for measurement of isotopically labeled pyrimidine intermediates.
a,b, Schematic diagrams of DTB (a), or NOC block (b) cell synchronization procedures employed. c, Schematic diagram showing experimental overview of cell cycle metabolomics. d,e, Metabolic pathways showing participation of extracellular glucose and glutamine into de novo pyrimidine biosynthesis, highlighted (red) shows exogenously supplemented amide-15N-glutamine. f,g, Estimation of relative concentrations (top) or fraction enrichments (bottom) of amide-15N-glutamine-derived metabolites shown in (e) upon DTB release (f), or NOC release (g). h, Measurement of thymine peak height by GC-MS. i, Diagram from (d) with highlighted (red) pathway, exogenously supplemented α-15N-glutamine. j, Estimation of relative concentrations (top) or fraction enrichments (bottom) of α-15N-glutamine-derived metabolites shown in (i) upon the time course sampling of cells post-DTB release. 1 h isotopic labeling. k, Diagram from (d) with highlighted (red) pathway, exogenously supplemented uniformly labeled-13C-glucose. 3 h isotopic labeling. l, Estimation of relative concentrations (top) or fraction enrichments (bottom) of uniformly labeled-13C-glucose-derived metabolites shown in (k) upon the time course sampling of cells post-DTB release. m, A 2-dimensional plot of cells distributed by intracellular complexity (granularity; the Side Scatter (SSC) parameter measured by flow cytometer) and DAPI-stained genomic DNA. 7 different gates constructed for acquisition of cells at different phases of the cell cycle as shown in (n). o, An experimental diagram showing a cell sorting strategy for acquisition of cells at different phases of the cell cycle, primarily targeting cells through the 4 gates (#1, 3, 4 and 7). This strategy includes asynchronous cells supplemented with amide-15N-glutamine (1 h) prior to a fluorescence-activated cell sorting (FACS; Hoechst 33342, 10 µM, 20 h). p, Estimation of relative concentrations of amide-15N-glutamine-derived de novo pyrimidine biosynthetic intermediates. Data are the mean ± s.d. (n = 3, biological replicates). Two-tailed Student’s t-test. *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001, or NS.
a, western blots of canonical cell cycle indicating proteins, cyclin F and phosphorylated histone H3, and de novo pyrimidine proteins of interest, CAD and DHODH. b, Measurements of cell parameters after DTB release: cell number, mean cell volume, and total cell/culture volume in absolute or relative values. Data are the mean ± s.d. (n = 3, technical replicates, 5 independent experiments). c, EdU incorporation of cells synchronized by DTB. d, Volumetric distribution of cells synchronized by DTB.
a,b, Positive ion (a), or negative ion (b) lipidomics of wild-type HeLa cells synchronized by DTB. c, 3–6 h post-DTB-specific accumulations of lipid species. Data are the mean ± s.d. (n = 3, biological replicates). d, Differential accumulation of di- (left) or triglycerides (right) of wild-type HeLa cells synchronized by DTB.
Extended Data Fig. 4 Cell cycle-independent transcription of CAD and CAD co-essential pyrimidine metabolic genes.
a, Top 100 CAD co-dependent genes (DepMap, Broad Institute). b, CRISPR-mediated knock-out effect of CAD co-essential pyrimidine genes on numerous cancer cell lines (DepMap, Broad Institute). UCK1, a cell non-essential gene, and RRM2, a cell-essential gene, are also laid out as extreme examples. c, A curated human pyrimidine metabolism map (modified KEGG). Allosteric activation by PRPP is noted in a green dotted-arrow whereas allosteric inhibition by UTP is in a red dotted-pole. d, Relative transcription levels of CAD co-dependent pyrimidine genes (Cyclebase 3.0). RRM2 and CCNF (cyclin F) are examples of genes known with cell cycle-dependent transcription level change.
Extended Data Fig. 5 Prediction of glycerol gradient-sedimented CAD oligomers, and observation of cell cycle-independent CAD oligomerization.
a,b, Coomassie blue- (a) or silver-stained (b) 4–12% Bis-Tris gel, loaded with a glycerol gradient molecular standard mixture of thyroglobulin, apoferritin, β-amylase, and bovine serum albumin differentially resolved through glycerol gradient rate zonal centrifugation. The results were reproducible over 5 times ((a) and (b) are independent). c, Relative abundance of the silver-stained standards in (b). d, Estimation of glycerol gradient fractions corresponding to CAD oligomers by interpolating CAD oligomer molecular sizes into a regression line generated by the sedimentation information of standard molecules. e, A crystal structure of aspartate transcarbamoylase (ATCase) domain of CAD with R2187 residue highlighted in red (PDB: 5G1O; apo). Each monomer of the ATCase trimer is distinctly colored. f, A crystal structure of dihydroorotase (DHOase) domain of CAD with M1601 residue highlighted in red and pink (PDB: 4C6D; N-carbamoyl-L-aspartate (substrate) bound). Each monomer of the DHOase dimer is distinctly colored. g, Western blots of CAD differentially expressed in R2187A, M1601E, or R2187A/M1601E CAD mutant-expressing sgCAD single cell clones selected. The single cell clones with a red asterisk were mainly tested for CAD activity estimation in this report. h, Western blots of CAD of DTB-synchronized wild-type HeLa cell lysates differentially sedimented throughout a 10–35 % glycerol gradient by rate-zonal centrifugation (left). Quantification of CAD protein intensity from the western blotting results (right). i, Western blots of CAD of NOC-synchronized wild-type HeLa cell lysates differentially sedimented throughout a 10–35 % glycerol gradient by rate-zonal centrifugation (left). Quantification of CAD protein intensity from the western blotting results (right).
Extended Data Fig. 6 Verification of cell cycle synchronization of G-Loop1 CAD mutant cells and estimation of their CAD activity during the cell cycle.
a,b, Cell parameter measurement (a) and EdU incorporation (b) of wild-type HeLa cells synchronized by NOC. c,d, Cell parameter measurement (c) and (d) EdU incorporation (d) of wild-type HeLa cells synchronized by DTB. e, Estimation of CAD activity of wild-type HeLa cells synchronized by DTB. f,g, Cell parameter measurement (f) and EdU incorporation (g) of G-Loop1 HeLa cells synchronized by NOC. h,i, Cell parameter measurement (h) and EdU incorporation (i) of G-Loop1 HeLa cells synchronized by DTB. j, Estimation of CAD activity of G-Loop1 HeLa cells synchronized by DTB. Data are the mean ± s.d. (n = 3, biological replicates). Two-tailed Student’s t-test. *P < 0.1, **P < 0.01, ***P < 0.001, ****P< 0.0001, or NS.
Extended Data Fig. 7 Verification of identical thermal shifts of wild-type and G-Loop1 CAD upon addition of ATP or CTP.
a, Cellular thermal shift assay (CETSA) of wild-type HeLa in the presence of different amounts of UTP (left), and relative CAD intensities are estimated (right). b, Cellular thermal shift assay (CETSA) of wild-type HeLa, wild-type CAD-reconstituted and G-Loop1-expressing sgCAD cells (left), and relative CAD intensities are estimated (right). No nucleotides were incubated with the cell lysates. c,d, Cellular thermal shift assay (CETSA) of wild-type HeLa or G-Loop1-expressing sgCAD cells in the presence of 30 mM of ATP (c; left) or CTP (d; left), and relative CAD intensities are estimated (c,d; right).
Extended Data Fig. 8 Verification of CAD mutant protein expression level in selected single cell clone cell lines, and estimation of their CAD activities.
a, Immunoblots of sgCAD single cell clone #1 and 8. Clones with red asterisk used in this report. b, Immunoblots of CAD in wild-type or mutant CAD-expressing sgCAD single cell clones. Wild-type HeLa cell lysate was loaded in titration in western blot for selection of clones with comparable CAD expression. Clones with red asterisk used in this report. c, e, Immunoblots (c) and relative activity (e) of CAD in single cell clones differentially expressing CAD mutants whose mutations correspond to UMP-interacting residues in E. Coli carB. d,f, Immunoblots (d) and relative activity (f) of CAD in single cell clones differentially expressing CAD mutants whose allosteric domain is partially or entirely truncated or glycine-substituted. g,h, Immunoblots (g) and relative activity (h) of CAD in single cell clones differentially expressing CAD mutants whose Loop1 is partially replaced with alanine(s). k, Relative activity of CAD in single cell clones expressing CAD mutants whose three glutamates are substituted for alanines (3E-3A), ± uridine supplementation (100 µg ml-1, 3 h). i,j, Immunoblots (i) and relative activity (j) of CAD in single cell clones differentially expressing CAD mutants whose Loop2 is partially replaced with alanine(s). l,m, Immunoblots (l) and relative activity (m) of CAD in single cell clones differentially expressing CAD mutants whose Loop1 or 2 is duplicated (only glutamates/arginines were mutated; Loop1 3E-3R for Loop1 duplication; Loop2 3R-3E for Loop2 duplication (Loop2 3R-3E immunoblots not shown)) or switched (3E@3R). n, Relative 15N-dihydroorotate concentration of CAD in single cell clones whose three glutamates of Loop1 are replaced with arginines (Loop1 3E-3R). o, Immunoblots of CAD in single cell clones differentially expressing CAD mutants whose T′-loop is replaced with CPS 1T′-loop. Clones with red asterisk used in this report. Data are the mean ± s.d. (n = 3, biological replicates). Two-tailed Student’s t-test vs. wild-type HeLa. *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001, or NS.
Extended Data Fig. 9 Proposed mechanisms of UTP-mediated allosteric regulation in wild-type HeLa, G-Loop1, and T′-loop chimera CAD cells and verification of cell cycle synchronization of T′-loop chimera CAD mutant cell line.
a, Structural comparisons of human CAD allosteric domain structure(s) homology-modeled against E. coli carB or human CPS1. Crystal structure of E. coli carB allosteric domain (top left), two human CPS1 allosteric domains (top right; allosteric activator-bound (active state) and apo (inactive state)). Predicted human CAD allosteric domain structures homology-modeled against E. coli carB (bottom left), human CPS1 (bottom right). T′-loop colored green (active), red (inactive). Allosteric ligands shown. Annotated residues exemplify differences in structure between E. coli carB- and human CPS1-based models. b, CAD phylogenetic tree. Uniprot codes in parenthesis. Human CAD, CPS1, and E. coli carB, colored red. c, Overview of pairwise alignment. Percentage identity noted below. d, Docking simulations of UTP-relevant metabolites in human CPS1-based allosteric domain structure of human CAD. e, Alignment of human CAD CPS2 and CPS1 T′-loops. f, Relative frequencies of residue interactions between T′-loop and other domains of human CAD CPS2. Analyses based upon two structures of human CAD CPS2, predicted by homology modeling against human CPS1 structures (PDB: 5DOU, active; 5DOT, inactive). T′-loop-interacting residues in active state (top) or inactive state (bottom) model quantitated via counting neighboring residues of conserved or divergent residues of T′-loop. Human CAD T′-loop residues conserved in human CPS1 are green; divergent, red. Allosteric domain of human CAD, grey. g, Proposed mechanism of CAD allosteric regulation. When [UTP] < Kd of UTP for CAD allosteric domain, Loop1 and Loop2 interact with and stabilize T′-loop, leading to substrate channel formation; active CAD. When [UTP] > Kd, UTP binds to allosteric domain and recruits Loop1 and Loop2, destabilizing T′-loop and substrate channel; inactive CAD. In G-Loop1 mutant, Loop1 constantly interacts with T′-loop, inducing T′-loop stability and substrate channel formation; constitutively active CAD. In T′-loop chimera, [UTP] > Kd, UTP binds to the allosteric domain and recruits Loop1 and Loop2. Because of T′-Loop replacement, loop1-T’-loop interactions and stability regulation are perturbed; no UTP-mediated allosteric regulation. h,i, Cell parameter (h) and EdU incorporation (i) of T′-loop chimera, NOC synchronized. Data are the mean ± s.d. (n = 3, technical replicates, 2 independent experiments).
Extended Data Fig. 10 Measurements of uridine auxotrophic proliferation and uridine-dependent CAD activity change of HeLa, RPE-1, A549, and MDA-MB-231 cell lines established upon transduction of indicated CAD mutants.
a–c, HeLa. d–f, RPE-1. g–i, A549. j–l, MDA-MB-231. a,d,g,j, Immunoblots of CAD in the indicated cell lines expressing indicated CAD mutants. b,e,h,k, Proliferation of indicated CAD-mutant expressing cells in standard conditions, relative to uridine supplemented (100 µg ml-1). Data are the mean ± s.d. (n = 3, technical replicates, 2 independent experiments). Two-tailed Student’s t-test statistical analyses applied. Compared to wild-type counterpart. c,f,i,l, Estimation of 15N-dihydroorotate concentration of indicated CAD mutant cells in the absence and presence of uridine (100 µg ml-1, 1 h). 1 h tracing. Data are the mean ± s.d. (n = 3, biological replicates). Two-tailed Student’s t-test statistical analyses applied. *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001, or NS.
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Shin, J., Mir, H., Khurram, M.A. et al. Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle. Nat Metab 5, 277–293 (2023). https://doi.org/10.1038/s42255-023-00735-9
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