Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a , Model of the major 4-HA catabolism pathway and its integration into FAO as previously described , . Question marks indicate uncharacterized enzymatic steps in mammals. b , Multiple-sequence alignment demonstrating conservation between LvaA and LvaC of the lva operon and eukaryotic ACAD10/11 homologs. c , LC–MS analysis of in vitro enzyme reactions with 4-HV-CoA as the starting substrate. Top: the raw signal intensities and chemical structures of 4-HV-CoA, 4-PV-CoA and 2-pentenoyl-CoA. Bar graphs represent data from reactions with recombinant ACAD10 NΔ34 (top) and ACAD11 (bottom). The same no-enzyme control data are displayed on both sets of graphs for clearer comparison. Data are expressed as the mean ± s.d. ( n = 3 technical replicates). d , Ferrocenium ion assay with recombinant ACAD family members and unsubstituted acyl-CoA substrates of various lengths. Top: general structure of the final product, enoyl-CoA. Initial velocity of the enzyme reaction corresponds to the change in absorbance over time (mean ± s.d.; n = 4 technical replicates). e , UV–vis quantification of 2-hexenoyl-CoA produced from 4-PH-CoA substrate (mean ± s.d.; n = 3 technical replicates). f , g , Michaelis–Menten plots of ACAD11 kinase ( f ) and ACAD domain ( g ) activities against synthetic 4-HV-CoA and 4-PV-CoA, respectively. Data are normalized to K* and A* inactive mutant controls. Each data point represents the mean ± s.d. ( n = 3 technical replicates; n = 6 technical replicates for WT in f ; only averages are shown for clarity). Statistical analysis was conducted using a one-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test ( c ), two-way ANOVA with Šídák’s multiple-comparisons test comparing the activity of each enzyme against an acyl-CoA substrate to the respective no-enzyme control ( d ) or one-way ANOVA with Dunnett’s multiple-comparisons test comparing all reactions with the no-enzyme control ( e ). * P < 0.05, ** P < 0.01 and *** P < 0.001. ND, not detected.

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Sequencing, Liquid Chromatography with Mass Spectroscopy, In Vitro, Recombinant, Control, Comparison, Produced, Mutagenesis, Activity Assay

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a , Sequence identity of lva operon enzymes to human proteins as reported by UniProtKB database. b , Sequence similarity of LvaC relative to mouse ACAD family according to pairwise alignments. c , SDS-PAGE gel of recombinant mouse ACAD10 NΔ34 and ACAD11 protein preparations from P. pastoris or RIPL E. coli orthogonal expression systems, respectively. WT = wild type; K* = Kinase inactive mutant; A* = ACAD inactive mutant. d , Table describing the site-specific mutations to inactivate recombinant ACAD10 NΔ34 and ACAD11 kinase and ACAD catalytic domains. e , HPLC chromatogram of acyl-CoA standards. f , LC-MS analysis of in vitro enzyme reactions in Fig. lacking 4-HV-CoA substrate. Displayed are the raw signal intensities and chemical structures of 4-HV-CoA, 4-PV-CoA, and 2-pentenoyl-CoA (above corresponding graphs). Top and bottom bar graphs represent data from reactions with recombinant ACAD10 NΔ34 and ACAD11, respectively. The same “no enzyme” control data is displayed on both sets of graphs for clearer comparison. Data are expressed as mean -/+ SD ( n = 3 technical replicates). g , Representative HPLC trace of pentenoyl-CoA isomers formed from 500 µM 4-HV-CoA by recombinant wild-type ACAD10 NΔ34 and ACAD11 in vitro (brown and black traces, respectively) in comparison to LvaE-generated pentenoyl-CoA standards (dark purple and blue traces, respectively).

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Sequencing, SDS Page, Recombinant, Expressing, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, In Vitro, Control, Comparison, Generated

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a , Cryo-EM structure of the ACAD11 K* mutant incubated with 4-HV-CoA. FAD molecules are highlighted in yellow. b , Front and side angle view of full-length ACAD11 A* mutant cryo-EM structure when incubated with 4-PV-CoA. c , Sequence alignment of key regions of the kinase domain. The overall canonical PKL subdomain structure is shown at the top. ACAD10/11 homologs from H. sapiens , M. musculus , and B. taurus are compared to PKL consensus motifs, with key conserved residues highlighted. d , MD modeling of 4-hydroxyoctanoyl-CoA (4-HO-CoA; orange sticks) in the kinase domain relative to the catalytic D220 and a highly conserved N225 (blue sticks). e , Coomassie-stained gels of recombinant ACAD11 ACAD-domain mutants purified from E. coli . f , UV-Vis HPLC quantification (259 nm) of 4-PV-CoA and 2-pentenoyl-CoA in reactions containing wild-type ACAD10/11 and varying concentrations of FAD (mean -/+ SD; n = 3 technical replicates). Statistics: Two-way ANOVA with Šídák’s multiple comparisons test comparing each condition to the respective 0 µM FAD reaction control. P -values are represented as asterisks: * = P < 0.05, ** = P < 0.01, *** = P < 0.001. nd = not detected.

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Cryo-EM Sample Prep, Mutagenesis, Incubation, Sequencing, Staining, Recombinant, Purification, Control

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a , Assay testing the non-specific hydration activity in recombinant ACAD11 reactions. 2-pentenoyl-CoA (2-Pen-CoA) and 3-pentenoyl-CoA (3-Pen-CoA) were tested as substrates. Raw signal intensities of peaks corresponding to substrates and 3-hydroxyvaleryl-CoA product are depicted. Data represents mean -/+ SD ( n = 3 technical replicates). b , Table of calculated Km, Vmax, and Kcat/Km values for ACAD11 kinase and ACAD domain activities. Values were obtained by fitting to Michaelis-Menten curves in Figs. and . Data are expressed as mean -/+ 95% symmetric confidence interval. c , Expected labeling pattern of acyl-CoA catabolites derived from the catabolism of [ 13 C 5 ]-4-hydroxyvalerate (4-HV) by the “major” or “minor” pathways. M + 3 propionyl-CoA labeling reflects catabolism through the 4-phosphovaleryl-CoA intermediate, presumably catalyzed by ACAD10/11. Statistics: One-way ANOVA with Dunnett’s multiple comparisons test comparing all conditions to the respective “no enzyme” control. P -values are represented as asterisks: * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Activity Assay, Recombinant, Labeling, Derivative Assay, Control

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a , Immunofluorescence imaging of overexpressed human ACAD10–FLAG and FLAG–ACAD11 in COS7 cells. Localization of FLAG-tagged constructs (cyan) is indicated by overlap in fluorescence intensity with mitochondrial (HSP60; magenta) and peroxisomal (PEX14; yellow) markers. Top, natural positioning of MTS and PTS on the full-length isoforms of ACAD10/11. b , Experimental workflow to evaluate 4-HA metabolism in Hepa1-6 cells. c , 4-PV-CoA levels detected in Hepa1-6 cells treated with 0 mM or 10 mM unlabeled 4-HV. d , 4-Phospho-DHA-CoA levels detected in Hepa1-6 cells treated with unlabeled 100 µM 4-HDHA conjugated to 1% fatty-acid-free BSA (w/v). Results in c , d are expressed as the mean signal intensity (normalized to total protein content) ± s.d. ( n = 3 independent experiments). e , Model of organellar 4-HA catabolism. Statistical analysis was conducted using a one-way ANOVA with Dunnett’s multiple-comparisons test comparing all cell lines to the WT control ( c , d ). * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Immunofluorescence, Imaging, Construct, Fluorescence, Control

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a , Data processing flowcharts. b , FSC curves of non-uniform refinement of the ACAD11 K* mutant and local refinement of the ACAD11 A* mutant. c , Local resolution of the final maps.

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Mutagenesis

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a, b , Representative cryo-EM densities of the ACAD11 K* mutant ( a ) and A* mutant ( b ). c, d , Angular distribution plots of ACAD11 K* mutant ( c ) and A* mutant ( d ).

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Cryo-EM Sample Prep, Mutagenesis

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a , Cryo-EM map and model of the ACAD11 A* mutant tetramer incubated with 4-PV-CoA. Kinase and ACAD domains are colored blue and orange, respectively. One molecule of FAD (sticks) is present in each ACAD domain active site. b , MD modeling of 4-HO-CoA (orange sticks) in the kinase domain active site. Conserved residues implicated in substrate binding are presented as light-blue sticks. Catalytic D220 is shown as dark-blue sticks. c , Kinase activity of WT ACAD10/11 or K* mutants with 4-HV-CoA or nonacylated 4-HV substrates. d , Sequence alignments of M. musculus ACAD10/11 with P. putida LvaC and other M. musculus ACAD family members. Highly conserved residues involved in acyl-CoA stabilization, FAD binding and catalytic activity are colored brown, orange and red, respectively. Highly conserved ACAD domain residues possibly involved with 4-phosphoacyl-CoA substrate recognition are highlighted in blue. e , MD modeling of 4-PV-CoA (orange sticks) in the ACAD domain active site. Key interacting residues (R626, R637, D753 and H634) are presented as sticks. Right, representation of the ACAD domain active site pocket surface. f – h , UV–vis absorbance (259 nm) of 4-HV-CoA ( f ), 4-PV-CoA ( g ) and 2-pentenoyl-CoA ( h ) in reactions containing WT ACAD11 and catalytically inactive mutants. i – k , UV–vis absorbance (259 nm) of 4-HV-CoA ( i ), 4-PV-CoA ( j ) and 2-pentenoyl-CoA ( k ) in reactions containing 5 µM FAD or 5-deazaFAD (dFAD). Data in f – k are presented as the mean ± s.d. ( n = 3 technical replicates). Statistical analysis was conducted using a two-way ANOVA with Šídák’s multiple-comparisons test comparing all conditions to the respective no-enzyme control ( c ), one-way ANOVA with Dunnett’s multiple-comparisons test comparing all mutants to the WT control ( f – h ) or two-way ANOVA with Šídák’s multiple-comparisons test comparing all conditions to the respective FAD-containing reaction controls ( i – k ). * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Cryo-EM Sample Prep, Mutagenesis, Incubation, Binding Assay, Activity Assay, Sequencing, Control

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a , Immunofluorescence imaging of overexpressed FLAG-tagged constructs in COS7 and U2OS cells at 10 µm scale. b , Crop of merged images depicted in Fig. and fluorescence emission line quantification (right graphs). Localization overlap of ACAD10:FLAG (cyan; top row) and FLAG:ACAD11 (cyan; bottom row) are compared to mitochondrial HSP60 (magenta) and peroxisomal PEX14 (yellow). Scale bar = 2 µm. c , Pearson correlation coefficients of the overlap between ACAD10:FLAG or FLAG:ACAD11, HSP60, and PEX14 across n = 11 and n = 15 technical replicate regions of interest (ROIs), respectively (ROIs are defined as 300 ×300 pixel areas; each ROI captures a portion of one transiently transfected COS7 cell). Each data point represents one independent ROI. Data are shown as box-plots. Centerline, median; box limits, 25 th to 75 th percentiles; whiskers, minimum and maximum points. d , Immunofluorescence imaging of overexpressed constructs with FLAG-tags swapped to opposite termini in COS7 and U2OS cells at 10 µm scale. e, f , Signal intensity of peaks that likely correspond to 4-hydroxyacyl-CoAs of exogenously delivered 4-HAs. Data are represented as the mean signal intensity (normalized to total protein) -/+ SD ( n = 3 technical replicates). Statistics: Two-sided Wilcoxon Ranked Sum Test was used to obtain Pearson correlation coefficients ( c ), two-way ANOVA with Šídák’s multiple comparisons test comparing each treatment group to the respective “wild type” control ( e, f ). P -values are represented as asterisks: * = P < 0.05. nd = not detected.

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Immunofluorescence, Imaging, Construct, Fluorescence, Transfection, Control

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a , Genotyping of Acad11 KO mouse colony. b , Normalized mRNA expression of Acad11 in liver tissue. Data are normalized to Rps3 levels and expressed as mean -/+ SD. c , Mendelian birth ratios of Acad11 mice. d, e , Volcano plots of log 2 -transformed fold changes of 4-HAs and other hydroxylated lipids in mice fed standard chow relative to statistical significance. Male ( d ) and female ( e ) Acad11 KO mice were compared to respective littermate controls. f-i , Concentrations of 4-OH C10 (top) and 4-OH C12 (bottom) in comparison with their respective 3-OH and 5-OH fatty acid isomers in plasma across all genotypes and sex. Data are expressed as box-plots. Centerline, median; box limits, 25 th to 75 th percentiles; whiskers, minimum and maximum points. Mice fed a defined standard chow for 5 days ( f, g ). Mice fed a HFD for 12 weeks ( h, i ). For b and d-g : Male WT n = 5, Male KO n = 5; Female WT n = 3, Female KO n = 6. Mice were approximately 4-5 months old when sacrificed. For h and i : Male WT n = 6, Male KO n = 5; Female WT n = 10, Female KO n = 7. Mice were approximately 3 months old at the start of HFD feeding. Statistics: Two-sided Student’s t -test comparing Acad11 KO mice to respective littermate controls ( b and f-i ), two-sided multiple t -tests ( d, e ). P -values are represented as asterisks: * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Expressing, Transformation Assay, Comparison, Clinical Proteomics

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a , Top 25 traits associated with predicted loss-of-function (pLoF) rare coding variants of human ACAD11 (Genebass exome database). Traits are ranked by statistical significance as calculated by SKAT gene burden test (−log P ≥ 4 denotes a strong association). Orange circles are traits related to fat. adjBMI, adjusted to body mass index. b , c , Volcano plots depicting log 2 -transformed fold changes of 4-HAs and other hydroxylated lipids in HFD-fed Acad11- KO mice versus statistical significance. Male ( b ) and female ( c ) Acad11- KO mice were compared to littermate controls. Male WT, n = 6; male KO, n = 5; female WT, n = 10; female KO, n = 7. Mice were 3 months old at the start of HFD feeding. d , Body weight of female WT and Acad11- KO mice throughout 12 weeks of HFD feeding. Data are presented as the mean ± s.d. e , Changes in body weight after HFD feeding. f – j , Percentage body weight of iWAT ( f ), gWAT ( g ), liver ( h ), kidneys ( i ) and heart ( j ) of HFD-fed mice. In d – j , female WT, n = 9; female KO, n = 7. Data in e – j are shown as box plots. Center line, median; box limits, 25th to 75th percentiles; whiskers, minimum and maximum points. k , Immunofluorescence images of WT and Acad11- KO primary adipocytes 8 days after differentiation ex vivo (representative of n = 3 independent biological replicates). Hoechst (blue), nuclei; BODIPY 493/503 (green), lipid droplets. Magnification, ×100; scale bar, 60 µm. l – n , Quantification of lipid droplet quantity ( l ), lipid droplet size ( m ) and nucleus size ( n ) of WT and Acad11- KO differentiated primary adipocytes. Data are expressed as the mean ± s.e.m. ( n = 3 independent biological replicates; each replicate is the average of three technical replicate images, except for two WT samples, each of which is the average of two technical replicate images). The average lipid droplet diameter ( m ) and average nuclei diameter ( n ) are normalized to cell count. o – r , mRNA expression of Tle3 ( o ), Glut4 ( p ), AdipoQ ( q ) and Leptin ( r ) in WT and Acad11- KO primary adipocytes during differentiation. Data are normalized to Rps3 . Bars represent the mean ± s.e.m. ( n = 3 independent biological replicates). Statistical analysis was conducted using multiple two-sided t -tests ( b , c ), a two-way repeated-measures ANOVA with Šídák’s multiple-comparisons test comparing Acad11 -KO mice to WT mice at each time point ( d ), a two-sided Student’s t -test ( e – j , l – n ) or a two-way ANOVA with Šídák’s multiple-comparisons test ( o – r ). * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Transformation Assay, Immunofluorescence, Ex Vivo, Cell Counting, Expressing

Journal: Nature Structural & Molecular Biology

Article Title: ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism

doi: 10.1038/s41594-025-01596-4

Figure Lengend Snippet: a-d , Total body ( a ), liver ( b ), kidneys ( c ), and heart ( d ) weights of WT and Acad11 mice fed standard chow. Data are shown as box-plots (Male WT n = 5, Male KO n = 5; Female WT n = 3, Female KO n = 6). Mice were approximately 4-5 months old when sacrificed. e , NMR body composition analysis quantifying total fat mass. Data are shown as box-plots (Male WT n = 5, Male KO n = 8; Female WT n = 8, Female KO n = 7). Standard chow-fed mice were approximately 8 months old when sacrificed. f-k , Body weight over time (mean -/+ SD) ( f ), iWAT ( g ), eWAT ( h ), liver ( i ), kidneys ( j ), heart ( k ) weights of male WT and Acad11 KO mice after 12 weeks of HFD feeding. l-q , Plasma markers of liver and kidney pathology and function in HFD-fed WT and Acad11 KO mice. For f-q , data are expressed as box plots. Male WT n = 6, Male KO n = 5; Female WT n = 9, Female KO n = 7. One WT is excluded from weight analyses due to renal agenesis. Mice were approximately 3 months old at the start of HFD feeding. For box-plots: centerline, median; box limits, 25 th to 75 th percentiles; whiskers, minimum and maximum points. r-t , Normalized gene expression in female WT and Acad11 KO primary adipocytes at zero and six days of differentiation. Acad11 ( r ), Pparg ( s ), and Cd36 ( t ) expression were normalized to Rps3 levels. Data are expressed as mean -/+ SEM ( n = 3 independent biological replicates). Statistics: Two-sided Student’s t -test comparing Acad11 KO mice to respective WT littermate controls ( a-e and g-q ), two-way repeated measures ANOVA with Šídák’s multiple comparisons test comparing Acad11 KO mice to wild-type mice at each time point ( f ), and two-way ANOVA with Šídák’s multiple comparisons test ( r-t ). P -values are represented as asterisks: * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Article Snippet: Cryopreserved Acad11- KO (C57BL/6NJ) mouse sperm was purchased from The Jackson Laboratory (51098-JAX).

Techniques: Clinical Proteomics, Gene Expression, Expressing