Metabolon formation regulates branched-chain amino acid oxidation and homeostasis

Abstract

The branched-chain aminotransferase isozymes BCAT1 and BCAT2, segregated into distinct subcellular compartments and tissues, initiate the catabolism of branched-chain amino acids (BCAAs). However, whether and how BCAT isozymes cooperate with downstream enzymes to control BCAA homeostasis in an intact organism remains largely unknown. Here, we analyse system-wide metabolomic changes in BCAT1- and BCAT2-deficient mouse models. Loss of BCAT2 but not BCAT1 leads to accumulation of BCAAs and branched-chain α-keto acids (BCKAs), causing morbidity and mortality that can be ameliorated by dietary BCAA restriction. Through proximity labelling, isotope tracing and enzymatic assays, we provide evidence for the formation of a mitochondrial BCAA metabolon involving BCAT2 and branched-chain α-keto acid dehydrogenase. Disabling the metabolon contributes to BCAT2 deficiency-induced phenotypes, which can be reversed by BCAT1-mediated BCKA reamination. These findings establish a role for metabolon formation in BCAA metabolism in vivo and suggest a new strategy to modulate this pathway in diseases involving dysfunctional BCAA metabolism.

Extended Data Figure 1.. Distinct Expression Patterns of BCAT Isozymes in Human and Mouse Tissues

(a) Expression of BCAT1 mRNA in seven human organs across developmental time. (b) Expression of BCAT2 mRNA in seven human organs across developmental time. (c) Expression correlation of genes encoding enzymes involved in the BCAA metabolic pathway in human tissues across developmental times. The color and size of each circle indicate the Pearson’s correlation coefficient scores for positive (red) and negative (blue) correlations. P values by a two-sided t-distribution with n-2 degrees of freedom. (d) mRNA and protein expression for BCAT1 and BCAT2 across human tissues. Heatmap shows the normalized expression values for mRNA (pink) and protein (brown) in the GTEx database. (e) Expression of Bcat2, Bckdha, Bckdhb, Bckdk and Ppm1k mRNA in fractionated liver cell types including hepatocytes (HC), hepatic stellate cells (HSC), Kupffer cells (KC), and liver sinusoidal endothelial cells (LSEC). mRNA expression values by RNA-seq (FKPM) are shown. (f) Expression of BCAT2, BCKDHA, BCKDHB, BCKDK and PPM1K protein in fractionated liver cell types. Protein expression values by quantitative proteomics (fraction of total) are shown.

Extended Data Figure 2.. Phenotypic Analysis of BCAT1 and BCAT2 Knockout Mice

(a) Schematic of the genotyping PCR for Bcat1^−/− and Bcat2^−/− constitutive KO mice. The locations and sizes of the genotyping primers and PCR products are shown. (b) Representative genotyping PCR and Sanger sequencing results for Bcat1^−/− and Bcat2^−/− mice. (c) Validation of Bcat1^−/− KO by Western blot analysis of mouse brain and heart. HEK293T cells with BCAT1 and BCAT2 double knockout (DKO) and DKO cells with BCAT1 or BCAT2 overexpression (BCAT1^OE and BCAT2^OE) were analyzed as controls. (d) Representative images for male and female mice of the indicated genotypes. (e) Representative H&E staining of epididymal adipose tissue in WT and Bcat2^−/− mice. Scale bar, 50 μm. (f) The organ weight relative to body weight is shown for heart, kidney and spleen of WT, Bcat1^−/− and Bcat2^−/− mice with the number (N) of samples shown. Results are mean ± SD and analyzed by one-way ANOVA. *P < 0.05, n.s. not significant. (g) Hindlimb skeletal muscle and epididymal adipose weight of the indicated genotypes. Results are mean ± SD (N = 4 WT and 3 Bcat2^−/− mice for muscle, and 4 WT and 4 Bcat2^−/− mice for fat) and analyzed by two-sided unpaired t-test. (h) Plasma AST and ALT levels in mice of the indicated genotypes. Results are mean ± SD (N = 5 WT and 4 Bcat2^−/− mice) and analyzed by two-sided unpaired t-test. (i-k) Representative H&E staining of heart (i), kidney (j), and liver (k) from WT or Bcat2^−/− mice. Scale bar, 50 μm.

Extended Data Figure 3.. Metabolomic Alterations Caused by BCAT1 or BCAT2 Deficiency

(a) Principle component analysis of metabolic profiles of liver, pancreas, plasma and muscle in WT, Bcat1^−/− and Bcat2^−/− mice. (b) Enriched pathways associated with increased or decreased metabolites across major organs in Bcat1^−/− mice. Color scale indicates negative log10 transformed P values of the pathways associated with increased (red) or decreased (blue) metabolites determined by MetaboAnalyst 5.0 pathway analysis using hypergeometric test. (c) Enriched pathways associated with increased or decreased metabolites across major organs in Bcat2^−/− mice. Color scale indicates negative log10 transformed P values of the pathways associated with increased (red) or decreased (blue) metabolites determined by MetaboAnalyst 5.0 pathway analysis using hypergeometric test.

Extended Data Figure 4.. Differentially Enriched Metabolites Caused by BCAT1 or BCAT2 Deficiency

Hierarchical clustering heatmaps are shown for the top 50 differentially enriched metabolites in each indicated tissue between WT, Bcat1^−/− and Bcat2^−/− mice from one-way ANOVA analysis. The red and blue color indicate higher and lower metabolite abundance by MetaboAnalyst 5.0, respectively.

Extended Data Figure 5.. Neuropathology of Bcat2^−/− mice

(a) Transverse relaxation time (T₂) maps of WT (left) and Bcat2^−/− (right) mouse brains. Color bar indicates T₂ values. (b) Relative abundance of L-DOPA, threonine, tryptophan, and tyrosine in brain tissue of Bcat2^−/− mice. Results are mean ± SD (N = 4 WT and 4 Bcat2^−/−) and analyzed by two-sided unpaired t-test. (c,d) Representative H&E staining of the striatum (c) and brainstem (d) in WT and Bcat2^−/− mice. Scale bar, 50 μm.

Extended Data Figure 6.. Generation of BCAT1 or BCAT2 Reconstituted Cell Models for Proximity Labeling

(a) Western blot analysis of enzymes in the BCAA metabolic pathway in major tissues of WT and Bcat2^−/− mice. (b) Western blot analysis and quantification of BCKDHA and P-BCKDHA in WT and Bcat2^−/− liver samples. Results shown as mean ± SD (N = 12 WT and 11 Bcat2^−/− mice) and analyzed by two-sided unpaired t-test. (c) Generation of BCAT1 or BCAT2 reconstituted HEK293T cells. Western blot analysis is shown for the mitochondrial location of mTD, BCAT1-mTD and BCAT2-mTD, and efficient biotinylation of mitochondrial proteins in the presence of doxycycline (Dox) and exogenous biotin. MT-CO2 and p70 S6 kinase (P70-S6K) were analyzed as controls for the fractionated mitochondria and cytoplasm, respectively. (d) Validation of efficient biotinylation of mitochondrial proteins by proximity labeling using mTD, BCAT1-mTD or BCAT2-mTD in the presence of Dox and exogenous biotin. (e) Co-immunoprecipitation of BCAT1 and BCKDH in mouse kidney mitochondrial lysates using α-BCKDHA antibody cross-linked Dynabeads Protein A. Input or immunoprecipitated samples were blotted with α-BCKDHA and α-BCAT2 antibodies, respectively. Co-immunoprecipitation was performed in the presence or absence of additives (Val, PLP, α-KG, thiamine diphosphate and Coenzyme A), whereas co-immunoprecipitation using Dynabeads Protein A without cross-linking with α-BCKDHA antibody was performed as a negative control.

Extended Data Figure 7.. Ectopic BCAT1 Expression Ameliorates BCAT2 Deficiency-Induces Metabolic Defects

(a) Western blot analysis to validate the cytosolic expression of BCAT1 in WT and Bcat1^OE kidney lysates. (b) Western blot results are shown for the validation of Bcat1^OE and Bcat1^OE;Bcat2^−/− mice and the expression of enzymes in the BCAA metabolic pathway in major tissues. (c) Fractional enrichment of KIV_M+5 in the plasma of the indicated genotypes during the 2-hour stable isotope infusion experiments. Results are mean ± SD (N = 4 WT, 4 Bcat2^−/−, 3 Bcat1^OE and 3 Bcat1^OE;Bcat2^−/− mice). (d) Relative abundance of Val_M+0,5 and KIV_M+0,5 in plasma samples before (0 hour) and after (2 hour) KIV_M+5 stable isotope infusion with the number (N) of independent samples shown. Results are mean ± SD and analyzed by two-way ANOVA. (e) Glutamate/α-KG ratios in major tissues of the indicated genotypes with the number (N) of independent samples shown. Results are mean ± SD and analyzed by one-way ANOVA. (f) NAD⁺/NADH ratio in major organs of the indicated genotypes with the number (N) of samples shown. Results are mean ± SD and analyzed by two-way ANOVA. (g) Insulin levels in the serum of the indicated genotypes. Results are mean ± SD (N = 5 WT, 4 Bcat2^−/−, 4 Bcat1^OE and 4 Bcat1^OE;Bcat2^−/− mice) and analyzed by one-way ANOVA.

Extended Data Figure 8.. BCAT1-Catalyzed BCKA Reamination Increases BCAA Transport and Protein Synthesis

(a) Western blot analysis of LAT1 (encoded by Slc7a5) in major tissues of WT, Bcat1^OE and Bcat1^OE;Bcat2^−/− mice. (b) Quantification of LAT1 expression in mice of the indicated genotypes relative to WT samples with the number (N) of samples shown. Results are mean ± SD and analyzed by two-sided unpaired t-test. (c) Representative Western blot analysis of 4EBP1, P-4EBP1, p70S6K, and P-p70S6K in WT or Bcat2^−/− liver lysates. Quantification is shown on the bottom. Results are mean ± SD (N = 3 WT and 3 Bcat2^−/− mice on BCAA-normal diet, and 3 Bcat2^−/− mice on BCAA-choice diet) and analyzed by one-way ANOVA. (d) Representative Western blot analysis of 4EBP1, P-4EBP1, p70S6K, and P-p70S6K in liver lysates from mice of the indicated genotypes. Quantification is shown on the bottom. Results are mean ± SD (N = 3 WT, 3 Bcat2^−/−, 3 Bcat1^OE and 3 Bcat1^OE;Bcat2^−/− mice) and analyzed by one-way ANOVA. (e) Amino acid levels in Bcat2^−/−, Bcat1^OE, and Bcat1^OE;Bcat2^−/− mouse tissues. Heatmap shows the log2 fold changes of the indicated metabolites in each mutant sample relative to WT controls (N = 3 Bcat2^−/−, 3 Bcat1^OE, and 4 Bcat1^OE;Bcat2^−/− mice). (f) Representative Western blot analysis of puromycin incorporation in major tissues of WT, Bcat1^OE and Bcat1^OE;Bcat2^−/− mice.

Figure 1.. Generation of BCAT1 and BCAT2 Knockout Mice

(a) Diagram of the BCAA metabolic pathway. BCAT isozymes catalyze the transamination of BCAAs to generate BCKAs. BCKAs can be reaminated by BCAT or irreversibly oxidized by BCKDH, forming R-CoA compounds that are further oxidized to enter the TCA cycle. (b) Expression correlation of genes encoding enzymes involved in the BCAA metabolic pathway in mouse tissues across developmental times. The color and size of each circle indicate the Pearson’s correlation coefficient scores for positive (red) and negative (blue) correlations. P values by a two-sided t-distribution with n-2 degrees of freedom. (c) mRNA and protein expression for BCAT1 and BCAT2 across mouse tissues. Heatmap shows the normalized expression values for mRNA (red) and protein (orange) in the GTEx database. (d) Schematic of Bcat1^−/− and Bcat2^−/− constitutive knockout (KO) mouse models. (e) Validation of Bcat1^−/− and Bcat2^−/− KO by representative Western blot analysis of major tissue types. HEK293T cells with BCAT1 and BCAT2 double knockout (DKO) and DKO cells stably expressing BCAT1 or BCAT2 (BCAT1^OE and BCAT2^OE) were analyzed as controls. Note that BCAT1 protein expression was low or undetectable in mouse heart, kidney, liver and spleen, and BCAT2 expression was low or undetectable in mouse liver. (f) Body weight growth curves of the indicated genotypes up to 12 weeks. Results are mean ± SD (N = 5 WT, 9 Bcat1^−/−, and 11 Bcat2^−/− mice) and analyzed by two-way ANOVA mixed-effect analysis with multiple comparisons. (g) Kaplan-Meier survival curves of WT, Bcat1^−/− and Bcat2^−/− mice (N = 16 WT, 11 Bcat1^−/−, and 7 Bcat2^−/− mice). P values by a log-rank Mantel-Cox test.

Figure 2.. Distinct Metabolomic Profiles of BCAT1 and BCAT2 Knockout Mice

(a) Workflow for targeted metabolomic profiling of 11 tissues from 6 to 8 weeks old WT, Bcat1^−/− or Bcat2^−/− mice (N = 4 mice per genotype). (b) Fraction (%) of differentially enriched metabolites in Bcat1^−/− or Bcat2^−/− tissues relative to WT controls (|fold change| ≥ 1.5, P ≤ 0.05; N = 4 mice per genotype). (c) Significantly changed metabolites in Bcat1^−/− or Bcat2^−/− relative to WT tissues. The x- and y-axis denote the negative log10 transformed P values calculated by two-sided linear model covariate analysis of MetaboAnalyst 5.0 ^, for differentially enriched metabolites among tissues and genotypes, respectively (N = 4 mice per genotype). (d) Metabolic pathways associated with enriched or depleted metabolites in Bcat2^−/− mice. Heatmap shows the negative log10 transformed P values from the pathway enrichment analysis of increased (red) or decreased (blue) metabolites (N = 4 mice per genotype) which were identified by two-sided unpaired t-test in the indicated tissues. (e) Relative abundance of metabolites associated with the top enriched pathways in Bcat1^−/− and Bcat2^−/− tissues. Heatmap shows the log2 fold changes of the indicated metabolites in each tissue of Bcat1^−/− or Bcat2^−/− mice relative to WT controls (N = 4 mice per genotype) which were identified by two-sided unpaired t-test in the indicated tissues.

Figure 3.. BCAT2 Deficiency Induces Metabolic Phenotypes Resembling MSUD

(a) BCAT2 deficiency induces phenotypes similar to MSUD caused by mutations in the BCKDH enzyme complex. (b) Total food intake normalized to mouse body weight, measured weekly for 12 weeks. Results are mean ± SD (N = 15 WT and 14 Bcat2^−/− mice) and analyzed by two-way ANOVA mixed-effect analysis with multiple comparisons. (c) Fraction (%) of BCAA-low diet consumed by the indicated genotypes. Results are mean ± SD (N = 6 WT and 8 Bcat2^−/− mice) and analyzed by two-way ANOVA mixed-effect analysis with multiple comparisons. (d) Body weight growth curves of indicated genotypes up to 12 weeks. Results are mean ± SD (N = 5 WT and 5 Bcat2^−/− mice on normal diet and 13 Bcat2^−/− mice on BCAA-choice diet) and analyzed by two-way ANOVA mixed-effect analysis with multiple comparisons. (e) Kaplan-Meier survival curves of WT or Bcat2^−/− mice with BCAA normal or choice diet (N = 11 WT and 6 Bcat2^−/− mice on normal diet and 10 Bcat2^−/− mice on BCAA-choice diet). P values by a log-rank Mantel-Cox test. (f) Relative abundance of Leu and Ile and their respective BCKAs, KIC and KMV, across different tissues in Bcat2^−/− mice with normal or choice diet. Results are mean ± SEM (N = 3 thymus samples and 4 samples for other tissues with normal diet, N = 3 samples for Leu and Ile measurements with choice diet, and N = 2 lung samples and 3 samples for other tissues for KIC and KMV measurements with choice diet) and analyzed by two-way ANOVA.

Figure 4.. Formation of BCAT2-BCKDH Complexes in Living Cells

(a) Schematic of the proximity labeling assays to determine BCAT2-interacting proteins in the mitochondria of living cells. (b) Schematic for the generation of BCAT1 and BCAT2 DKO HEK293T cells and the Western blot validation of single cell-derived clones. (c) Representative Western blot analysis of in vivo biotinylated proteins purified by streptavidin IP in DKO cells reconstituted with mTD, BCAT1-mTD or BCAT2-mTD. The BCKDH subunits E1 (BCKDHA and BCKDHB), E2 (DBT), E3 (DLD) and GLUD1 were specifically labeled by BCAT2-mTD and confirmed by mass spectrometry-based proteomics. (d) Validation of BCAT2-interacting proteins by Western blot analysis of streptavidin-purified mitochondrial proteins using antibodies specific to the indicated protein.

Figure 5.. BCAT2 Deficiency Impairs BCKDH-Catalyzed BCKA Oxidation

(a) Schematic of stable isotope tracing with [U-¹³C]-KIV in WT and Bcat2^−/− mice. [U-¹³C]-KIV can be reaminated to Val_M+5 by BCAT1 or BCAT2 in WT mice, or by BCAT1 in Bcat2^−/− mice. [U-¹³C]-KIV is oxidized by BCKDH to generate HIB_M+4. Succinate_M+3,2,1 are generated by the first, second and third turns of the TCA cycle, respectively. (b) Quantification of Val_M+5 and HIB_M+4 and fraction labeling of succinate_M+3,2,1 are shown. Values were normalized to infusion rate and tissue weight relative to WT samples for the indicated genotypes and tissues with the number (N) of independent samples shown. Results are mean ± SEM and analyzed by two-sided unpaired t-test. (c) Schematic of the radiochemical measurement of total BCKDH activity. BCKDH oxidizes [¹⁴C]-KIV, releasing ¹⁴CO₂ molecules which are trapped in the NaOH soaked wick and quantified by a scintillation counter. (d) Total BCKDH activity in heart, kidney and liver from WT or Bcat2^−/− mice with the number (N) of independent samples shown. Results are mean ± SD and analyzed by two-sided unpaired t-test. (e) Loss of BCAT2 impairs BCKDH activity to cause BCKA accumulation in Bcat2^−/− mice.

Figure 6.. Ectopic BCAT1 Expression Restores BCAT2-Deficiency-Induced Metabolic Defects

(a) Schematic of the Bcat1 knockin (Bcat1^LSL KI) and constitutive overexpression (Bcat1^OE) mouse models. (b) Body weight growth curves of the indicated genotypes. Results are mean ± SD (N = 4 WT, 6 Bcat1^OE, 6 Bcat2^−/−, and 5 Bcat1^OE;Bcat2^−/− mice) and analyzed by two-way ANOVA mixed-effects analysis with multiple comparisons. (c) Kaplan-Meier survival curves of WT, Bcat1^OE, Bcat2^−/−, and Bcat1^OE;Bcat2^−/− mice (N = 8 WT, 8 Bcat1^OE, 5 Bcat2^−/−, and 8 Bcat1^OE;Bcat2^−/− mice). P values by a log-rank Mantel-Cox test. (d) BCAA and BCKA levels in Bcat1^OE, Bcat2^−/−, and Bcat1^OE;Bcat2^−/− mouse tissues. Heatmap shows the log2 fold changes of the indicated metabolites in each mutant tissue relative to WT controls (N = 3 WT, 3 Bcat1^OE, 3 Bcat2^−/−, and 4 Bcat1^OE;Bcat2^−/− mice) which were identified by two-sided unpaired t-test in the indicated tissues. (e) Quantification of Val_M+5 and HIB_M+4 and fractional labeling of succinate_M+3,2,1 are shown. Values were normalized to infusion rate and tissue weight relative to WT in the indicated genotypes and tissues with the number (N) of independent samples shown. Results are ± SEM and analyzed by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s. not significant. The exact P values are provided in Supplementary Table 5. (f) Total BCKDH activity in liver, heart and kidney with the number (N) of independent samples shown. Results are mean ± SD and analyzed by two-way ANOVA. (g) Quantification of puromycin incorporation in mice of the indicated genotypes relative to WT samples with the number (N) of independent samples shown. Results are mean ± SD and analyzed by two-sided unpaired t-test. (h) [¹³C] Val_M+5 incorporation into protein in the indicated tissues and genotypes relative to WT samples with the number (N) of independent samples shown. Values were normalized to infusion rate and tissue weight. Results are mean ± SD and analyzed by two-way ANOVA. (i) Quantification of urinary 3-methyl-histidine and 3-methyl-histidine/creatinine ratio in the indicated genotypes with the number (N) of independent samples shown. Results are mean ± SD and analyzed by ordinary one-way ANOVA. (j) Schematic of the working model.