Regulation of purine metabolism connects KCTD13 to a metabolic disorder with autistic features

Abstract

Genetic variation of the 16p11.2 deletion locus containing the KCTD13 gene and of CUL3 is linked with autism. This genetic connection suggested that substrates of a CUL3-KCTD13 ubiquitin ligase may be involved in disease pathogenesis. Comparison of Kctd13 mutant (Kctd13 ^-/- ) and wild-type neuronal ubiquitylomes identified adenylosuccinate synthetase (ADSS), an enzyme that catalyzes the first step in adenosine monophosphate (AMP) synthesis, as a KCTD13 ligase substrate. In Kctd13 ^-/- neurons, there were increased levels of succinyl-adenosine (S-Ado), a metabolite downstream of ADSS. Notably, S-Ado levels are elevated in adenylosuccinate lyase deficiency, a metabolic disorder with autism and epilepsy phenotypes. The increased S-Ado levels in Kctd13 ^-/- neurons were decreased by treatment with an ADSS inhibitor. Lastly, functional analysis of human KCTD13 variants suggests that KCTD13 variation may alter ubiquitination of ADSS. These data suggest that succinyl-AMP metabolites accumulate in Kctd13 ^-/- neurons, and this observation may have implications for our understanding of 16p11.2 deletion syndrome.

Keywords: Metabolomics; Molecular Neuroscience; Proteomics.

Graphical abstract

Figure 1

ADSS is a putative target of a KCTD13 ubiquitin ligase (A) A Kctd13 deletion mouse was created by targeting the first exon with CRIPSR/Cas9. A 47-nucleotide deletion was introduced in the first exon leading to a frameshift. Error bars indicate standard deviation of the mean. (B) Western blots of whole-cell lysates from the cerebral cortex, hippocampus and striatum showed that in homozygous deletion mice, the KCTD13 protein was absent. In the cortex of heterozygous mice, the protein levels were significantly reduced (0.81 sd ± 0.3, t test p-val = 0.007). (C) A schematic depicting the workflow for SILAC labeling of wild-type and Kctd13^−/− neurons and subsequent quantitation of ubiquitin and total protein levels by global proteomics. (D) Volcano plot of log2 fold-change of protein changes in Kctd13^−/− vs. WT neurons (LogFC(Kctd13^−/−/WT)). The dotted line indicates an adjusted p value = 0.05 followed by a one-sample moderated t-test. The two significantly decreased sites of ADSS ubiquitination are indicated. The gradient hue (white [p-val = 0.99] to dark blue [p-val = 0.0035]) of the volcano plot reflects the adjusted p values resulting from the moderated t test. (E) Volcano plot of ubiquitination sites normalized by corresponding protein level changes in Kctd13^−/− vs. WT neurons (LogFC(Kctd13^−/−/WT)). The dot corresponding to ADSS is indicated in the figure. The dotted line indicates an adjusted p value = 0.05 followed by a one-sample moderated t-test. The gradient hue (white [p-val = 0.99] to dark blue [p-val = 0.00019]) of the volcano plot reflects the adjusted p values resulting from the moderated t test. CTX, cortex; HIP, hippocampus; STR, striatum.

Figure 2

ADSS is associated with and ubiquitinated by a CUL3-KCTD13 complex (A) Western blots show that levels of neuronal ADSS were increased in Kctd13^−/− mice relative to wild-type mice in dissociated neurons grown in vitro for 21 days. ∗ indicates significance, t test p = 0.01. Error bars indicate standard deviation of the mean. (B) ADSS-CUL3-KCTD13 proteins were immunoprecipitated together. ADSS-myc, HA-CUL3, and KCTD13 were transfected into HEK cells. Immunoprecipitates were collected using antibodies specific for the MYC epitope for ADSS-myc, KCTD13, and for the HA epitope for CUL3-HA. IgG immunoprecipitates were run as a negative control. Western blots were run and probed with the indicated antibodies. Additional immunoprecipitations were carried out in which one of the components was left out as indicated in the figure. (C) ADSS ubiquitination was KCTD13 dependent in a His-ubiquitin assay. ADSS-myc, KCTD13, HA-CUL3, and 6xhistidine-ubiquitin plasmids were transfected into HEK cells in the combinations indicated. His-ubiquitin conjugated proteins were collected and Western blots with myc antibody were performed to identify His-ubiquitinated ADSS. Total whole-cell lysates were probed on Western blots with the indicated antibodies to verify the expression of the indicated proteins. ∗ indicates significance, t test p < 0.005. Error bars indicate standard deviation of the mean.

Figure 3

Succinyl-adenosine (S-Ado) is increased in Kctd13^−/− neurons (A) A schematic of AMP biosynthesis is shown. (B) Purine metabolites were increased in Kctd13^−/− neurons. Core purine metabolism is shown with relative levels of each metabolite summarized. S-Ado, ADP, ATP, and AMP were significantly altered. ∗ indicates significance, t test, p < 0.05. Error bars indicate standard deviation of the mean. (C) Titration of L-alanosine in HEPES in increasing concentration into mouse neuronal cultures resulted in increased relative levels of IMP. Error bars indicate standard deviation of the mean. (D) S-Ado levels were reduced to wild-type levels by L-alanosine in Kctd13^−/− neurons. Data from 3 experiments were summarized for wild-type and Kctd13^−/− neurons treated with L-alanosine. The two-way ANOVA found the interaction to be significant (F(2,66) = 3.192, p = 0.05). Error bars indicate standard deviation of the mean. (E) Model for KCTD13 regulation of ADSS and purine metabolism. (Left panel) KCTD13 recruits ADSS to a CULLIN3 ubiquitin ligase where it is ubiquitylated. (Right panel) KCTD13 functionally acts as a negative regulator of ADSS levels. Deletion of KCTD13 resulted in elevated ADSS levels (red arrow). Elevated ADSS levels resulted in increased S-Ado levels which were decreased by the ADSS inhibitor L-alanosine.

Figure 4

Specific KCTD13 mutations reduce ADSS ubiquitination (A) A raster diagram indicates the KCTD13 variant positions that were analyzed. The top row (indigo tick marks) indicates all residue positions that were mutated. The second (magenta tick marks) row shows gnomAD variants. The third row (red tick marks) indicates a set of residues identified by the MTR viewer. The fourth row (purple tick marks) indicates a set of residues nominated by structural analysis. (B) A representative Western blot is shown of His6-Ubiquitin assays of KCTD13-dependent ubiquitination of ADSS for different KCTD13 mutations and wild-type KTD13. Western blots of total protein lysates indicate the levels of input proteins for each assay. (C) Summary data of each KCTD13 mutation for n = 3 independent assays. ∗ indicates significance compared to wild type, t test p < 0.05. Ubiquitination levels for each independent assay were normalized to KCTD13 levels. Data for each mutant are normalized to the level of wild-type KCTD13 ubiquitination of ADSS-myc. (D) Loss-of-function mutations (orange balls) and gain-of-function mutations (cyan balls) that were identified as significantly different from wild type (Student's t-test p < 0.05) are indicated on a KCTD13 homology model.