Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity

Abstract

Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism.

Fig. 1. SIRT4 interacts with the pyruvate dehydrogenase complex

(A) Density gradient-based cellular fractionation of MRC5 cells isolates SIRT4-EGFP with mitochondrial marker COX IV. (B) Functional network of SIRT4 interactions reveals association with dehydrogenase complexes. The E2 components in each complex (diamonds) contain lipoamide modifications (red circle). (C) SIRT4-EGFP (green) co-localizes with DLAT and PDHX (white) within mitochondria (MitoTracker Red). (D) Immunoaffinity purification of SIRT4-EGFP co-isolates DLAT and PDHX. (E) Reciprocal immunoaffinity purification of DLAT co-isolates endogenous SIRT4 in wild-type fibroblasts. See also Figure S1.

Fig. 2. SIRT4 hydrolyzes lipoyl-, biotin-, and acetyl-lysine modifications in vitro

(A) Recombinant SIRT4 (5 μM) was incubated with various acyl-modified H3K9 peptides (10 μM) with or without NAD (1 mM), and product and residual substrate peptides detected by LC-MS after reaction. Representative extracted ion chromatograms show unreacted acyl-modified H3K9 substrates (S), and unmodified H3K9 products (P, ~16.5 min). +NAD chromatograms are offset for clarity. (B) The percentage of product (unmodified peptide) formed as a function of increasing concentration of wild-type SIRT4 (±NAD) (mean ± S.E.M.; n=3). (C) Same as (B), except product formation from H3K9 substrates after reaction with increasing concentration of catalytically inactive SIRT4 H161Y. (D) Same as (A), except SIRT4 was incubated with putative biological DLAT and PDHX lipoyl lysine peptides (10 μM). Representative extracted ion chromatograms of unreacted DLAT and PDHX lipoyl peptide substrates (S) and unmodified products (P). (E) Scheme depicting the NAD+-dependent delipoylation of lipoyl-lysine mediated by SIRT4 lipoamidase activity. See also Figure S2.

Fig. 3. Steady-state kinetics reveals SIRT4 has the highest catalytic efficiency for lipoyl-modified substrates among mitochondrial SIRTs

(A–B) SIRT4 (5 μM) initial velocity (v₀) versus substrate concentration [S] for (A) H3K9- and (B) DLAT-acyl peptides (mean ± S.E.M.; n=3). [SIRT4] = 5 μM. v₀ vs. [S] were linear for acetyl substrates and were re-plotted to estimate k_cat/K_m (see Fig S3A). (C–D) Comparison of SIRT3 (0.5 μM) and SIRT4 (0.5 μM) initial velocity vs. [S] for DLAT K259 (C) acetyl and (D) lipoyl peptide (mean ± S.E.M.; n=3). SIRT4 v₀ vs. [S] was linear for DLAT acetyl and was re-plotted to estimate k_cat/K_m (see Fig S3B). If no error bars are displayed, errors were smaller than the data point size.See also Figure S3.

Fig. 4. Elevated SIRT4 expression decreases the activity and lipoylation of the pyruvate dehydrogenase complex in cultured cells

(A) PDH activity in fibroblasts expressing mitochondrial SIRT proteins vs. GFP-expressing cells (CTL) (mean ± S.E.M.; n=3 SIRTs 3–5; n=5 GFP; ****p<0.0001) measured by a PDH immunocapture colorimetric assay, (B) Western blot analysis of endogenous, full length lipoylated DLAT in cells overexpressing mitochondrial SIRTs. DLAT and COX IV – loading controls. (C) Western blot analysis of regulatory PDH-E1α phosphorylation (pS232, pS293, pS300) upon overexpression of SIRT4, catalytically inactive mutant H161Y, or GFP (CTL). E1 is loading control. (D) Relative PDH activity of untreated (control) or “activated” (+pyruvate dehyrogenase phosphatase, PDP1) purified porcine PDH complex incubated with wild-type or H161Y SIRT4. Western blot analysis of PDH-E1α phosphorylation sites; E1 – loading control. (E) Representative MS/MS spectra of K132 lipoyl peptide detected from endogenous DLAT immunopurified from mitochondria of fibroblasts and digested with endoproteinase GluC. K*, reduced and di-carbamidomethylated lipoyl-lysine (Δm = 304 amu vs. unmodified lysine). (F) SRM quantification of endogenous DLAT lipoyl K132 and K259 in fibroblasts (left) and HEK293 cells (right) (mean ± S.E.M; n=3, *p=0.03, ***p=0.0003). See also Figure S4.

Fig. 5. Endogenous SIRT4 inhibits PDH in cultured fibroblasts and in vivo, in mouse liver

(A) PDH activity time course in wild type fibroblasts stimulated with glutamax (4mM) for 2, 3, and 8 days, versus unstimulated cells (mean ± S.E.M.; n=4 2D and 3D, p<0.0001; n=3 8D, p=0.0007). (B) Western blot analysis of regulatory PDH-E1α phosphorylation sites and total E1 (loading control), and endogenous SIRT4, DLAT, and COX IV (loading control) levels, following glutamax stimulation. (C) SRM quantification of DLAT lipoyl levels (K132 and K259) in cells stimulated with glutamax versus unstimulated (mean ± S.E.M.; n=3) for 2D (ns), 3D (*p=0.015), and 8D (**p=0.007, *p=0.018). (D) Relative SIRT4 mRNA expression measured by qRT-PCR in fibroblast stably expressing either non-targeting control shRNA (shCTL) or one of five different constructs targeting SIRT4 (shSIRT4 #1–5). (E) Western blot analysis of SIRT4 and COX IV (loading control) from mitochondria purified from fibroblasts expressing shRNA constructs shCTL, shSIRT4 #1, and shSIRT4 #5. (F) PDH activity in fibroblasts with knock-down levels of endogenous SIRT4 (shSIRT4 #1 or #5, mean ± S.E.M; n=4) treated with glutamax (4 mM for 8D), versus control shCTL cells (mean ± S.E.M; n=7, ***p<0.0001). (G) PDH activity, lipoyl levels of endogenous DLAT (lipoic acid), and total DLAT levels (DLAT) from mouse liver mitochondria of Sirt4^−/− mice (mean ± S.E.M, n = 3, **p<0.039) versus wild-type control (n = 4). See also Figure S5.