Succinate dehydrogenase is a direct target of sirtuin 3 deacetylase activity - PubMed (original) (raw)

Succinate dehydrogenase is a direct target of sirtuin 3 deacetylase activity

Lydia W S Finley et al. PLoS One. 2011.

Abstract

Background: Sirtuins (SIRT1-7) are a family of NAD-dependent deacetylases and/or ADP-ribosyltransferases that are involved in metabolism, stress responses and longevity. SIRT3 is localized to mitochondria, where it deacetylates and activates a number of enzymes involved in fuel oxidation and energy production.

Methodology/principal findings: In this study, we performed a proteomic screen to identify SIRT3 interacting proteins and identified several subunits of complex II and V of the electron transport chain. Two subunits of complex II (also known as succinate dehydrogenase, or SDH), SDHA and SDHB, interacted specifically with SIRT3. Using mass spectrometry, we identified 13 acetylation sites on SDHA, including six novel acetylated residues. SDHA is hyperacetylated in SIRT3 KO mice and SIRT3 directly deacetylates SDHA in a NAD-dependent manner. Finally, we found that SIRT3 regulates SDH activity both in cells and in murine brown adipose tissue.

Conclusions/significance: Our study identifies SDHA as a binding partner and substrate for SIRT3 deacetylase activity. SIRT3 loss results in decreased SDH enzyme activity, suggesting that SIRT3 may be an important physiological regulator of SDH activity.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. SIRT3 interacts with subunits of complex II and complex V.

FLAG-IPs were performed on HEK293T cells transiently expressing FLAG-tagged sirtuins (T1-7). (A) SIRT1-7 IPs were immunoblotted with antibodies against SDHA, OSCP and FLAG. (B) SIRT3-5 IPs were immunoblotted with an antibody cocktail recognizing the SDHA and SDHB subunits of complex II and the F1α subunit of complex V (top) or a cocktail containing antibodies against representative subunits of complexes I–V (middle). SDHB is the subunit recognized by the complex II antibody.

Figure 2

Figure 2. SDHA is acetylated at 13 lysine residues.

(A) Schematic of SDHA summarizing the four domains (FAD-binding, capping, helical and C-terminal) with 13 identified acetylated residues shown in red (novel) or white (previously identified). (B) The 13 acetylated lysines were mapped on to the corresponding residues of the crystal structure of avian complex II.

Figure 3

Figure 3. SIRT3 deacetylates SDHA.

Complex II (A) and acetylated proteins (B) were immunoprecipitated from liver mitochondria isolated from SIRT3 WT and KO mice. IPs were immunoblotted with antibodies against acetyl-lysine (AcK), SDHA, SDHB and GDH. (C) Complex II immunoprecipitated from mouse liver mitochondria was incubated with recombinant SIRT3 or catalytically inactive SIRT3 (SIRT3 H248Y) in the presence or absence of NAD and NAM, a sirtuin inhibitor. After deacetylation, IPs were immunoblotted using antibodies against acetylated proteins, SDHA and SIRT3. In all panels, antibodies against GFP were used as negative controls.

Figure 4

Figure 4. SIRT3 regulates SDH activity.

Succinate dehydrogenase activity was measured in (A) SIRT3 WT and KO MEF extracts (n = 4), (B) liver mitochondria (n = 3) and (C) brown adipose tissue (BAT) mitochondria (n = 6–7). SDH activity was normalized to sample protein content and expressed as a ratio of WT levels. Values are expressed as mean ±SEM. *, P<0.05.

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