SIRT4 omaa lipoamidaasi ominaisuutta säätäen pyruvaattidehydrogenaasikompleksin PDH aktiivisuustilaa

Volume 159, Issue 7, 18 December 2014, Pages 1615-1625

Article

Sirtuin 4 Is a Lipoamidase Regulating Pyruvate Dehydrogenase Complex Activity

Author links open overlay panel 
Rommel A.Mathias¹²Todd M.Greco¹AdamOberstein¹Hanna G.Budayeva¹RumelaChakrabarti¹Elizabeth A.Rowland¹YibinKang¹ThomasShenk¹Ileana M.Cristea¹https://doi.org/10.1016/j.cell.2014.11.046

Highlights SIRT4 is a lipoamidase that functions in cells and mouse liver mitochondria Lipoamidase activity of SIRT4 is superior to its deacetylase activity SIRT4 inhibits PDH activity via enzymatic hydrolysis of the lipoamide cofactor Endogenous SIRT4 lipoamidase activity can be induced by glutamine stimulation

Summary

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.

 Introduction---
Sirtuins (SIRTs) are a family of seven mammalian nicotinamide adenine dinucleotide (NAD⁺)-dependent enzymes that regulate diverse biological processes, including genome regulation, stress response, metabolic homeostasis, and aging (Guarente, 2000,Imai et al., 2000).
 SIRTs display widespread subcellular distributions, as SIRT1, SIRT6, and SIRT7 are nuclear, SIRT2 is predominantly cytoplasmic, and SIRTs3–5 are mitochondrial (Haigis et al., 2006,Michishita et al., 2005).
As all SIRTs have a conserved deacetylase domain, these enzymes are generally known as lysine deacetylases, acting in opposition to acetyltransferases to remove acetyl-modifications from lysine residues (Imai et al., 2000). However, SIRTs exhibit varying catalytic efficiencies to this modification.
 SIRTs1–3 display robust deacetylase activity, in contrast to SIRTs4–5 that show little to no activity (Haigis et al., 2006,Michishita et al., 2005,Schuetz et al., 2007).
 Emerging evidence has revealed that several SIRTs can hydrolyze alternative lysine modifications more efficiently than acetyl. Specifically, SIRT5 preferentially desuccinylates and demalonylates protein substrates (Du et al., 2011,Peng et al., 2011), while SIRT6 can hydrolyze long-chain fatty acyl lysine modifications (Jiang et al., 2013).
These studies have highlighted the functionally dynamic nature of this family of proteins, which are able to perform different enzymatic reactions and regulate a wide range of cellular processes.Mitochondrial SIRTs 3–5 regulate ATP production, apoptosis, and cell signaling (Verdin et al., 2010) through distinct enzymatic functions. SIRT3 is considered to be the major deacetylase of the mitochondria, as SIRT3-deficient mice exhibit significant protein hyperacetylation (Lombard et al., 2007).
 The desuccinylase activity of SIRT5 was shown to target proteins involved in fatty acid β-oxidation and ketone body synthesis pathways, with SIRT5-deficient mice exhibiting an accumulation of acylcarnitines and a decrease in β-hydroxybutyrate production (Rardin et al., 2013). More recently, SIRT5 was reported to regulate lysine glutarylation levels, thereby modulating the activity of carbamoyl phosphase synthase 1, a critical enzyme in the urea cycle (Tan et al., 2014). In contrast to SIRT3 and SIRT5, SIRT4 enzymatic functions have generally remained more elusive (Newman et al., 2012). SIRT4 has been reported to regulate glutamine metabolism (Csibi et al., 2013, Jeong et al.,2013) and fatty acid oxidation via PPAR-α activity (Laurent et al., 2013a). To date, the enzymatic activity of SIRT4 is largely based on its ability to ADP-ribosylate glutamate dehydrogenase (GLUD1), which regulates amino-acid-dependent insulin secretion (Haigis et al., 2006). The deacylase activities of SIRT4 have remained less well characterized. Initial studies reported limited deacetylation activity (Lin et al., 2012,Michishita et al., 2005), yet SIRT4 was recently reported to control lipid catabolism through deacetylation of malonyl-CoA decarboxylase (MCD) (Laurent et al., 2013b). Additionally, acetylated SIRT4 substrate candidates have been identified in vitro via peptide microarrays (Rauh et al., 2013) and by screening the activity of recombinant SIRTs against various acyl-histone peptides Feldman et al., 2013).

 Unfortunately, these efforts may have been hampered by difficulty in maintaining soluble and active recombinant SIRT4. Therefore, reconciliation of in vitro enzymatic activities with in vivo biological substrates and downstream physiological functions remains a challenge.

Here, we characterized SIRT4 protein interactions within mitochondria, identifying its association with proteins containing lipoyl and biotinyl modifications. In agreement with this, we demonstrate that SIRT4 removes lipoyl- and biotinyl-lysine modifications more efficiently than acetylations. We discover a physical and functional interaction between SIRT4 and the components of the pyruvate dehydrogenase complex (PDH). 

 PDH is a mitochondrial complex comprised of 

• three catalytic subunits 

 (E1, pyruvate decarboxylase; 

E2, dihydrolipoyllysine acetyltransferase [DLAT];

E3, dihydrolipoyl dehydrogenase),

( a structural subunit (PDH-binding component X [PDHX])

 and two regulatory subunits (PDH kinase and PDH phosphatase) (Zhou et al., 2001).

 The complex catalyzes the decarboxylation of pyruvate to generate acetyl CoA, and links glycolysis to the TCA cycle. Its activity is known to be regulated by phosphorylation of the E1 subunit, phosphorylation that can be also impacted by E1 acetylation (Fan et al., 2014,Jing et al., 2013,Linn et al., 1969,Wieland and Jagow-Westermann, 1969). 

Here, we show that SIRT4 provides a previously unrecognized, phosphorylation-independent, mechanism of PDH regulation. SIRT4 hydrolyzes lipoamide cofactors from the DLAT E2 component of the PDH complex, thereby inhibiting PDH activity.

 Finally, as glutamine stimulation in rat liver is also known to inhibit the PDH (Häussinger et al.,1982), we investigated whether SIRT4 may play a role in this process.

 Indeed, we show that glutamine stimulation induces endogenous SIRT4 lipoamidase activity, triggering a reduction in both DLAT lipoyl levels and PDH activity. 

As the PDH controls pyruvate decarboxylation, fueling multiple downstream pathways, our findings highlight SIRT4 as a critical regulator of cellular metabolism.

 RESULTS

SIRT4 Interacts with the Three Mitochondrial Dehydrogenase Complexes

To investigate potential cellular substrates of SIRT4, we used proteomics to define its mitochondrial protein interactions. We constructed MRC5 fibroblasts stably expressing SIRT4-EGFP. Using density-based organelle fractionation (coisolation with mitochondrial COX IV, Figure 1A) and direct fluorescence microscopy (colocalization with MitoTracker, Figure 1C and Figure S1A available online), we confirmed its mitochondrial localization. Mitochondria were isolated and the interactions of SIRT4-EGFP were characterized by immunoaffinity purification-mass spectrometry (IP-MS) (

Joshi et al., 2013). Interaction specificity was computationally assessed using SAINT (

Choi et al., 2011), and 106 significant SIRT4 candidate interactions were identified (Table S1), including the known interactions and substrates, GLUD1, IDE and MLYCD (Ahuja et al., 2007,Haigis et al., 2006,Laurent et al., 2013b). 

 We hypothesized that as yet unrecognized substrates were also identified, and interrogated SIRT4 interactions using bioinformatics to extract enriched metabolic pathways and assemble functional protein networks. Notably, pyruvate metabolism, the TCA cycle, branched-chain amino acid catabolism, and biotin metabolism were significantly enriched pathways (Figure S1). Interaction of SIRT4 with biotin-dependent carboxylases has been reported (Wirth et al., 2013), validating the reliability of our data set. Interestingly, we found that SIRT4 associated with all three of the multimeric mammalian dehydrogenase complexes—PDH, oxoglutarate dehydrogenase (OGDH), and branched-chain alpha-keto acid dehydrogenase (BCKDH) (Figure 1B). These complexes occupy discrete positions within the cellular metabolic landscape, regulating TCA cycle activity and amino acid metabolism (Figure S1C).

 Given its relative prominence within SIRT4 interactions, we focused on PDH.

 The PDH complex is known to be regulated by reversible phosphorylation of its E1 component (Linn et al., 1969,Wieland and Jagow-Westermann, 1969), with acetylation of E1 also impacting its phosphorylation levels (

Fan et al., 2014,Jing et al., 2013). We confirmed that SIRT4-EGFP colocalized (Figure 1C) and immunoisolated (Figure 1D) with DLAT and PDH component X (PDHX), the E2 and E3 subunits of PDH, respectively (Figure 1B). Furthermore, in wild-type (WT) human fibroblast cells, we confirmed that DLAT interacts with endogenous SIRT4 by reciprocal IP (Figure 1E).