Sirtuins and reversible acetylation

Sirtuin Mechanism

Mechanism of deacetylation by the Sirtuins.

Accumulating data indicate that lysine acetylation is a prevalent regulatory mechanism of protein function, with thousands of acetylated proteins identified by mass spectrometry. Silent information regulator 2 (Sir2 or Sirtuin) protein deacetylases are a class of evolutionarily conserved enzymes that function in critical cellular processes such as transcription, DNA repair, metabolism and stress resistance. Among the major classes of lysine deacetylases, the sirtuins utilize a unique catalytic mechanism that consumes NAD+, providing a direct connection between protein deacetylation and central metabolic pathways.

We are examining the central hypothesis that reversible protein acetylation is a major regulatory mechanism for controlling diverse metabolic processes, and that at the molecular level, site-specific acetylation alters the intrinsic activity of targeted proteins. To address these questions we are using a breadth of approaches that involve biochemistry,  proteomics, enzymology, and utilize mammalian tissue culture system and mouse models.

Current research highlights:

1.) Quantitative acetyl-proteomics to investigate SIRT3 function during caloric restriction.

SIRT3 is a mitochondrial NAD+-dependent deacetylase that regulates mitochondrial metabolism. Sirt3 exhibits tissue specific expression patterns, differentially regulating global mitochondrial protein acetylation, and subsequent metabolic pathways. Previously, we investigated the mitochondrial-wide changes to the acetyl-proteome during caloric restriction (CR) in mice and concomitantly examined the function of SIRT3 in mediating the CR response. Building upon these previous results, we recently employed a quantitative mass spectrometry method to analyze the acetyl-proteome of brain, heart, kidney, liver, and skeletal muscle in wild type mice or mice lacking SIRT3 to further our understanding of the tissue specific functions of SIRT3.

Multi-tissue acetylome

Quantitative acetyl-proteomics workflow.

2.) Long-chain fatty acids activate the deacetylase activity of SIRT6.

SIRT6 is a nuclear localized mammalian protein deacylase. Mice deficient in SIRT6 have metabolic defects and genomic instability, leading to shortened life span. Despite in vivo evidence indicating that SIRT6 is an efficient histone deacetylase, SIRT6 displays very weak deacetylase activity in vitro. Our early work suggested that the inefficient activity is due to a less active conformation of the enzyme. We recently demonstrated that SIRT6 is directly activated by free long-chain fatty acids, including ones linked to the health benefits of dietary polyunsaturated fatty acids. These results suggest the SIRT6 is stimulated to down-regulate carbohydrate and lipid metabolism during conditions that increase levels of particular omega-3 and omega-6 fatty acids, such fasting and dietary supplement intake. In addition, these observations suggest that the development of small-molecule activators of SIRT6 holds therapeutic potential to treat cancer, inflammation, and metabolic diseases. Current work in the lab is focusing on the the in vivo effects that omega fatty have on the deacetylase activity of SIRT6. Additionally, we are interested in identifying more potent activators, which could hold therapeutic potential.

grantfig

SIRT6 is activated by free long-chain fatty acids.

 

SIRT6 grant figure

 

Papers to get started:

Here, we show that long-chain free fatty acids, including omega-3 and omega-6 fatty acids stimulate SIRT6 deacetylase activity. In addition, we provide evidence that long-chain deacylation is a general feature of sirtuins and sirtuins display distinct, but overlapping specificity for diverse acylated peptides. Discovery of endogenous, small-molecule activators of SIRT6 demonstrates the therapeutic potential of compounds that promote SIRT6 function.

Here, we describe a quantitative acetyl-proteomic method that combines isobaric tagging for multiplexed quantification, immunoenrichment, two-dimensional chromatography, and high-resolution, high-accuracy MS. We applied this approach to examine the mitochondrial-wide changes to the acetyl-proteome during CR in mice and concomitantly examined the function of resident protein deacetylase SIRT3 in mediating the CR response. This quantitative acetylation atlas provides a key resource for understanding mitochondrial regulation and reversible protein acetylation. With these data, we establish that SIRT3 is a major regulator of the mitochondrial acetylome in response to CR and discover new processes controlled by SIRT3.

In this study, we provide detailed cellular and in vitro evidence for the direct regulation of glycolysis by sirtuin-mediated deacetylation of PGAM1. Prior to this study, detailed investigations of several metabolic enzymes indicated that acetylation inhibits activity. Here, we show that Sirt1-dependent deacetylation of PGAM1 reduces the rate of catalysis. In addition, the study provides biochemical evidence that glycolysis is modulated by reversible acetylation and demonstrates that PGAM1 deacetylation and activity are directly controlled by Sirt1.

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