Protein Post-Translational Acetylation Controls Lifespan through Regulation of Metabolism-Related Enzymes
Date Issued
2011
Date
2011
Author(s)
Lu, Jin-Ying
Abstract
In this essay we report the use of whole proteome screen in eukaryotes to discover the first non-histone substrates of the only essential lysine acetyltransferase, and the first non-chromatin acetylation sites in yeast.
In the first study, we used the yeast proteome microarray to identify 13 in vivo substrates of the only essential lysine acetyltransferase NuA4 complex (containing the Esa1 catalytic subunit) in yeast. Many of the in vivo substrates are metabolic enzymes and stress-response proteins, including phosphoenolpyruvate carboxykinase (Pck1), a well-characterized enzyme catalyzing the rate-limiting step in gluconeogenesis in the cytoplasm, indicating a surprising extranuclear function of NuA4 in regulating metabolism besides its well-studied regulation of chromatin-related processes.
Using mass spectrometry, we identified lysine residue 514 (K514) as the Esa1-dependent acetylation site in Pck1, and further showed the same site was deacetylated by lysine deacetylase Sir2. Moreover, we found that mutations of K514 affected enzymatic activity of Pck1 to conduct gluconeogenesis and hence the ability of cells to grow on non-fermentable carbon sources such as ethanol. When K514 was mutated to arginine (pck1-K514R) to abolish acetylation at this critical site, the enzymatic activity of purified Pck1 was markedly decreased in vitro, and the cells lost the ability to grow in ethanol. By contrast, substituting K514 with glutamine (pck1-K514Q, a mutation mimicking constitutive acetylation) could almost completely rescue the lethality of mutants with defective Esa1 function in ethanol. These results suggested that K514 is the only lysine residue of Pck1 targeted by Esa1 and Sir2.
Interestingly, both pck1-K514Q and sir2∆ mutants enhance the viability of cells on high concentration of ethanol. Loss of Pck1 activity by either deletion of the encoding gene or by mutation of K514 to arginine blocked the extension of chronological life span under calorie restriction. Furthermore, this activating acetylation might be conserved in mammalian system since human Pck1 could rescue the lethality of yeast cells lacking PCK1 in ethanol, despite a low sequence homology, and hPck1 acetylation and glucose production was dependent on TIP60 in human hepatocellular carcinoma (HepG2) cells. In summary, we have found novel extracellular functions of yeast NuA4 complex in regulating gluconeogenesis and chronological life span.
Aging is a plastic phenotype determined partially by the cellular metabolism and energy expenditure. Our second study describes a novel regulatory cascade mediated by acetylation and phosphorylation that modulates cellular metabolism, growth and replicative life span in yeast. At the top of the pathway is the essential lysine acetyltransferase NuA4, which acetylates a regulatory subunit of the Snf1 complex (yeast AMPK), called Sip2. This acetylation blocks kinase action by stabilizing Sip2-Snf1 protein interaction; in response to deacetylation, Snf1 is released from Sip2 inhibition and then activates a life span shortening pathway by phosphorylating Sch9.
Snf1 kinase is a key regulator of energy homeostasis required for transcription of glucose-repressed genes and certain stress-response genes. A previously unsolved mystery is why Snf1 activity increases with aging and exhibits negative effects on life span extension. We show that Sch9, the yeast homologue of mammalian Akt/S6K, a known life span antagonist, is phosphorylated and activated by Snf1. Acetylation of Sip2 enhances physical interaction with Snf1, antagonizes its catalytic activity and suppresses detrimental effects of Snf1 on life span extension. Decreased Sip2 acetylation during aging enhances activity of Snf1 and the downstream Sch9 kinase. We further demonstrate that Sip2 acetylation decreases intracellular trehalose level, a stress indicator, and increases resistance to aging-associated oxidative stress.
Whether calorie restriction is the only pathway and approach for life span extension is unclear. Previous reports suggested that calorie restriction might lead to unwelcome health concerns in humans, especially elderly and non-obese subjects. Our study provides evidence of a potential “intrinsic aging pathway” mediated by an acetylation-phosphorylation cascade that is largely unresponsive to calorie restriction. Although calorie restriction increases the life span of non-acetylatable Sip2 mutants, or when SIP2 is deleted (both mimicking the aging status); the benefit of glucose limitation to constitutively acetylated Sip2 mutants is limited, indicating a partially overlapping common downstream pathway for calorie restriction and intrinsic aging.
Our study confirmed the relationship between non-histone protein acetylation and both chronological and replicative life span in yeast. We hope that in the future, the results can be applied to aging-related diseases, such as diabetes mellitus and cancer, in higher organisms and also humans.
Subjects
Proteome microarray
lysine acetylation
gluconeogenesis
life span
SDGs
Type
thesis
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