Michelle F. Green

Lysine Glutarylation Is a Protein Posttranslational Modification Regulated by SIRT5

We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed that Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.

Hypothalamic CaMKK2 Contributes to the Regulation of Energy Balance

Detailed knowledge of the pathways by which ghrelin and leptin signal to AMPK in hypothalamic neurons and lead to regulation of appetite and glucose homeostasis is central to the development of effective means to combat obesity. Here we identify CaMKK2 as a component of one of these pathways, show that it regulates hypothalamic production of the orexigenic hormone NPY, provide evidence that it functions as an AMPKalpha kinase in the hypothalamus, and demonstrate that it forms a unique signaling complex with AMPKalpha and beta. Acute pharmacologic inhibition of CaMKK2 in wild-type mice, but not CaMKK2 null mice, inhibits appetite and promotes weight loss consistent with decreased NPY and AgRP mRNAs. Moreover, the loss of CaMKK2 protects mice from high-fat diet-induced obesity, insulin resistance, and glucose intolerance. These data underscore the potential of targeting CaMKK2 as a therapeutic intervention.

Characterization of the CaMKKβ-AMPK signaling complex.

The AMP-activated protein kinase (AMPK) is a critical regulator of energy homeostasis, and is a potential target for treatment of metabolic diseases as well as cancer. AMPK can be phosphorylated and activated by the tumor suppressor LKB1 or the Ca(2+)/CaM-dependent protein kinase kinase β (CaMKKβ). We previously identified a physical complex between CaMKKβ and AMPK (Anderson, K. A., Ribar, T. J., Lin, F., Noeldner, P. K., Green, M. F., Muehlbauer, M. J., Witters, L. A., Kemp, B. E., and Means, A. R. (2008) Cell Metabolism 7, 377-388). Here we expand our analysis of the CaMKKβ-AMPK signaling complex and show that whereas CaMKKβ can form a complex with and activate AMPK, CaMKKα cannot. In addition, we show that CaMKKβ and AMPK associate through their kinase domains, and CaMKKβ must be in an active conformation in order to bind AMPK but not to associate with an alternative substrate, Ca(2+)/Calmodulin-dependent protein kinase IV (CaMKIV). Our results demonstrate that CaMKKβ and AMPK form a unique signaling complex. This raises the possibility that the CaMKKβ-AMPK complex can be specifically targeted by small molecule drugs to treat disease.

SnapShot: Mammalian Sirtuins

The mammalian sirtuins have emerged as critical regulators of cellular stress resistance, energy metabolism, and tumorigenesis. In some contexts, they delay the onset of age-related diseases and promote a healthy lifespan. The seven mammalian sirtuins, SIRT1-7, share a highly conserved NAD+-binding catalytic core domain although they exhibit distinct expression patterns, catalytic activities, and biological functions. This SnapShot provides an overview of these properties, with an emphasis on their relevance to aging.

Measurement of Fatty Acid Oxidation Rates in Animal Tissues and Cell Lines

While much oncological research has focused on metabolic shifts in glucose and amino acid oxidation, recent evidence suggests that fatty acid oxidation (FAO) may also play an important role in the metabolic reprogramming of cancer cells. Here, we present a simple method for measuring FAO rates using radiolabeled palmitate, common laboratory reagents, and standard supplies. This protocol is broadly applicable for measuring FAO rates in cultured cancer cells as well as in both malignant and nontransformed animal tissues.

Ethanol Metabolism Modifies Hepatic Protein Acylation in Mice

Mitochondrial protein acetylation increases in response to chronic ethanol ingestion in mice, and is thought to reduce mitochondrial function and contribute to the pathogenesis of alcoholic liver disease. The mitochondrial deacetylase SIRT3 regulates the acetylation status of several mitochondrial proteins, including those involved in ethanol metabolism. The newly discovered desuccinylase activity of the mitochondrial sirtuin SIRT5 suggests that protein succinylation could be an important post-translational modification regulating mitochondrial metabolism. To assess the possible role of protein succinylation in ethanol metabolism, we surveyed hepatic sub-cellular protein fractions from mice fed a control or ethanol-supplemented diet for succinyl-lysine, as well as acetyl-, propionyl-, and butyryl-lysine post-translational modifications. We found mitochondrial protein propionylation increases, similar to mitochondrial protein acetylation. In contrast, mitochondrial protein succinylation is reduced. These mitochondrial protein modifications appear to be primarily driven by ethanol metabolism, and not by changes in mitochondrial sirtuin levels. Similar trends in acyl modifications were observed in the nucleus. However, comparatively fewer acyl modifications were observed in the cytoplasmic or the microsomal compartments, and were generally unchanged by ethanol metabolism. Using a mass spectrometry proteomics approach, we identified several candidate acetylated, propionylated, and succinylated proteins, which were enriched using antibodies against each modification. Additionally, we identified several acetyl and propionyl lysine residues on the same sites for a number of proteins and supports the idea of the overlapping nature of lysine-specific acylation. Thus, we show that novel post-translational modifications are present in hepatic mitochondrial, nuclear, cytoplasmic, and microsomal compartments and ethanol ingestion, and its associated metabolism, induce specific changes in these acyl modifications. These data suggest that protein acylation, beyond protein acetylation, contributes to the overall metabolic regulatory network and could play an important role in the pathogenesis of alcoholic liver disease.

Ca2+/Calmodulin-dependent Protein Kinase Kinase β Is Regulated by Multisite Phosphorylation

Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a serine/threonine-directed kinase that is activated following increases in intracellular Ca2+. CaMKKβ activates Ca2+/calmodulin-dependent protein kinase I, Ca2+/calmodulin-dependent protein kinase IV, and the AMP-dependent protein kinase in a number of physiological pathways, including learning and memory formation, neuronal differentiation, and regulation of energy balance. Here, we report the novel regulation of CaMKKβ activity by multisite phosphorylation. We identify three phosphorylation sites in the N terminus of CaMKKβ, which regulate its Ca2+/calmodulin-independent autonomous activity. We then identify the kinases responsible for these phosphorylations as cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3). In addition to regulation of autonomous activity, we find that phosphorylation of CaMKKβ regulates its half-life. We find that cellular levels of CaMKKβ correlate with CDK5 activity and are regulated developmentally in neurons. Finally, we demonstrate that appropriate phosphorylation of CaMKKβ is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKKβ-dependent signaling cascades.

Oxygen Flux Analysis to Understand the Biological Function of Sirtuins

The sirtuins are a family of highly conserved NAD(+)-dependent lysine deacylases with important roles in metabolic regulation. Of the seven mammalian sirtuins, three localize to the mitochondria: SIRT3, SIRT4, and SIRT5. Mitochondrial sirtuins are crucial regulators of the metabolic network that controls energy homeostasis and impacts cancer, obesity, diabetes, mitochondrial diseases, metabolic disorders, and many other human diseases of aging. To best study the mitochondrial function of the sirtuins, we have employed an oxygen flux analyzer as a tool to track and record the extracellular oxygen consumption rate and acidification rate that reflects mitochondrial respiration and glycolysis, respectfully. Here we described the methods using this assay to study the substrate utilization and mitochondrial function in a human hepatocellular carcinoma cell line, Huh7. Additionally, we have generated a stable SIRT4 knocked-down Huh7 cell line. With this cell line, we evaluated how the absence of SIRT4 affects mitochondrial function, glucose utilization, glutamine oxidation, and fatty acid oxidation in these cells.