Statin therapy inhibits fatty acid synthase via dynamic protein modifications.

Statins are a class of drug widely prescribed for the prevention of cardiovascular disease, with pleiotropic cellular effects. Statins inhibit HMG-CoA reductase (HMGCR), which converts the metabolite HMG-CoA into mevalonate. Recent discoveries have shown HMG-CoA is a reactive metabolite that can non-enzymatically modify proteins and impact their activity.

Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice.

A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be nonenzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways.

β-Cell-specific ablation of sirtuin 4 does not affect nutrient-stimulated insulin secretion in mice.

Sirtuins are a family of proteins that regulate biological processes such as cellular stress and aging by removing posttranslational modifications (PTMs). We recently identified several novel PTMs that can be removed by sirtuin 4 (SIRT4), which is found in mitochondria. We showed that mice with a global loss of SIRT4 [SIRT4-knockout (KO) mice] developed an increase in glucose- and leucine-stimulated insulin secretion, and this was followed by accelerated age-induced glucose intolerance and insulin resistance.

Metabolic control by sirtuins and other enzymes that sense NAD, NADH, or their ratio.

NAD is a dinucleotide cofactor with the potential to accept electrons in a variety of cellular reduction-oxidation (redox) reactions. In its reduced form, NADH is a ubiquitous cellular electron donor. NAD, NADH, and the NAD/NADH ratio have long been known to control the activity of several oxidoreductase enzymes.

SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion.

Sirtuins are NAD-dependent protein deacylases that regulate several aspects of metabolism and aging. In contrast to the other mammalian sirtuins, the primary enzymatic activity of mitochondrial sirtuin 4 (SIRT4) and its overall role in metabolic control have remained enigmatic. Using a combination of phylogenetics, structural biology, and enzymology, we show that SIRT4 removes three acyl moieties from lysine residues: methylglutaryl (MG)-, hydroxymethylglutaryl (HMG)-, and 3-methylglutaconyl (MGc)-lysine.

Detection of the Previously Unobserved Stereoisomers of Thujone in the Essential Oil and Consumable Products of Sage (Salvia officinalis L.) Using Headspace Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry.

The discovery of the (+)-α-thujone and (-)-β-thujone stereoisomers in the essential oil of sage (Salvia officinalis L.) and dietary supplements is documented for the first time. The detection was accomplished using a chiral resolution protocol of racemic α-/β-thujone on headspace solid-phase microextraction-gas chromatography-mass spectrometry.

Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH.

Protein lysine posttranslational modification by an increasing number of different acyl groups is becoming appreciated as a regulatory mechanism in cellular biology. Sirtuins are class III histone deacylases that use NAD(+)as a co-substrate during amide bond hydrolysis. Several studies have described the sirtuins as sensors of the NAD(+)/NADH ratio, but it has not been formally tested for all the mammalian sirtuinsin vitro To address this problem, we first synthesized a wide variety of peptide-based probes, which were used to identify the range of hydrolytic activities of human sirtuins.

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.

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.

Mitochondrial protein acetylation regulates metabolism.

Changes in cellular nutrient availability or energy status induce global changes in mitochondrial protein acetylation. Over one-third of all proteins in the mitochondria are acetylated, of which the majority are involved in some aspect of energy metabolism. Mitochondrial protein acetylation is regulated by SIRT3 (sirtuin 3), a member of the sirtuin family of NAD+-dependent protein deacetylases that has recently been identified as a key modulator of energy homoeostasis.

Deletion of CaMKK2 from the liver lowers blood glucose and improves whole-body glucose tolerance in the mouse.

Ca(2+)/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca(2+)/CaM-dependent protein kinase family that is expressed abundantly in brain. Previous work has revealed that CaMKK2 knockout (CaMKK2 KO) mice eat less due to a central nervous system -signaling defect and are protected from diet-induced obesity, glucose intolerance, and insulin resistance. However, here we show that pair feeding of wild-type mice to match food consumption of CAMKK2 mice slows weight gain but fails to protect from diet-induced glucose intolerance, suggesting that other alterations in CaMKK2 KO mice are responsible for their improved glucose metabolism.

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.

BDNF-mediated cerebellar granule cell development is impaired in mice null for CaMKK2 or CaMKIV.

The Ca(2+)/calmodulin-activated kinases CaMKK2 and CaMKIV are highly expressed in the brain where they play important roles in activating intracellular responses to elevated Ca(2+). To address the biological functions of Ca(2+) signaling via these kinases during brain development, we have examined cerebellar development in mice null for CaMKK2 or CaMKIV. Here, we demonstrate that CaMKK2/CaMKIV-dependent phosphorylation of cAMP response element-binding protein (CREB) correlates with Bdnf transcription, which is required for normal development of cerebellar granule cell neurons.

Central control of feeding.

The rising rate of obesity in Western countries has led to intensified efforts to understand the molecular mechanisms underlying the central control of appetite and feeding behavior. This report highlights studies published from 2006 to 2008 revealing novel centrally acting anorexigenic hormones, the continued unraveling of complex hypothalamic intracellular signaling pathways that regulate feeding, and insights into leptin resistance.

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.

Regulation of AMP-activated protein kinase by multisite phosphorylation in response to agents that elevate cellular cAMP.

The AMP-activated protein kinase (AMPK) and cAMP signaling systems are both key regulators of cellular metabolism. In this study, we show that AMPK activity is attenuated in response to cAMP-elevating agents through modulation of at least two of its alpha subunit phosphorylation sites, viz. alpha-Thr(172) and alpha1-Ser(485)/alpha2-Ser(491), in the clonal beta-cell line INS-1 as well as in mouse embryonic fibroblasts and COS cells.

The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases.

The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKalphaThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs.

The autonomous activity of calcium/calmodulin-dependent protein kinase IV is required for its role in transcription.

Calcium/calmodulin-dependent kinase IV (CaMKIV) is a multifunctional serine/threonine kinase that is positively regulated by two main events. The first is the binding of calcium/calmodulin (Ca(2+)/CaM), which relieves intramolecular autoinhibition of the enzyme and leads to basal kinase activity. The second is activation by the upstream kinase, Ca(2+)/calmodulin-dependent kinase kinase.

Regulation and function of the calcium/calmodulin-dependent protein kinase IV/protein serine/threonine phosphatase 2A signaling complex.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a member of the broad substrate specificity class of Ca(2+)/calmodulin (CaM)-dependent protein kinases and functions as a potent stimulator of Ca(2+)-dependent gene expression. Activation of CaMKIV is a transient, tightly regulated event requiring both Ca(2+)/CaM binding and phosphorylation of the kinase on T200 by an upstream CaMK kinase (CaMKK). Previously, CaMKIV was shown to stably associate with protein serine/threonine phosphatase 2A (PP2A), which was proposed to play a role in negatively regulating the kinase.

Catalytic activity is required for calcium/calmodulin-dependent protein kinase IV to enter the nucleus.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a nuclear protein kinase that responds to acute rises in intracellular calcium by phosphorylating and activating proteins involved in transcription. Consistent with these roles, CaMKIV is found predominantly in the nucleus of cells in which it is expressed. Here we evaluate nuclear entry of CaMKIV and demonstrate that the protein kinase homology domain is both necessary and sufficient for nuclear localization.

Defective signaling in a subpopulation of CD4(+) T cells in the absence of Ca(2+)/calmodulin-dependent protein kinase IV.

Ca(2+)/calmodulin-dependent protein kinase IV-deficient (CaMKIV(-/-)) mice have been used to investigate the role of this enzyme in CD4(+) T cells. We identify a functional defect in a subpopulation of CD4(+) T cells, characterized by a cell surface marker profile usually found on memory phenotype CD4(+) T cells. Upon T-cell receptor engagement, the mutant cells produce diminished levels of interleukin-2 (IL-2), IL-4, and gamma interferon protein and mRNA.