Regulation of S-formylglutathione hydrolase by the anti-aging gene klotho.

Klotho is an aging-suppressor gene. The purpose of this study is to investigate the binding sites (receptors) and function of short-form Klotho (Skl). We showed that Skl physically bound to multiple proteins. We found physical and functional interactions between Skl and S-formylglutathione hydrolase (FGH), a key enzyme in the generation of the major cellular anti-oxidant GSH, using co-immunoprecipitation-coupled mass spectrometry. We further confirmed the colocalization of Skl and FGH around the nucleus in kidney cells using immunofluorescent staining. Skl positively regulated FGH gene expression via Kid3 transcription factor. Overexpression of Skl increased FGH mRNA and protein expression while silencing of Skl attenuated FGH mRNA and protein expression. Klotho gene mutation suppressed FGH expression in red blood cells and kidneys resulting in anemia and kidney damage in mice. Overexpression of Skl increased total GSH production and the GSH/GSSG ratio, an index of anti-oxidant capacity, leading to a decrease in intracellular H2O2 and superoxide levels. The antioxidant activity of Skl was eliminated by silencing of FGH, indicating that Skl increased GSH via FGH. Interestingly, Skl directly interacted with FGH and regulated its function. Site-directed mutagenesis of the N-glycan-modified residues in Skl abolished its antioxidant activity, suggesting that these N-glycan moieties are important features that interact with FGH. Specific mutation of Asp to Ala at site 285 resulted in a loss of anti-oxidant activity of Skl, suggesting that N-glycosylation at site 285 is the key mechanism that determines Skl activity. Therefore, this study demonstrates, for the first time, that Skl regulates anti-oxidant GSH generation via interaction with FGH through N-glycosylation.

Induction of anti-aging gene klotho with a small chemical compound that demethylates CpG islands.

Klotho (KL) is described as an anti-aging gene because mutation of Kl gene leads to multiple pre-mature aging phenotypes and shortens lifespan in mice. Growing evidence suggests that an increase in KL expression may be beneficial for age-related diseases such as arteriosclerosis and diabetes. It remains largely unknown, however, how Kl expression could be induced. Here we discovered novel molecular mechanism for induction of Kl expression with a small molecule 'Compound H', N-(2-chlorophenyl)-1H-indole-3-caboxamide. Compound H was originally identified through a high-throughput screening of small molecules for identifying Kl inducers. However, how Compound H induces Kl expression has never been investigated. We found that Compound H increased Kl expression via demethylation in CpG islands of the Kl gene. The demethylation was accomplished by activating demethylases rather than inhibiting methylases. Due to demethylation, Compound H enhanced binding of transcription factors, Pax4 and Kid3, to the promoter of the Kl gene. Pax4 and Kid3 regulated Kl promoter activity positively and negatively, respectively. Thus, our results show that demethylation is an important molecular mechanism that mediates Compound H-induced Kl expression. Further investigation is warranted to determine whether Compound H demethylates the Kl gene in vivo and whether it can serve as a therapeutic agent for repressing or delaying the onset of age-related diseases.

Molecular basis of Klotho: from gene to function in aging.

The discovery of the Klotho (KL) gene, which was originally identified as a putative aging-suppressor gene, has generated tremendous interest and has advanced understanding of the aging process. In mice, the overexpression of the KL gene extends the life span, whereas mutations to the KL gene shorten the life span. The human KL gene encodes the α-Klotho protein, which is a multifunctional protein that regulates the metabolism of phosphate, calcium, and vitamin D. α-Klotho also may function as a hormone, although the α-Klotho receptor(s) has not been found. Point mutations of the KL gene in humans are associated with hypertension and kidney disease, which suggests that α-Klotho may be essential to the maintenance of normal renal function. Three α-Klotho protein types with potentially different functions have been identified: a full-length transmembrane α-Klotho, a truncated soluble α-Klotho, and a secreted α-Klotho. Recent evidence suggests that α-Klotho suppresses the insulin and Wnt signaling pathways, inhibits oxidative stress, and regulates phosphatase and calcium absorption. In this review, we provide an update on recent advances in the understanding of the molecular, genetic, biochemical, and physiological properties of the KL gene. Specifically, this review focuses on the structure of the KL gene and the factors that regulate KL gene transcription, the key sites in the regulation of α-Klotho enzyme activity, the α-Klotho signaling pathways, and the molecular mechanisms that underlie α-Klotho function. This current understanding of the molecular biology of the α-Klotho protein may offer new insights into its function and role in aging.

Glycosylation of Skp1 promotes formation of Skp1-cullin-1-F-box protein complexes in dictyostelium.

O(2) sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1-F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1-cullin-1-F-box protein-Rbx1 subcomplex of E3(SCF)Ub ligases. E3(SCF)Ub ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O(2) availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA-Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding, consistent with an effect on the abundance of potential substrate proteins. We propose that O(2)-dependent posttranslational glycosylation of Skp1 promotes association with F-box proteins and their engagement in functional E3(SCF)Ub ligases that regulate O(2)-dependent developmental progression.

Role of the Skp1 prolyl-hydroxylation/glycosylation pathway in oxygen dependent submerged development of Dictyostelium.

BACKGROUND: Oxygen sensing is a near universal signaling modality that, in eukaryotes ranging from protists such as Dictyostelium and Toxoplasma to humans, involves a cytoplasmic prolyl 4-hydroxylase that utilizes oxygen and α-ketoglutarate as potentially rate-limiting substrates. A divergence between the animal and protist mechanisms is the enzymatic target: the animal transcriptional factor subunit hypoxia inducible factor-α whose hydroxylation results in its poly-ubiquitination and proteasomal degradation, and the protist E3SCF ubiquitin ligase subunit Skp1 whose hydroxylation might control the stability of other proteins. In Dictyostelium, genetic studies show that hydroxylation of Skp1 by PhyA, and subsequent glycosylation of the hydroxyproline, is required for normal oxygen sensing during multicellular development at an air/water interface. Because it has been difficult to detect an effect of hypoxia on Skp1 hydroxylation itself, the role of Skp1 modification was investigated in a submerged model of Dictyostelium development dependent on atmospheric hyperoxia. RESULTS: In static isotropic conditions beneath 70-100% atmospheric oxygen, amoebae formed radially symmetrical cyst-like aggregates consisting of a core of spores and undifferentiated cells surrounded by a cortex of stalk cells. Analysis of mutants showed that cyst formation was inhibited by high Skp1 levels via a hydroxylation-dependent mechanism, and spore differentiation required core glycosylation of Skp1 by a mechanism that could be bypassed by excess Skp1. Failure of spores to differentiate at lower oxygen correlated qualitatively with reduced Skp1 hydroxylation. CONCLUSION: We propose that, in the physiological range, oxygen or downstream metabolic effectors control the timing of developmental progression via activation of newly synthesized Skp1.

The Skp1 protein from Toxoplasma is modified by a cytoplasmic prolyl 4-hydroxylase associated with oxygen sensing in the social amoeba Dictyostelium.

In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O(2) and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O(2) regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3(SCF) ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O(2) sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O(2) levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O(2) requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O(2). The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O(2) by detecting the levels of O(2)-regulated metabolites such as α-ketoglutarate.

Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in dictyostelium.

The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O(2) regulation of development, suggesting the existence of an ancient O(2)-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O(2) signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. (1)H NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3(SCF)ubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3(VBC)ubiquitin ligases.