Young-Ki Paik et al. (2005) International Worm Meeting "Role of Daumone and Cholesterol in the Metabolic Aging of Caenorhabditis elegans"

Mankil Jung, Pan-Young Jeong, Heekyeong Kim, David J Chitwood, Yong-hyeon Yim, Young-Ki Paik

[International Worm Meeting, 2005]

We will present data on two key metabolic regulators of C. elegans: dauer-inducing pheromone (daumone) and cholesterol. First, we will focus on daumone, which signals C. elegans to enter the dauer stage, an enduring and non-ageing stage of the nematode life cycle with distinctive adaptive features and extended lifespan. To elucidate a daumone-mediated signaling pathway, we isolated natural daumone from C. elegans by large-scale purification, then synthesized and characterized daumone, which was found to be a novel glycolipid (ascarylose-fatty acid). We will show that both natural and chemically synthesized daumones equally induce dauer larva formation in C. elegans (N2 strain) and certain dauer mutants, and also result in competition between food and daumone. Second, we will discuss a role of cholesterol in metabolic aging. C. elegans, lacking the de novo sterol biosynthetic pathway, requires sterol as an essential nutrient. We microinjected a human 7-dehydrocholesterol reductase (DHCR) expression vector into C. elegans, which was then chromosomally incorporated by 47;-radiation. This transgenic C. elegans (cholegans, i.e., cholesterol producing C. elegans) showed 31% longer lifespan when grown in sterol-deficient medium and contained 80% more cholesterol than wild-type control. Brood size of cholegans was reduced 40% compared to wild-type control, although growth rate was not significantly altered. The biochemical basis for lifespan extension of cholegans appears to partly result from its acquired resistance against both UV irradiation and thermal stress. Taken together, our results should help to elucidate roles for daumone and cholesterol in ageing. (Supported by BioGreen21 Project [to YKP])

Young-Ki Paik et al. (2006) East Asia C. elegans Meeting "A Comprehensive Proteomic Analysis of Dauer and Cholesterol Signaling Pathway in Caenorhabditis elegans"

Mi Jeong Jeong, Young-Ki Paik, Keun Na, Pan-Young Jeong

[East Asia C. elegans Meeting, 2006]

With the availability of its complete genome sequence and unique biological features relevant to human disease, C. elegans has become an invaluable model organism for the studies of proteomics, leading to the elucidation of nematode gene function. We have focused on two key metabolic regulators of C. elegans: dauer-inducing pheromone (daumone; Jeong et al., 2005, Nature, 433, 541-545) and cholesterol (Choi et al, 2003, MCP, 2, 1086-95). First, using various proteomic tools including 2D-LC-MS/MS and DIGE, we analyzed proteomic profiles upon treatment of daumone, which signals C. elegans to enter the dauer stage, an enduring and non-ageing stage of the nematode life cycle. We found that there are multi-level changes in protein expression in the area of energy metabolism (decreased) and oxidative defense system (increased), suggesting that differential changes in various metabolic pathways appear to accommodate this non-aged state. This proteomic data was also paralleled with DNA microarray studies, which provided a window for direct comparison of proteomic and genomic profiles in dauer state. Second, we have launched a global C. elegans proteome project (CEPP) in order to analyze a comprehensive map of proteins involved in cholesterol signaling pathway in embryonic stage of C. elegans. In this talk, we will discuss a few lessons as well as some progress obtained from the pilot phase with respect to data management and functional annotation in C. elegans

Mo Xu et al. (2008) "Regulation of fat metabolism in Caenorhabditis elegans by lipid binding proteins"

Mo Xu, Young-Ki Paik

[2008]

"In mammals, fatty acid binding proteins transport fatty acids and other hydrophobic ligands, making them essential for processes such as fatty acid uptake, transport, and oxidation. In Caenorhabditis elegans, the lbp gene family (lbp-1 to -9) encodes the FABP homologs. Here, the role of each of these genes in fat metabolism was investigated using RNA-mediated interference (RNAi). Disruption of only lbp-5, -6, or -9 by RNAi feeding elevated relative fat content, as determined by Nile Red staining. Analysis of the expression of these three genes at different developmental stages indicated that low levels of LBP might have reduced less fatty acid oxidation, resulting in fat accumulation as seen in dauer larvae. Next, we investigated the relationship between these three genes and nhr-49, a key regulator of fat usage that is known to modulate pathways controlling fat consumption and fatty acid levels. Quantitative RT-PCR revealed that nhr-49 knockdown decreased the expression of lbp-5 and lbp-9, but not lbp-6. In support of this finding, nhr-49 RNAi to lbp-5::gfp decreased the intensity of lbp-5 florescence. Finally, bacterially-expressed LBP proteins were purified and assayed for fatty acid-binding activity. Interestingly, although LBP-5 and -6 have about 69% amino acid sequence similarity, they exhibit opposite binding preferences, suggesting that these proteins have divergent regulatory functions. Taken together, our findings suggest that each of the LBP proteins play differential roles in the regulation in fat metabolism in C. elegans and may provide insights into the regulation in fat metabolism in mammals. (Supported by a grant from a Forest Science & Technology Project [No. S110707L0501501 to YKP] through the Korea Forest Service)"

Y Kimura et al. (1999) International C. elegans Meeting "Ca2+/CaM-dependent protein kinases (CaM-K)-mediated signal cascade is conserved in C. elegans"

K Eto, H Tokumitsu, Y Kimura, M Muramatsu

[International C. elegans Meeting, 1999]

In mammals, Ca 2+ /CaM protein kinases (CaM-K) is thought to be important for Ca 2+ -dependent signal transduction. This cascade consists of the upstream CaM-kinase kinase (CaM-KK) and downstream CaM kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV). In the present study, we have identified CaM-KK and CaM-KI orthologue of C. elegans (named Ce CaM-KK and Ce CaM-KI, respectively) and analyzed their biochemical activities. Ce CaM-KK has two unusual features in the catalytic domain which is conserved between species, that is, a lack of acidic residues important for substrate recognition and an Arg-Pro rich insert (RP-domain) which is essential for the recognition and activation of CaM-KIV and CaM-KI. Indeed, Ce CaM-KK activated mammalian CaM-KIV in vitro . Ce CaM-KI is highly homologous to mammalian CaM-KI and is activated by Ce CaM-KK through phosphorylation of Thr179 in a Ca 2+ /CaM-dependent manner. Truncation at residue 295 in CeCaM-KI generates a constitutively active enzyme, suggesting that COOH-terminal at residue 295 contains autoinhibitory and CaM-binding domains. Unlike mammalian CaM-KI, Ce CaM-KI mainly localized in the nucleus of transfected mammalian cells by the virtue of the NH 2 -terminal six residues containing a functional nuclear localization signal. Taken together, these results suggest that CaM-KK/CaM-KI cascade in C. elegans is conserved and operated both in vitro and intact cells. Current research aims to the physiological functions of Ce CaM-KK and Ce CaM-KI cascade in vivo . Analyses for the expression patterns and isolation of mutant worms for both genes are underway.

Hyun Ah Sun et al. (2006) East Asia C. elegans Meeting "Molecular Cloning of Caenorhabditis elegans HMG-CoA Synthase Gene and Its Regulation in the Dauer Stage"

Young-Ki Paik, Hyun Ah Sun

[East Asia C. elegans Meeting, 2006]

HMG-CoA synthase (HMGS) has been known as one of the key enzymes involved in cholesterol biosynthesis (in cytosol) and ketone body formation (in mitochondrion) in animals, depending on the physiological condition. In C. elegans, it has not previously been well established as to the presence of this enzyme or its regulation under various physiological states of this nematode. In the course of our study for the daumone-related biology, we were interested in cloning and characterization of HMGS gene. Since one of the characteristic physiological features of dauer larvae of C. elegans is believed to be an yet to be characterized efficient fat mobilization and ketone body formation to meet the energy demands for survival, we wanted to investigate how this enzyme might be involved in energy metabolism in the daumone-induced dauer larvae. To understand a functional role of this enzyme in dauer state, we first isolated F25B4.6 clone, an ortholog of human HMGS gene, by searching through the C. elegans genome database. Our data from DNA sequencing, RNAi and RT-PCR experiments suggest that this clone indeed encodes an authentic C. elegans HMGS and its mRNA level appears to be regulated developmentally, which peaks the early L2 stage. With the availability of the over-expressed recombinant protein (Mr.. 51.4 kDa), functional characterization and molecular regulation of HMGS is now underway ([Supported by a grant from BioGreen 21 Project to YKP]).

Eto K et al. (1999) J Biol Chem "Ca(2+)/Calmodulin-dependent protein kinase cascade in Caenorhabditis elegans. Implication in transcriptional activation."

Kimura Y, Eto K, Tokumitsu H, Arai K, Takahashi N, Muramatsu M, Masuho Y

[J Biol Chem, 1999]

We have recently demonstrated that Caenorhabditis elegans Ca(2+)/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803-15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CeCaM-KI), which has approximately 60% identity to mammalian CaM-KI. CeCaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by CeCaM-KK through phosphorylation of Thr(179) in a Ca(2+)/CaM-dependent manner, resulting in a 30-fold decrease in the K(m) of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-KI, CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH(2)-terminal six residues ((2)PLFKRR(7)) contain a functional nuclear localization signal. We have also demonstrated that CeCaM-KK and CeCaM-KI reconstituted a signaling pathway that mediates Ca(2+)-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of CeCaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved in C. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca(2+)-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.

Pan-Young Jeong et al. (2005) International Worm Meeting "Comprehensive proteomic analysis of dauer larva state in Caenorhabditis elegans."

Young-Ki Paik, Pan-Young Jeong

[International Worm Meeting, 2005]

The dauer is a developmentally arrested third stage larva of the nematode C. elegans. Entry into dauer arrest is promoted by lack of food, high temperature, and high concentration of dauer pheromone, a constitutively secreted substance serving as an indicator of population density. Dauer larva has been described as non-aging, as post dauer lifespan is not affected by the duration that an animal spends in the dauer state. In order to determine protein changes during the dauer larva stage, we performed proteomic analysis for the dauer larva and mixed normal N2 stage using 2-dimensional electrophoresis (2DE) and mass spectrometry (MS). We were able to identify more than 170 protein spots out of 460 spots that are differentially expressed in dauer larva stage by 2DE. Our MS results indicate that the expression levels of proteins involved in energy metabolic pathway including glycolysis, gluconeogenesis, TCA, oxidative phosphorylation and fatty acid 46;-oxidation pathways were notably decreased in dauer larva, as compared with the normal worms. However, the structurally related proteins (galactin and LEV-11) and detoxification related proteins (SCP-like extracellular protein, DHP-2, CAH-4 and Peroxiredoxin) were increased in dauer larva. The lipid (fatty acid) binding protein, some ATP biosynthesis and transport system and pentosphosphate pathway related proteins remained unchanged in dauer larva stage, suggesting dauer larva stage may cause differential changes in expression of proteins involved in various metabolic pathways to accommodate this non-aged state when faced with adverse environmental condition.

E Jin Shin et al. (2006) East Asia C. elegans Meeting "PDK, a Molecular Switch for Energy Metabolism in Caenorhabditis elegans"

E Jin Shin, Young-Ki Paik

[East Asia C. elegans Meeting, 2006]

When faced with an adverse environment such as depletion of food, constitutively secreted C. elegans daumone signals this nematode enter dauer stage, an enduring and non-aged condition. We hypothesized that like hibernating animal C. elegans might also have the same molecular machine which controls an energy consumption during dauer stage. To understand a molecular mechanism by which dauer larvae manage energy metabolism, we were interested in cloning and characterizing C. elegans pyruvate dehydrogenase kinase (CePDK), which has been known as molecular switch for hibernating animal by preventing the flow of glycolytic products into the tricarboxylic acid cycle in the adverse condition. Through the extensive homologue search and molecular cloning works, we isolated ZK370.5 clone which is believed to encode CePDK. Having established that the over-expressed recombinant CePDK in E. coli exhibited the anticipated molecular characteristics such as molecular size and catalytic function, mRNA expression pattern was examined using quantitative RT-PCR. Our preliminary data showed that CePDK mRNA is highly expressed in either dauer or starved state but suppressed in other states, suggesting the presence of regulatory mechanism by which the nutritional state can be sensitized by CePDK in this nematode. When examined mRNA distribution in the several mutants that are grown at fed, starved and dauer condition, the levels of CePDK gene expression were higher in both daf-2 and age-1 than those in either daf-16 or nhr-49. In particular, RNAi of CePDK in daf-2 mutants resulted in a reduced lifespan, indicating that PDK may also be regulated by DAF-16. (Supported by a grant from BioGreen 21 Project to YKP).

Ying-Xiu Li et al. (2008) "A potential role for acetyl-CoA carboxylase in molting of Caenorhabditis elegans"

Young-Ki Paik, Ying-Xiu Li

[2008]

"Acetyl-CoA carboxylase (ACC) is a key enzyme in biosynthesis of fatty acids, which serve essential cellular functions in energy storage, membrane structure, and signaling. In Caenorhabditis elegans, W09B6.1 is known ACC homologous gene whereas its point mutant pod-2 is known to produce a embryonic lethal phenotype, polarity defects, and an osmotic-sensitive phenotype. Here, we further characterized the role of ACC in C. elegans through expression of transgenic ACC (WO9B6.1::GFP) and WO9B6.1 RNAi feeding. Transgenic ACC was primarily localized to the hypodermis and intestine, where it was present throughout all four developmental stages. Electron microscopic analysis revealed that W09B6.1 RNAi worms contained double cuticles, likely due to incomplete ecdysis during the molting process. This RNAi-induced molting defect was accompanied by down regulation of several protease genes (e.g., Acn-1, Cpz-1, Nas-37), as determined by a quantitative RT-PCR. Inhibition of fatty acid synthase also caused down regulated expression of these proteases. These findings suggest that ACC plays an important role in molting and that compromised fatty acid metabolism induces crucial defects in molting process through alterations in protease activity. (Supported by a grant from a Forest Science & Technology Project [No. S110707L0501501 to YKP] through the Korea Forest Service)."

Byung-Kwon Choi et al. (2001) International C. elegans Meeting "A C.elegans Sterol reductase homologue involved in vulva development"

Young-Ki Paik, Byung-Kwon Choi

[International C. elegans Meeting, 2001]

Sterol metabolism in Caenorhabditis elegans ( C.elegans ) is different from the other species such as mammals and yeast. It is unable to synthesize sterol through de novo pathway but is able to use sterols such as cholesterol, sitosterol and stigmasterol from the environment. Until now, a few data has been reported about sterol metabolism in C. elegans . However, research on the cloning of cDNA and functional studies of any enzyme involved in sterol metabolism has not been reported. Through the search of Sanger database, the B0250 cosmid clone, which contained a homologous gene to human 7-dehydrocholesterol reductase, human lamin B receptor and yeast ERG24 was identified. From the sequence data, C. elegans sterol reductase homologous cDNA (CeSR) was cloned by screening of embryo cDNA library followed by 5'-RACE. The deduced peptide was 457 amino acids and exhibited identities of 29, 28 and 27%, and similarity of 47, 43 and 45 % with those of human 7-dehydrocholesterol reductase, human Lamin B receptor, and yeast ERG24, respectively. The function of CeSR is not known at present. To examine the localization of CeSR expression, 5' flanking region of CeSR was fused with green fluorescent protein (GFP) and injected into gonad. CeSR expression was appeared in the head neurons, hypodermis and body wall muscles during the larva stages (L1 to L3). CeSR was also expressed in the vulva muscle beginning in late larva stage (especially L4) and maintained through the adult stage. But the expression in the body wall muscle was disappeared after L4 stage. Our results indicate that CeSR is a nerve cell- and vulva-specific protein. We are in process of RNA mediated interference by transgene.