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ABSTRACT: Thioredoxin reductase (TrxR) is a member of the pyridine nucleotide-disulfide reductase family, which mainly functions in the thioredoxin system. TrxR is found in all living organisms and exists in two major ubiquitous isoenzymes in higher eukaryotic cells; One is cytosolic and the other mitochondrial. Mitochondrial TrxR functions to protect mitochondria from oxidative stress, where reactive oxidative species are mainly generated, while cytosolic TrxR plays a role to maintain optimal oxido-reductive status in cytosol. In this study, we report differential physiological functions of these two TrxRs in C. elegans. trxr-1, the cytosolic TrxR, is highly expressed in pharynx, vulva and intestine, whereas trxr-2, the mitochondrial TrxR, is mainly expressed in pharyngeal and body wall muscles. Deficiency of the non-selenoprotein trxr-2 caused defects in longevity and delayed development under stress conditions, while deletion mutation of the selenoprotein trxr-1 resulted in interference in acidification of lysosomal compartment in intestine. Interestingly, the acidification defect of trxr-1(jh143) deletion mutant was rescued, not only by selenocystein-containing wild type TRXR-1, but also cysteine-substituted mutant TRXR-1. Both trxr-1 and trxr-2 were up-regulated when worms were challenged by environmental stress such as heat shock. These results suggest that trxr-1 and trxr-2 function differently at organismal level presumably by their differential sub-cellular localization in C. elegans.
Molecules and Cells 07/2012; 34(2):209-18. · 2.18 Impact Factor
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ABSTRACT: The nematode C. elegans is an ideal organism to study the in vivo genetic and biochemical functions of calreticulin. In vitro studies show that the
C. elegans CRT-1 protein, like other calreticulins, is a calcium-binding molecular chaperone. Mutants of crt-1 in C. elegans mutants are viable and fertile, offering the opportunity for scientists to study in vivo functions of calreticulin at more
depth, crt-1 null mutants showed temperature-sensitive fertility defects, and transcription of crt-1 was upregulated in stress conditions such as high temperature and ethanol treatment suggesting that calreticulin may be functioning
in stress response. Mutants of the calreticulin gene were also shown to suppress necrotic cell death in neurons. The use of
pharmological agents and the genetic application of mutants involved in ER calcium homeostasis showed that calreticulin was
critical in the regulation of ER calcium levels during the neuronal degeneration process. Double mutants of crt-1 mutants and itr-1 IP3 receptor mutants displayed synergistic severity in defecation rhythm defects further suggesting the role of calreticulin
in ER calcium homeostasis. Further genetic analysis in C. elegans between crt-1 and other components involved in ER calcium regulation should deepen our understanding of calreticulin and calcium homeostasis
at both the cellular and organism level.
07/2011: pages 248-257;
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Le Tho Son,
Kyung-Min Ko,
Jeong Hoon Cho,
Gunasekaran Singaravelu,
Indrani Chatterjee,
Tae-Woo Choi,
Hyun-Ok Song,
Jae-Ran Yu, Byung-Jae Park,
Sun-Kyung Lee,
Joohong Ahnn
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ABSTRACT: Dicarbonyl/L-xylulose reductase (DCXR) converts l-xylulose into xylitol, and reduces various α-dicarbonyl compounds, thus performing a dual role in carbohydrate metabolism and detoxification. In this study, we identified DHS-21 as the only DCXR ortholog in Caenorhabditis elegans. The dhs-21 gene is expressed in various tissues including the intestine, gonadal sheath cells, uterine seam (utse) cells, the spermathecal-uterus (sp-ut) valve and on the plasma membrane of spermatids. Recombinant DHS-21 was shown to convert L-xylulose to xylitol using NADPH as a cofactor. Dhs-21 null mutants of C. elegans show defects in longevity, reproduction and egg-laying. Knock-down of daf-16 and elt-2 transcription factors affected dhs-21 expression. These results suggest that DHS-21 is a bona fide DCXR of C. elegans, essential for normal life span and reproduction.
FEBS letters 05/2011; 585(9):1310-6. · 3.54 Impact Factor
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ABSTRACT: Autophagy and endocytic pathway are highly regulated catabolic processes. Both processes are crucial for cell growth, development, differentiation, disease and homeostasis and exhibit membrane rearrangement for their function. Autophagy and endocytic pathway represent branches of the lysosomal digestive system, autophagy being responsible for degradation of cytoplasmic components and endocytic pathway for degradation of exogenous substances. Here we report that autophagy is activated when endocytic pathway regulatory genes such as rab-5 and rabx-5 are disrupted. Defects in the ubiquitin binding domain of RABX-5 are critical in activating autophagy. We also observed that the elevated autophagy level does not contribute to lifespan extension of rabx-5 mutant. Our results suggest that autophagy may compensate for the endocytic pathway when regulatory genes for the endocytic pathway malfunction, providing a case of complementation between two functionally related cellular processes.
Molecules and Cells 05/2011; 31(5):477-81. · 2.18 Impact Factor
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ABSTRACT: Shank protein is one of the postsynaptic density (PSD) proteins which play a major role in proper localization of proteins at membranes. The shn-1, a homolog of Shank in Caenorhabditis elegans, is expressed in neurons, pharynx, intestine, vulva and sperm. We have previously reported a possible genetic interaction between Shank and IP₃ receptor by examining shn-1 RNAi in IP₃ receptor (itr-1) mutant background. In order to show the direct interaction of Shank and IP₃ receptor as well as to show the direct in vivo function of Shank, we have characterized two different mutant alleles of shn-1, which have different deletions in the different domains. shn-1 mutants were observed for Ca²+-related behavioral defects with itr-1 mutants. We found that only shn-1 mutant defective in ANK repeat-domain showed significant defects in defecation, pharyngeal pumping and fertility. In addition, we found that shn-1 regulates defecation, pharyngeal pumping and probably male fertility with itr-1. Thus, we suggest that Shank ANK repeat-domain along with PDZ may play a crucial role in regulating Ca²+-signaling with IP₃ receptor.
Molecules and Cells 12/2010; 31(1):79-84. · 2.18 Impact Factor
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ABSTRACT: C. elegans coelomocytes are macrophage-like scavenger cells that provide an excellent in vivo system for the study of clathrin-mediated endocytosis. Using this in vivo system, several genes involved in coelomocyte endocytosis have been identified previously. However, the detailed mechanism of endocytic pathway is still unknown. Here, we report a new function of calcineurin, an evolutionarily conserved Ca(2+)/calmodulin-dependent Ser/Thr protein phosphatase, in coelomocyte endocytosis. We found that calcineurin mutants show defective coelomocyte endocytosis. Genetic analysis suggests that calcineurin and a GTPase, dynamin (DYN-1), may function upstream of an orphan receptor, CUP-4, to regulate endocytosis. Therefore, we propose a model in which calcineurin may regulate coelomocyte endocytosis via DYN-1 and CUP-4 in C. elegans.
Molecules and Cells 09/2010; 30(3):255-62. · 2.18 Impact Factor
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ABSTRACT: Caenorhabditis elegans is a free living soil nematode and thus in its natural habitat, C. elegans encounters many different species of soil bacteria. Although some soil bacteria may be excellent sources of nutrition for the worm, others may be pathogenic. Thus, we undertook a study to understand how C. elegans can identify their preferred food using a simple behavioral assay. We found that there are various species of soil bacteria that C. elegans prefers in comparison to the standard laboratory E. coli strain OP50. In particular, two bacterial strains, Bacillus mycoides and Bacillus soli, were preferred strains. Interestingly, the sole feeding of these bacteria to wild type animals results in extended lifespan through the activation of the autophagic process. Further studies will be required to understand the precise mechanism controlling the behavior of identification and selection of food in C. elegans.
Molecules and Cells 09/2009; 28(3):209-13. · 2.18 Impact Factor
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ABSTRACT: Nramp1 (natural resistance-associated macrophage protein-1) is a functionally conserved iron-manganese transporter in macrophages. Manganese (Mn), a superoxide scavenger, is required in trace amounts and functions as a cofactor for most antioxidants. Three Nramp homologs, smf-1, smf-2, and smf-3, have been identified thus far in the nematode Caenorhabditis elegans. A GFP promoter assay revealed largely intestinal expression of the smf genes from early embryonic through adult stages. In addition, smf deletion mutants showed increased sensitivity to excess Mn and mild sensitivity to EDTA. Interestingly, these smf deletion mutants demonstrated hypersensitivity to the pathogen Staphylococcus aureus, an effect that was rescued by Mn feeding or knockdown of the Golgi calcium/manganese ATPase, pmr-1, indicating that Mn uptake is essential for the innate immune system. This reversal of pathogen sensitivity by Mn feeding suggests a protective and therapeutic role of Mn in pathogen evasion systems. We propose that the C. elegans intestinal lumen may mimic the mammalian macrophage phagosome and thus could be a simple model for studying Mn-mediated innate immunity.
Biochemical and Biophysical Research Communications 09/2009; 390(1):136-41. · 2.48 Impact Factor
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ABSTRACT: Accumulation of unfolded proteins in the endoplasmic reticulum triggers the unfolded protein response (UPR) pathway, which increases the expression of chaperones to maintain the homeostasis. Calreticulin is a calcium-binding chaperone located in the lumen of endoplasmic reticulum (ER). Here we show that in response to a UPR inducing reagent, tunicamycin, the expression of calreticulin (crt-1) is specifically up-regulated in Caenorhabditis elegans. Tunicamycin (TM) induced expression of the crt-1 requires IRE-1 and XBP-1 but is ATF-6 and PEK-1 independent. Analysis of the crt-1 promoter reveals a putative XBP-1 binding site at the -284 to -278 bp region, which was shown to be necessary for TM-mediated induction. Genetic analysis of crt-1 mutants and mutants of UPR pathway genes show various degrees of developmental arrest upon TM treatment. Our results suggest that the TM-induced UPR pathway culminates in the up-regulation of crt-1, which protects the worm from deleterious accumulation of unfolded proteins in the ER. Knockdown of the crt-1, pdi-2, or pdi-3 increased the crt-1 expression, whereas knockdown of the hsp-3 or hsp-4 did not have any effect on crt-1 expression, indicating the existence of complex compensatory networks to cope up with ER stress.
Journal of Molecular Biology 10/2007; 372(2):331-40. · 4.00 Impact Factor
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Jeong Hoon Cho,
Kyung Min Ko,
Gunasekaran Singaruvelu,
Wonhae Lee,
Gil Bu Kang,
Seong-Hwan Rho, Byung-Jae Park,
Jae-Ran Yu,
Hiroaki Kagawa,
Soo Hyun Eom,
Do Han Kim,
Joohong Ahnn
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ABSTRACT: Dual roles of calsequestrin (CSQ-1) being the Ca2+ donor and Ca2+ acceptor make it an excellent Ca2+-buffering protein within the sarcoplasmic reticulum (SR). We have isolated and characterized a calsequestrin (csq-1)-null mutant in Caenorhabditis elegans. To our surprise, this mutant csq-1(jh109) showed no gross defects in muscle development or function but, however, is highly sensitive to perturbation of Ca2+ homeostasis. By taking advantage of the viable null mutant, we investigated the domains of CSQ-1 that are important for polymerization and cellular localization, and required for its correct buffering functions. In transgenic animals rescued with various CSQ-1 constructs, the in vivo patterns of polymerization and localization of several mutated calsequestrins were observed to correlate with the structure-function relationship. Our results suggest that polymerization of CSQ-1 is essential but not sufficient for correct cellular localization and function of CSQ-1. In addition, direct interaction between CSQ-1 and the ryanodine receptor (RyR) was found for the first time, suggesting that the cellular localization of CSQ-1 in C. elegans is indeed modulated by RyR through a physical interaction.
Journal of Cell Science 06/2007; 120(Pt 9):1551-8. · 6.11 Impact Factor
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ABSTRACT: Calcineurin is a Ca2+/Calmodulin activated Ser/Thr phosphatase that is well conserved from yeast to human. In Caenorhabditis elegans, tax-6 and cnb-1 encode catalytic and regulatory subunits of calcineurin, respectively. We performed yeast two-hybrid screening using TAX-6 as a bait to identify calcineurin interacting proteins. KIN-29 is one of proteins that specifically interacted with TAX-6. KIN-29 is a Ser/Thr kinase previously shown to be involved in regulating gene expression of a subset of chemoreceptors in specific neurons. Both TAX-6 and KIN-29 are expressed in hypodermis, muscles, and neurons. Moreover, both calcineurin and kin-29 mutants exhibit similar phenotypes, namely small body size, small brood size, and slow growth. Here we describe specific genetic interaction between tax-6 and kin-29 in regulating body size, serotonin mediated egg laying, and chemoreceptor expression.
Biochemical and Biophysical Research Communications 02/2007; 352(1):29-35. · 2.48 Impact Factor
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ABSTRACT: Vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump, which transports protons across the membrane. It is a multi-protein complex which is composed of at least 13 subunits. The Caenorhabditis elegans vha-8 encodes the E subunit of V-ATPase which is expressed in the hypodermis, intestine and H-shaped excretory cells. VHA-8 is necessary for proper intestinal function likely through its role in cellular acidification of intestinal cells. The null mutants of vha-8 show a larval lethal phenotype indicating that vha-8 is an essential gene for larval development in C. elegans. Interestingly, characteristics of necrotic cell death were observed in the hypodermis and intestine of the arrested larvae suggesting that pH homeostasis via the E subunit of V-ATPase is required for the cell survival in C. elegans.
FEBS Letters 06/2006; 580(13):3161-6. · 3.54 Impact Factor
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ABSTRACT: Proper folding and maintenance of the native structure are central to protein function and are assisted by a family of proteins called chaperones. Calreticulin and calnexin are ER resident chaperones well conserved from worm to human. Calreticulin/calnexin knock-out mice exhibit a severe phenotype, whereas in Caenorhabditis elegans, calreticulin [crt-1(jh101)]- and calnexin [cnx-1(nr2009)]-null mutant worms exhibit only a mild phenotype, suggesting the possible existence of alternative chaperone machinery that can compensate for the deficiency of calreticulin and/or calnexin. In order to rapidly identify the compensatory chaperone components involved in this process, we analyzed the proteome of crt-1(jh101) mutants and [crt-1(jh101);cnx-1(nr2009)] double mutants. When grown at 20 degrees C, we found that five proteins were up-regulated and two proteins were down-regulated in crt-1(jh101) mutants; nine proteins were up-regulated and five proteins were down-regulated in [crt-1(jh101);cnx-1(nr2009)] double mutants. In addition, elevation of the cultivation temperature to 25 degrees C, which is still permissive to growth but causes specific defects in mutants, led to the identification of several additional proteins. Interestingly, the consistent increment of heat shock protein-70 family members (hsp70) together with protein disulfide isomerase (PDI) at all the examined conditions suggests the possible compensatory function imparted by hsp70 and PDI family members in the absence of calreticulin and/or calnexin.
PROTEOMICS 03/2006; 6(4):1329-39. · 4.51 Impact Factor
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ABSTRACT: Thioredoxin, an oxidoreductase, is a multifunction protein. The thioredoxin system is composed of NADPH, thioredoxin reductase and thioredoxin. This enzyme is highly conserved from bacteria to humans. We have characterized TRX-1, a thioredoxin homolog in C. elegans, which has about 36% identity in amino acid sequence with human thioredoxin. By gfp reporter system, trx-1 has been shown to be restrictedly expressed in ASI and ASJ neurons and in intestine. Immunostaining confirmed the intestinal expression. Full-length cDNA of trx-1 has been isolated by cDNA library PCR and subsequently cloned and sequenced. We have shown that the encoded protein functions as a reductase in the insulin reducing assay. Moreover, we have isolated a deletion mutant by PCR-based TMP-UV mutagenesis method. Mutant animals have reduced life span and are sensitive to oxidative stress. Reintroduction of trx-1 into mutant worms fully restored the wild-type phenotype. Our results suggest that trx-1 has important functions in life span regulation and oxidative stress response in C. elegans.
Genes to Cells 01/2006; 10(12):1203-10. · 2.68 Impact Factor
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ABSTRACT: Calnexin, a type I integral Ca(2+)-binding protein in the endoplasmic reticulum (ER) membrane, has been implicated in various biological functions including chaperone activity, calcium homeostasis, phagocytosis, and ER stress-induced apoptosis. Caenorhabditis elegans CNX-1 is expressed in the H-shaped excretory cell, intestine, dorsal and ventral nerve cord, spermatheca, and head and tail neurons throughout development. A cnx-1 null mutant displays temperature-sensitive developmental and reproductive defects, and retarded growth under stress. Moreover, a double knockout mutant of calnexin and calreticulin exhibits additive severe defects. Interestingly, both cnx-1 transcript and protein levels are elevated under stress conditions suggesting that CNX-1 may be important for stress-induced chaperoning functions in C. elegans. Glycosidase treatment and site-directed mutagenesis confirmed that CeCNX-1 is N-glycosylated at two asparagine residues of Asn(203) and Asn(571). When transgenic animals from cnx-1 mutant were generated, a glycosylation defective construct failed to rescue phenotypes of cnx-1 mutant suggesting that glycosylation is important for calnexin's functions in C. elegans.
Biochemical and Biophysical Research Communications 01/2006; 338(2):1018-30. · 2.48 Impact Factor
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ABSTRACT: Ca(2+)/calmodulin-dependent calcineurin has been shown to have important roles in various Ca(2+) signaling pathways. We have previously reported that cnb-1(jh103) mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in Caenorhabditis elegans. Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1(null) mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, which is required for maintenance of normal body size in C.elegans. Genetic interactions between tax-6 and several mutants including egl-30 and egl-10, which are known to be involved in G-protein signaling pathways suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Goalpha) and egl-30(Gqalpha). Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C.elegans.
Journal of Molecular Biology 12/2004; 344(2):585-95. · 4.00 Impact Factor
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ABSTRACT: Protein localization in the postsynaptic density (PSD) of neurons is mediated by scaffolding proteins such as PSD-95 and Shank, which ensure proper function of receptors at the membrane. The Shank family of scaffolding proteins contain PDZ (PSD-95, Dlg, and ZO-1) domains and have been implicated in the localizations of many receptor proteins including glutamate receptors in mammals. We have identified and characterized shn-1, the only homologue of Shank in Caenorhabditis elegans. The shn-1 gene shows approximately 40% identity over 1000 amino acids to rat Shanks. SHN-1 protein is localized in various tissues including neurons, pharynx and intestine. RNAi suppression of SHN-1 did not cause lethality or developmental abnormality. However, suppression of SHN-1 in the itr-1 (sa73) mutant, which has a defective inositol-1,4,5-trisphosphate (IP(3)) receptor, resulted in animals with altered defecation rhythm. Our data suggest a possible role of SHN-1 in affecting function of IP(3) receptors in C. elegans.
FEBS Letters 04/2004; 561(1-3):29-36. · 3.54 Impact Factor
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Byung-Jae Park,
Duk-Gyu Lee,
Jae-Ran Yu,
Sun-Ki Jung,
Kyuyeong Choi,
Jungsoo Lee,
Jiyeon Lee,
Sik Kim,
Jin Il,
Lee, [......],
Junho Lee,
Andrew Singson,
Woo,
Keun Song,
Hyun Soo,
Eom,
Chul-Seung Park,
Han Kim,
Jaya Bandyopadhyay,
Joohong Ahnn
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ABSTRACT: Calreticulin (CRT), a Ca 2 -binding protein known to have many cellular functions, including regulation of Ca 2 homoeostasis and chaperone activity, is essential for heart and brain devel-opment during embryogenesis in mice. Here, we report the functional characterization of Caeno-rhabditis elegans calreticulin (crt-1). A crt-1 null mutant does not result in embryonic lethality but shows temperature-dependent reproduction defects. In C. elegans CRT-1 is expressed in the intestine, pharynx, body-wall muscles, head neurons, coelomocytes, and in sperm. crt-1 males exhibit reduced mating efficiency and defects late in sperm development in addition to defects in oocyte development and/or somatic gonad function in hermaphrodites. Furthermore, crt-1 and itr-1 (inositol triphosphate receptor) together are required for normal behavioral rhythms. crt-1 transcript level is elevated under stress conditions, suggesting that CRT-1 may be important for stress-induced chaperoning function in C. elegans.
Molecular Biology of the Cell. 09/2001; 12:2835-2845.
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ABSTRACT: Two putative homologues of large conductance Ca2+-activated K+ channel alpha-subunit gene (slowpoke or slo) were revealed by C. elegans genome sequencing. One of the two genes, F08B12.3 (Ce-slo-2), shows a relatively low amino acid sequence similarity to other Slo sequences and lacks key functional motifs, which are important for calcium and voltage sensing. However, its overall structure and regions of homology, which are conserved in all Slo proteins, suggest that Ce-SLO-2 should belong to the Slo channel family. We have cloned a full-length cDNA of the Ce-slo-2, which encodes a protein containing six putative transmembrane segments with a K+-selective pore and a large C-terminal cytosolic domain. Green fluorescent protein (GFP) and whole-mount immunostaining analyses revealed that Ce-slo-2 is specifically expressed in neuronal cells at the nerve ring, at the ventral nerve cord of the mid-body, and at the tail region. We have also identified a putative human counterpart of Ce-slo-2 from a human brain EST database, which shows a stretch of highly conserved amino acid residues. Northern blot and mRNA dot blot analyses revealed a strong and specific expression in brain and skeletal muscle. Taken together, our data suggest that Ce-slo-2 may constitute an evolutionarily conserved gene encoding a potassium channel that has specific functions in neuronal cells.
Gene.
