Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney

In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lung-specific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for alpha-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and l-xylulose, and were inhibited competitively by n-butyric acid. Therefore, the proteins are designated as dicarbonyl/l-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and l-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC ) are identical to l-xylulose reductase (EC ), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.     

An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans.

Mutations in daf-2 and age-1 cause a dramatic increase in longevity as well as developmental arrest at the dauer diapause stage in Caenorhabditis elegans. daf-2 and age-1 encode components of an insulin-like signaling pathway. Both daf-2 and age-1 act at a similar point in the genetic epistasis pathway for dauer arrest and longevity and regulate the activity of the daf-16 gene. Mutations in daf-16 cause a dauer-defective phenotype and are epistatic to the diapause arrest and life span extension phenotypes of daf-2 and age-1 mutants. Here we show that mutations in this pathway also affect fertility and embryonic development. Weak daf-2 alleles, and maternally rescued age-1 alleles that cause life span extension but do not arrest at the dauer stage, also reduce fertility and viability. We find that age-1(hx546) has reduced both maternal and zygotic age-1 activity. daf-16 mutations suppress all of the daf-2 and age-1 phenotypes, including dauer arrest, life span extension, reduced fertility, and viability defects. These data show that insulin signaling, mediated by DAF-2 through the AGE-1 phosphatidylinositol-3-OH kinase, regulates reproduction and embryonic development, as well as dauer diapause and life span, and that DAF-16 transduces these signals. The regulation of fertility, life span, and metabolism by an insulin-like signaling pathway is similar to the endocrine regulation of metabolism and fertility by mammalian insulin signaling.     

Bioinformatic and biochemical characterization of DCXR and DHRS2/4 from Caenorhabditis elegans

Several reductases belonging to the large enzyme superfamily of the short-chain dehydrogenases/reductases (SDR) are involved in the reductive metabolism of carbonyl containing xenobiotics. In order to characterize the human enzymes dicarbonyl/l-xylulose reductase (DCXR), and dehydrogenase/reductase members 2 and 4 (DHRS2, DHRS4) in terms of metabolism of xenobiotics, orthologues from the model organism Caenorhabditis elegans (C. elegans) were identified by using hidden Markov models that were developed in the present study. Accordingly, we describe the characterization of proteins from C. elegans as orthologous to the human enzymes DCXR and DHRS2/4 using a combined approach of bioinformatic and biochemical methods. With the hidden Markov model based system we identified the C. elegans proteins SDR20C18, SDR25C21 and SDR25C22 as being homologous to the human enzymes DCXR, and DHRS2 or DHRS4, respectively. After cloning and overexpression of these three C. elegans genes in Escherichia coli we could purify SDR20C18 and SDR25C22 as soluble proteins by Ni-affinity chromatography, whereas recombinant SDR25C21 was only found in inclusion bodies. Both SDR20C18 (UniProtAcc: Q21929) and SDR25C22 (UniProtAcc: Q93790) were tested with a variety of xenobotic carbonyl compounds as substrates. A comparison of the catalytic activities of SDR20C18 and SDR25C22 with well-known substrates of the human forms revealed that SDR20C18 is the DCXR-orthologue enzyme to the human enzyme and that SDR25C22 might be a DHRS2/4 homologue. Due to their high sequence identity, it was so far not possible to distinguish between SDR25C22 and the human DHRS2/4 proteins by means of sequence analysis alone. However, the study of homologue genes in the model organism C. elegans can provide valuable information on the putative physiological role of the corresponding human form.     

P26h and dicarbonyl/L-xylulose reductase are two distinct proteins present in the hamster epididymis

We have previously identified a 34 kDa protein (P34H) on the human sperm surface covering the acrosome. Using the hamster, we have also described a sperm protein, P26h, which is acquired by spermatozoa during epididymal transit. Both P34H and P26h belong to the carbonyl reductase family. Using molecular tools derived from P34H, we searched in the hamster epididymis for another protein related to the human sperm protein. Cloning and sequencing of P31h cDNA revealed 100% homology with the kidney DCXR (Dicarbonyl/L-Xylulose reductase). Northern Blot experiments revealed a single mRNA that was more expressed in the caput than in the corpus and cauda segment of adult epididymides. In situ hybridization was performed on sexually mature hamsters showing that the mRNA was localized in the principal cells throughout the epididymis. Using an anti-P34H antibody we have identified a P34H related protein named P31h (for 31 kDa). This protein showed 2D-electrophoretic behavior different from P26h and was detectable all along the epididymis (caput, corpus, and cauda) by Western Blot analysis. Immunohistochemistry techniques showed that P31h was localized in the perinuclear region of the principal cells of the epididymal epithelium within the three sections, both in sexually mature and immature animals. Results are discussed with regards to the potential function of DCXR in the epididymis.     

A novel kinase regulates dietary restriction‐mediated longevity in Caenorhabditis elegans

Although dietary restriction (DR) is known to extend lifespan across species, from yeast to mammals, the signalling events downstream of food/nutrient perception are not well understood. In Caenorhabditis elegans, DR is typically attained either by using the eat‐2 mutants that have reduced pharyngeal pumping leading to lower food intake or by feeding diluted bacterial food to the worms. In this study, we show that knocking down a mammalian MEKK3‐like kinase gene, mekk‐3 in C. elegans, initiates a process similar to DR without compromising food intake. This DR‐like state results in upregulation of beta‐oxidation genes through the nuclear hormone receptor NHR‐49, a HNF‐4 homolog, resulting in depletion of stored fat. This metabolic shift leads to low levels of reactive oxygen species (ROS), potent oxidizing agents that damage macromolecules. Increased beta‐oxidation, in turn, induces the phase I and II xenobiotic detoxification genes, through PHA‐4/FOXA, NHR‐8 and aryl hydrocarbon receptor AHR‐1, possibly to purge lipophilic endotoxins generated during fatty acid catabolism. The coupling of a metabolic shift with endotoxin detoxification results in extreme longevity following mekk‐3 knock‐down. Thus, MEKK‐3 may function as an important nutrient sensor and signalling component within the organism that controls metabolism. Knocking down mekk‐3 may signal an imminent nutrient crisis that results in initiation of a DR‐like state, even when food is plentiful.     

The L-isoaspartyl-O-methyltransferase in Caenorhabditis elegans larval longevity and autophagy

The protein L-isoaspartyl-O-methyltransferase, coded by the pcm-1 gene in Caenorhabditis elegans, participates in the repair of age-damaged proteins. We tested the ability of pcm-1-deficient nematodes to survive starvation stress as developmentally-arrested L1 larvae. We found that pcm-1 mutant L1 larvae do not survive as well as wild-type L1 larvae when incubated in M9 medium without nutrients. We then tested whether the starved L1 larvae could continue development when allowed access to food in a recovery assay. A loss of recovery ability with age was observed for all larvae, with little or no difference between the pcm-1 mutant and wild-type N2 larvae. Interestingly, when L1 larvae were starved in cholesterol-containing S medium or M9 medium supplemented with cholesterol, the survival rates of both mutant and wild-type animals nearly doubles, with pcm-1 larvae again faring more poorly than N2 larvae. Furthermore, L1 larvae cultured in these cholesterol-containing media show an increase in Sudan Black staining over animals cultured in M9 medium. The longevity defects of pcm-1 mutants previously seen in dauer larvae and here in L1 larvae suggest a defect in the ability of pcm-1 mutants to recycle and reuse old cellular components in pathways such as autophagy. Using an autophagosomal marker, we found evidence suggesting that the pcm-1 mutation may inhibit autophagy during dauer formation, suggesting that the absence of protein repair may also interfere with protein degradation pathways.     

Two pleiotropic classes of daf-2 mutation affect larval arrest,adult behavior,reproduction and longevity in Caenorhabditis elegans.

The nematode Caenorhabditis elegans responds to overcrowding and scarcity of food by arresting development as a dauer larva, a nonfeeding, long-lived, stress-resistant, alternative third-larval stage. Previous work has shown that mutations in the genes daf-2 (encoding a member of the insulin receptor family) and age-1 (encoding a PI 3-kinase) result in constitutive formation of dauer larvae (Daf-c), increased adult longevity (Age), and increased intrinsic thermotolerance (Itt). Some daf-2 mutants have additional developmental, behavioral, and reproductive defects. We have characterized in detail 15 temperature-sensitive and 1 nonconditional daf-2 allele to investigate the extent of daf-2 mutant defects and to examine whether specific mutant traits correlate with each other. The greatest longevity seen in daf-2 mutant adults was approximately three times that of wild type. The temperature-sensitive daf-2 mutants fell into two overlapping classes, including eight class 1 mutants, which are Daf-c, Age, and Itt, and exhibit low levels of L1 arrest at 25.5 degrees. Seven class 2 mutants also exhibit the class 1 defects as well as some or all of the following: reduced adult motility, abnormal adult body and gonad morphology, high levels of embryonic and L1 arrest, production of progeny late in life, and reduced brood size. The strengths of the Daf-c, Age, and Itt phenotypes largely correlated with each other but not with the strength of class 2-specific defects. This suggests that the DAF-2 receptor is bifunctional. Examination of the null phenotype revealed a maternally rescued egg, L1 lethal component, and a nonconditional Daf-c component. With respect to the Daf-c phenotype, the dauer-defective (Daf-d) mutation daf-12(m20) was epistatic to daf-2 class 1 alleles but not the severe class 2 alleles tested. All daf-2 mutant defects were suppressed by the daf-d mutation daf-16(m26). Our findings suggest a new model for daf-2, age-1, daf-12, and daf-16 interactions.     

Genetic, behavioral and environmental determinants of male longevity in Caenorhabditis elegans

Males of the nematode Caenorhabditis elegans are shorter lived than hermaphrodites when maintained in single-sex groups. We observed that groups of young males form clumps and that solitary males live longer, indicating that male-male interactions reduce life span. By contrast, grouped or isolated hermaphrodites exhibited the same longevity. In one wild isolate of C. elegans, AB2, there was evidence of copulation between males. Nine uncoordinated (unc) mutations were used to block clumping behavior. These mutations had little effect on hermaphrodite life span in most cases, yet many increased male longevity even beyond that of solitary wild-type males. In one case, the neuronal function mutant unc-64(e246), hermaphrodite life span was also increased by up to 60%. The longevity of unc-4(e120), unc-13(e51), and unc-32(e189) males exceeded that of hermaphrodites by 70-120%. This difference appears to reflect a difference in sex-specific life span potential revealed in the absence of male behavior that is detrimental to survival. The greater longevity of males appears not to be affected by daf-2, but is influenced by daf-16. In the absence of male-male interactions, median (but not maximum) male life span was variable. This variability was reduced when dead bacteria were used as food. Maintenance on dead bacteria extended both male and hermaphrodite longevity.     

Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism

Background

Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages.

Results

We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2 acts in dauer at least partly through modulation of daf-2/IIS and TGF-β signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers.

Conclusions

Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity and lipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.     

UNC-108/Rab2 regulates postendocytic trafficking in Caenorhabditis elegans

After endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways is differentially regulated. Here, we identify UNC-108/Rab2 as a regulator of postendocytic trafficking in both neurons and coelomocytes. Mutations in the Caenorhabditis elegans Rab2 gene unc-108, caused the green fluorescent protein (GFP)-tagged glutamate receptor GLR-1 (GLR-1::GFP) to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, postendocytic trafficking of the marker Texas Red-bovine serum albumin was delayed. These results demonstrate that UNC-108/Rab2 regulates postendocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative postendocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking.     

FLR-4, a novel serine/threonine protein kinase, regulates defecation rhythm in Caenorhabditis elegans

The defecation behavior of the nematode Caenorhabditis elegans is controlled by a 45-s ultradian rhythm. An essential component of the clock that regulates the rhythm is the inositol trisphosphate receptor in the intestine, but other components remain to be discovered. Here, we show that the flr-4 gene, whose mutants exhibit very short defecation cycle periods, encodes a novel serine/threonine protein kinase with a carboxyl terminal hydrophobic region. The expression of functional flr-4::GFP was detected in the intestine, part of pharyngeal muscles and a pair of neurons, but expression of flr-4 in the intestine was sufficient for the wild-type phenotype. Furthermore, laser killing of the flr-4-expressing neurons did not change the defecation phenotypes of wild-type and flr-4 mutant animals. Temperature-shift experiments with a temperature-sensitive flr-4 mutant suggested that FLR-4 acts in a cell-functional rather than developmental aspect in the regulation of defecation rhythms. The function of FLR-4 was impaired by missense mutations in the kinase domain and near the hydrophobic region, where the latter allele seemed to be a weak antimorph. Thus, a novel protein kinase with a unique structural feature acts in the intestine to increase the length of defecation cycle periods.     

Refractory dissolved organic matter can influence the reproduction of Caenorhabditis elegans (Nematoda)

1. We investigated the effect of refractory dissolved organic matter (refractory DOM: fulvic acids (FAs) and ultrafiltrates (UFs)), isolated from five different sources, on the reproduction of the bacterivorous nematode Caenorhabditis elegans. Nematodes were exposed to DOM (0.5–64 mg L?1 dissolved organic carbon) during a whole life cycle (72 h). At the end of the test, the number of offspring per worm was determined.
2. We also studied the effect of refractory DOM on abundance, cell size, and activity of the bacteria (Escherichia coli) that were used as a food source for the nematodes, to assess possible indirect effects of DOM via the food organisms.
3. The effects of DOM on the reproduction of C. elegans varied, depending on the origin and concentration of DOM. FAs isolated from a soil leachate and from the effluent of a waste water plant, as well as UFs from a humic lake and from a marsh, stimulated the reproduction of C. elegans. FAs from ground water had no effect, while FAs from a humic lake inhibited the reproduction of the nematodes. All effects occurred at ecologically relevant DOM concentrations and showed clear dose–response relationships.
4. Neither bacterial abundance nor cell size were influenced by refractory DOM. Bacterial activity was unaffected by four types of DOM. Only FAs from the humic lake caused a significant decrease in bacterial activity over 72 h.
5. The negative effect of FAs from the humic lake on nematode reproduction may be a consequence of a lower bacterial activity in this treatment. The positive effects of refractory DOM, however, could not be related to bacterial parameters. Therefore, we assume that the DOM directly influenced the reproduction of C. elegans. We speculate that refractory DOM can potentially be an additional carbon source or a source of trace nutrients influencing the reproduction of C. elegans. Adsorption of refractory DOM on bacterial cells, serving as food for the nematodes, may have been an important factor for the availability of DOM for C. elegans.