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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 22, 2003 as doi:10.1096/fj.02-1022fje. |
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Laboratorio Andaluz de Biología, Departamento de Ciencias Ambientales, Universidad Pablo de Olavide, E-41013 Sevilla, Spain
3Correspondence: Laboratorio Andaluz de Biología, Departamento de Ciencias Ambientales, Universidad Pablo de Olavide, Ctra. Utrera, Km. 1, E-41013 Sevilla, Spain. E-mail: pnavas{at}dex.upo.es
SPECIFIC AIM
Ubiquinone (coenzyme Q9, Q9) is synthesized in Caenorhabditis elegans mitochondria, and recent results indicate that Q9 and dietary coenzyme Q8 (Q 8) regulate the aging process in this nematode. We have studied the effect of the interference by double-stranded RNA (dsRNA) of gene sequences homologous to those of Q6 biosynthesis in Saccharomyces cerevisiae in Q9 biosynthesis and Q8 intake. We also studied respiratory chain properties in mitochondria of silenced worms and some aspects of phenotype, with special attention to life span.
PRINCIPAL FINDINGS
1. Eight genes have been identified by dsRNA interference to participate in Q9 biosynthesis in C. elegans
S. cerevisiae coq genes, whose function has been demonstrated to participate in Q6 biosynthesis in yeast, were compared with C. elegans genome using BLAST search, run on NCBI (NIH) electronic resources. Eight sequences were selected showing 46-60% homology and 30-47% identity with those of S. cerevisiae coq genes. These sequences were subcloned into pL4440 vector and used to transform the Escherichia coli HT115 strain, which were able to produce dsRNA. Wild-type (N2) nematodes were fed with pL4440-coq-transformed bacteria and Q9 biosynthesis was followed in nematode populations by HPLC analysis. Q9 contents were rapidly decreased in all interfered populations, reaching steady-state levels after about 2 wk, but Q9 levels did not reach zero even after 3 wk (Fig. 1
). Q9 depletion reversed when the interfered worm populations were fed back on HT115 pL4440 bacteria for 10 to 14 days.
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2. Silencing genes of Q9 biosynthesis in C. elegans induces a decrease of Q8 biosynthesis in pL4440-coq transformed E. coli strains
Q8 content in nematode membranes was significantly diminished after interference of coq-1 to coq-8 genes (Fig. 1)
. As pharyngeal pumping was unaltered in silenced worms, this decrease in N2 Q8 levels was not due to diminished HT115 bacteria intake. Q8 levels measured in HT115 dsRNA-producing bacteria displayed significantly lower Q8 content than that of control strains transformed with the empty pL4440 plasmid.
3. The interference of genes responsible of Q9 biosynthesis extends life span in C. elegans
Silenced worms did not show changes in phenotype features of wild-type nematodes, such as pharyngeal pumping or brood size. However, the analysis of survival curves of interfered populations showed an extended life span compared with non-interfered populations (Fig. 2
). This difference became evident as early as percentile 25, and both mean and maximum life span determined in coq-silenced nematodes were significantly higher than those observed in non-interfered worms.
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4. Respiratory chain is unaltered in mitochondria of silenced nematodes but produces fewer superoxide anions
Mitochondria-enriched fractions were isolated from interfered nematodes and Q-dependent electron transport activities of the respiratory chain were studied. Both complexes I+III and II+III showed no significant differences in coq-interfered mitochondria compared with control population. However, mitochondria isolated from silenced animals produced significantly fewer superoxide anions than those purified from control populations. This difference was > 30% in most cases, reaching a maximum of 50% in mitochondria of coq-4 gene-interfered nematodes.
CONCLUSIONS
Q biosynthesis genes have been identified as a set of eight complementation groups in bacteria and yeast, but in C. elegans knowledge is restricted to clk-1 (ZC395.2), homologue to S. cerevisiae coq-7/cat-5 that encodes a hydroxylase. coq-3 (Y57G11C.11) null mutant strains are lethal, and recently the participation of its gene product (homologue to S. cerevisiae Coq-3p) in Q9 biosynthesis has been suggested. The functions of the remaining six additional genes interfered here have not previously been demonstrated in C. elegans.
Global Q9 content was rapidly decreased in N2 strains cultured during the first days of interference reaching steady levels after about 2 wk, probably the time needed to equilibrate protein synthesis and turnover rates. However, Q9 levels did not reach zero even after 3 wk, probably because this technique either does not completely block protein translation or does not affect all cells of the nematode to the same extent. Reversion of Q9 depletion was achieved when the interfered worm populations were fed on HT115 pL4440 bacteria again, demonstrating that reexpression of specific coq genes led to recovery of control Q9 levels. Thus, the involvement of C. elegans C24A11.9 (coq-1), F57B9.4 (coq-2), Y57G11C.11 (coq-3), T03F1.2 (coq-4), ZK652.9 (coq-5), K07B1.2 (coq-6), and C35D10.4 (coq-8) gene sequences together with ZC395.2 (clk-1) on Q9 biosynthesis is clearly demonstrated.
Among the genes silenced, yeast homologue to coq-1 (C24A11.9) codifies a polyprenyl synthetase and represents a branch-point enzyme to several metabolic pathways related to lipid synthesis such as dolichol and some sterols. Yeast Coq-2p homologue is a polyprenyltransferase that catalyzes the prenylation of 4-hydroxybenzoic acid. The gene products of yeast and rat coq-3 homologues catalyze two O-methyltransferase steps in Q biosynthesis essential for the final structure of ubiquinone ring. Yeast coq-6 gene product shares identities with a large family of proteins that function as flavopro-tein monooxygenases, although its catalytic participation in Q biosynthesis is unknown. Yeast coq-5 encodes a C-methyltransferase, but a direct demonstration of its function is not available. No catalytic function on Q6 biosynthesis has been appointed for Coq-4p and Coq-8p in yeast; however, deletion of yeast coq-8 prevents the organization of complex III in the respiratory chain.
Nematode dietary Q8 isoform content was significantly diminished after interference of coq-1 to coq-8 genes. This decrease was not due to an altered pharyngeal pumping but was dependent on the lower levels of Q8 observed in HT115 dsRNA-producing bacteria. This phenomenon may reflect the nature of E. coli ubi genes, which have a high homology with yeast coq genes used here for C. elegans selection. Thus, it is not surprising that dsRNA expressed in E. coli may cause interference with its own ubi genes mRNA.
Interfered animals displayed an extended life span compared with control. Recent reports show that endogenous oxidative stress is important in longevity. Thus, a mutation in complex II succinate dehydrogenase gene increases superoxide levels and premature aging in C. elegans. A mutation in isp-1 gene, a structural component of complex III, decreases endogenous oxidative stress and extends life span in C. elegans. Life span of C. elegans is also extended by caloric restriction, probably by slowing down the production of reactive oxygen species. In general, genes important for mitochondrial function are included among those genes whose interference affects life span in C. elegans. The proteins codified by six genes identified here (coq-3 to coq-8) function in the mitochondria and, following the latter idea, must be included in the group whose interference modifies life span. The final product of the biosynthesis pathway, coenzyme Q, is an essential electron carrier in the respiratory chain.
Analysis of mitochondria purified from interfered worms showed that superoxide anion production was decreased, but Q-dependent respiratory chain activities of complexes I+III and II+III were not significantly different from those observed in mitochondria of non-interfered worms, even though global Q9 and Q8 levels were significantly lower. Two pools of Q have been described in mitochondria. One is bound to proteins and correlates negatively with superoxide production. The other is represented by free Q, which positively correlates with superoxide production. A low concentration of Q in interfered mitochondria would decrease the free Q pool and lower superoxide production, as observed here. The remaining Q concentration in silenced mitochondria would be in the range of respiratory chain requirements. Thus, there is an apparent equilibrium between an undamaged respiratory chain and Q concentration to prevent the leak of electrons such as superoxide (Fig. 3
), but keeping an efficient respiration.
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Our results demonstrate that at least the eight genes identified in this work participate in Q9 biosynthesis and that its interference extends life span in C. elegans. Q concentration decreased by silencing experiments leads to a lower superoxide production in the mitochondrial electron transport chain, contributing to extend life span. Although extended life span in C. elegans with compromised mitochondria cannot be explained solely by a lower free radical formation, some contribution must be assigned to a decreased endogenous oxidative stress because it exerts lower damage in macromolecules. These findings support the endogenous oxidative stress hypothesis to explain at least partially the extension of life span in C. elegans.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1022fje; to cite this article, use FASEB J. (April 22, 2003) 10.1096/fj.02-1022fje 
2 These author contributed equally to this work.
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