Defining Caenorhabditis elegans as a model system to investigate lipoic acid metabolism

Abstract

Lipoic acid (LA) is a sulfur-containing cofactor that covalently binds to a variety of cognate enzymes that are essential for redox reactions in all three domains of life. Inherited mutations in the enzymes that make LA, namely lipoyl synthase, octanoyltransferase, and amidotransferase, result in devastating human metabolic disorders. Unfortunately, because many aspects of this essential pathway are still obscure, available treatments only serve to alleviate symptoms. We envisioned that the development of an organismal model system might provide new opportunities to interrogate LA biochemistry, biology, and physiology. Here we report our investigations on three Caenorhabditis elegans orthologous proteins involved in this post-translational modification. We established that M01F1.3 is a lipoyl synthase, ZC410.7 an octanoyltransferase, and C45G3.3 an amidotransferase. Worms subjected to RNAi against M01F1.3 and ZC410.7 manifest larval arrest in the second generation. The arrest was not rescued by LA supplementation, indicating that endogenous synthesis of LA is essential for C. elegans development. Expression of the enzymes M01F1.3, ZC410.7, and C45G3.3 completely rescue bacterial or yeast mutants affected in different steps of the lipoylation pathway, indicating functional overlap. Thus, we demonstrate that, similarly to humans, C. elegans is able to synthesize LA de novo via a lipoyl-relay pathway, and suggest that this nematode could be a valuable model to dissect the role of protein mislipoylation and to develop new therapies.

Keywords: Caenorhabditis elegans (C. elegans); RNA interference; RNA interference (RNAi); development; energy metabolism; fatty acid metabolism; inborn error of metabolism; lipoic acid; mitochondrial metabolism; oxidative stress; post-translational modification.

Figure 1.

The LipB-LipA pathway, present in E. coli, is the simplest pathway characterized, because it only requires two enzyme activities to get all proteins lipoylated (octanoyltransferase and lipoate synthase). On the other hand, in the lipoyl-relay pathway found in Gram-positive bacteria, yeasts, and humans, two extra protein activities are required: GcvH, which is an obligate intermediary, and an amidotransferase, which allows lipoylation of E2 subunits after modification of GcvH. During lipoate uptake, lipoate ligases activate lipoate with an ATP molecule and then catalyze the transfer reaction to E2 or GcvH subunits. In the case of B. subtilis, this reaction is restricted to GcvH and ODH-E2.

Figure 2.

C. elegans development under depleted LA conditions. A, worms were grown in LA-free medium (M9sup) and fed with E. coli OP50 or strains unable to synthesize LA, ΔlplA ΔlipA (TM131) and ΔlplA ΔlipB (TM136). Pictures were taken on the fourth day of the second generation of worms grown on LA-depleted conditions. Scale bars represent 200 µm. B, protein extracts from nematodes fed with TM131 were analyzed by Western blotting using antibodies against LA. DLAT-1, DLST-1, and DBT-1 are the lipoylated E2 subunits of PDH, ODH, and BKDH, respectively.

Figure 3.

Depletion of M01F1.3 causes a larval arrest phenotype. N2 worms grown in M9sup or with the addition of the indicated supplements and fed with E. coli AL100 (A, B, D, and F) or grown in NGM and fed with HT115 (E). Photographs were taken with 100× magnification and correspond to the fourth day of the second-generation animals. Scale bars represent 100 µm. C, protein extracts from N2 control animals and from worms of the first and second generations treated with M01F1.3 RNAi were analyzed by Western blotting using antibodies against LA. The same blot was probed with an antibody against actin to serve as loading control. G and H, Nomarski photographs taken on the fourth day of the experiment of rrf-3 worms grown in M9sup fed with E. coli AL100. The triangle indicates a typical embryo before being laid. Scale bars represent 10 µm.

Figure 4.

Characterization of M01F1.3 RNAi treatment. A, comparison of the content of mmBCFAs between N2 treated (subjected to M01F1.3 RNAi) and control (stage L3) worms. Bar graph represents the content of mmBCFAs. Each bar is the mean ± S.D. from three independent experiments. *p < 0.05; **p < 0.005. B, control and M01F1.3 RNAi rrf-3 worms were transferred as young adults to plates containing 15 mm Fe(II) sulfate. Viability was scored every 30 min and represented as percent survival of worms. C, rrf-3 worms were grown in M9sup plates and fed with E. coli ΔlplA ΔlipA (AL103) strain transformed with either the empty vector (control, dashed lane) or the M01F1.3 dsRNA-producing vector (black solid lane). M01F1.3 RNAi treatment was also performed in the presence of 25 µm LA (gray solid lane). Worms were transferred to fresh plates when necessary to avoid generation mixes. 50 worms were used for each treatment in each assay. Data were collected from three independent experiments. D, four young adult worms subjected to RNAi treatment with E. coli AL100 strain were transferred to fresh plates and permitted to lay eggs for 4.5 h. Progeny was scored after 2 days. Each graph is the mean ± S.D. from four independent experiments. ***p < 0.001; NS, no significant difference between counted progeny from control and treated worms. Worm strains used were WM28 (rde-1, insensitive to RNAi treatment), XE1581, XE1582, XE1474, and XE1375 (sensitive to RNAi treatment in cholinergic, glutamatergic, dopaminergic, and GABAergic neurons, respectively). E, pictures taken with 25× magnification on the fourth day of RNAi treatment in strain XE1581. Scale bars represent 1 mm.

Figure 5.

M01F1.3 complements a lipA mutant in B. subtilis. A, growth of B. subtilis WT (JH642) or ΔlipA (CM37) strains transformed with either the empty vector (EV) or pAL14, a plasmid with the cDNA coding for mature M01F1.3 under a xylose-inducible promoter (mM01F1.3). Strains were streaked onto minimal medium plates with the supplements indicated. Plates were incubated at 37 °C for 2 days. B, protein extracts from strain CM37 transformed with pAL14 and grown with or without xylose were analyzed by Western blotting with antibodies against LA. BCFAP, branched-chain fatty acid precursors.

Figure 6.

Characterization of ZC410.7. A, N2 worms grown in M9sup and fed with E. coli ΔlipA strain (AL100). Photographs were taken with 100× magnification and correspond to the fourth day of the second-generation animals. Scale bars represent 100 µm. B, yeast strains were grown overnight in synthetic glucose medium lacking uracil, and upon standardization by A₆₀₀, dilutions were spotted in complex medium plates containing glucose or glycerol as carbon sources. Parental strain BY4741 and lip2 mutant were transformed with the empty vector (EV) as positive and negative controls, respectively. The lip2 mutant was transformed with plasmid p426-GPD containing a WT copy of lip2 or C. elegans ZC410.7 coding sequence. C, protein extracts obtained from these cultures were incubated with antibodies against LA.

Figure 7.

C45G3.3 expression functionally complements yeast and bacterial mutants. A, yeast strains were grown overnight in synthetic glucose medium lacking uracil, and upon standardization by A₆₀₀, dilutions were spotted in complex medium plates containing glucose or glycerol as carbon sources. Parental strain BY4741 and lip3 mutant were transformed with the empty vector (EV) as positive and negative controls, respectively. lip3 mutant was transformed with plasmid p426-GPD containing a WT copy of lip3 or C. elegans C45G3.3 coding sequence. B, protein extracts obtained from these cultures were incubated with antibodies against LA. C, growth of B. subtilis WT (JH642) or ΔlipM ΔlplJ (NM65) strains transformed with either the empty vector (EV) or pAL18, a plasmid that allows expression of C. elegans C45G3.3 protein under the control of a xylose-inducible promoter. Strains were streaked onto minimal medium plates containing xylose and casamino acids vitamin-free, with the indicated supplements. Plates were incubated at 37 °C for 2 days. BCFAP, branched-chain fatty acid precursors.