The Caenorhabditis elegans K10C2.4 gene encodes a member of the fumarylacetoacetate hydrolase family: a Caenorhabditis elegans model of type I tyrosinemia

Abstract

In eukaryotes and many bacteria, tyrosine is degraded to produce energy via a five-step tyrosine degradation pathway. Mutations affecting the tyrosine degradation pathway are also of medical importance as mutations affecting enzymes in the pathway are responsible for type I, type II, and type III tyrosinemia. The most severe of these is type I tyrosinemia, which is caused by mutations affecting the last enzyme in the pathway, fumarylacetoacetate hydrolase (FAH). So far, tyrosine degradation in the nematode Caenorhabditis elegans has not been studied; however, genes predicted to encode enzymes in this pathway have been identified in several microarray, proteomic, and RNA interference (RNAi) screens as perhaps being involved in aging and the control of protein folding. We sought to identify and characterize the genes in the worm tyrosine degradation pathway as an initial step in understanding these findings. Here we describe the characterization of the K10C2.4, which encodes a homolog of FAH. RNAi directed against K10C2.4 produces a lethal phenotype consisting of death in young adulthood, extensive damage to the intestine, impaired fertility, and activation of oxidative stress and endoplasmic stress response pathways. This phenotype is due to alterations in tyrosine metabolism as increases in dietary tyrosine enhance it, and inhibition of upstream enzymes in tyrosine degradation with RNAi or genetic mutations reduces the phenotype. We also use our model to identify genes that suppress the damage produced by K10C2.4 RNAi in a pilot genetic screen. Our results establish worms as a model for the study of type I tyrosinemia.

FIGURE 1.

The tyrosine degradation pathway. Shown are the chemical structure of each compound and the names of the predicted worm genes.

FIGURE 2.

Inhibition of C. elegans fah produces a lethal phenotype and results in destruction of the intestine. Low power view of worms treated with control (A) or K10C2.4 RNAi (C). High power view of control (B) or K10C2.4 (D) RNAi-treated worms. Arrows, intestine. E, Western blot showing change in K10C2.4 protein levels following RNAi treatment. Thin arrow, K10C2.4. Thick arrow, K10C2.4:GFP fusion. Anti-actin served as a loading control. F, expression of a K10C2.4:GFP transgene in the intestine and hypodermis of worms.

FIGURE 3.

The K10C2.4 phenotype is enhanced by tyrosine or homogentisic acid. Plotted is the mean number of progeny produced by worms treated with control RNAi, K10C2.4 RNAi, or K10C2.4 RNAi supplemented with Tyr or HGA. The standard deviation is indicated by the error bar (p < 0.01 for all comparisons).

FIGURE 4.

The K10C2.4 phenotype depends on an intact tyrosine degradation pathway. Low power views of worms treated with GFP RNAi (A), K10C2.4 RNAi (B, I with high power Nomarski image), or 1:1 K10C2.4 RNAi and GFP RNAi (C), F42D1.2 RNAi (D), T21C12.2 RNAi (E, H with high-power image), W06D4.1 RNAi (F), or D1053.1 RNAi (G). Arrows in H and I, intestine.

FIGURE 5.

Inhibition of K08F8.4 can partially rescue worms treated with K10C2.4 RNAi. High power views of worms treated with K10C2.4 RNAi (C), or 1:1 K10C2.4 RNAi and K08F8.4 RNAi (A) or T21C12.2 RNAi (B).

FIGURE 6.

Treatment of worms with SA mimics the K10C2.4 phenotype. Low power views of worms treated with control RNAi (A) or control RNAi and SA (B). C–F, low and high power Nomarski images of similar controls (C and E) and SA-treated worms (D and F). G, SA produces dose-dependent toxicity to treated worms. Plotted are the percentages of each phenotype seen on plates supplemented with the indicated concentrations of SA.

FIGURE 7.

Inhibition of K10C2.4 activates specific cell stress pathways. Nomarski and fluorescence images of L3 larval worms, which carry the hsp16-2::GFP reporter (A), daf-16::GFP transgene (B), gst-4::GFP reporter (C), or hsp-4::GFP reporter (D) and are treated with control RNAi (top panels) or K10C2.4 RNAi (bottom panels). E, day 1 adult images of worms in D.

FIGURE 8.

A pilot genetic screen identifies mutations in tyrosine aminotransferase and homogentisate dioxygenase as suppressors of K10C2.4 RNAi toxicity. Shown are alignments around P224 in F42D1.2 (A) and G173 in W06D4.1 (B). P_deleterious cSNP prediction of negative effect on protein function.