The Caenorhabditis elegans K10C2.4 Gene Encodes a Member of the Fumarylacetoacetate Hydrolase Family

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.

Disruption of Fumarylacetoacetate Hydrolase Causes Spontaneous Cell Death under Short-Day Conditions in Arabidopsis

“…The HGO, an enzyme upstream of FAH in the Tyr degradation pathway, is essential for the catalysis of homogentisate into MAA and FAA (Lindblad et al, 1977). Inactivation of HGO in fungi, mice, and worms, therefore, was able to rescue the lethality associated with FAH mutations (Fernández-Cañón and Peñalva, 1995b;Manning et al, 1999;Fisher et al, 2008). Through generation of the double mutant sscd1 hgo, we also found that disruption of the Arabidopsis putative HGO is able to completely eliminate the spontaneous cell death in the sscd1 mutant (Fig.…”

“…In both human HT1 and mouse fah mutants, the metabolic intermediates (MAA, FAA, succinylacetoacetate, and succinylacetone) were highly accumulated, which are toxic to cells and tissues (Lindblad et al, 1977;Grompe et al, 1993;Aponte et al, 2001;Jorquera and Tanguay, 2001). Due to technical difficulties, it is so far not possible to detect these metabolic intermediates in entire worms (Fisher et al, 2008) and in Arabidopsis plants (data not shown). To verify that the cell death phenotype in the sscd1 mutant might also have resulted from the accumulation of succinylacetoacetate and succinylacetone, we followed the approach used in worms (Fisher et al, 2008) and treated Arabidopsis seedlings with succinylacetone, the unique product that is commercially available among these metabolic intermediates.…”

“…Due to technical difficulties, it is so far not possible to detect these metabolic intermediates in entire worms (Fisher et al, 2008) and in Arabidopsis plants (data not shown). To verify that the cell death phenotype in the sscd1 mutant might also have resulted from the accumulation of succinylacetoacetate and succinylacetone, we followed the approach used in worms (Fisher et al, 2008) and treated Arabidopsis seedlings with succinylacetone, the unique product that is commercially available among these metabolic intermediates. We found that treatment of wild-type seedlings with succinylacetone mimicked the sscd1 phenotypes: the treated wild-type seedlings exhibited wilted leaves and slow-growth symptoms under LD (Fig.…”

The Production of <em>C. elegans</em> Transgenes via Recombineering with the <em>galK</em> Selectable Marker

“…This can lead to the construction of a transgene which is more reflective of the native expression pattern, or the construction of a functional transgene when other approaches fail 5 . The resulting transgenes can carry a variety of epitope tags including GFP or a TAP tag.…”

“…While these transgenes are often successful with regards to producing a GFP reporter gene or expressing a cDNA in a desired pattern, these transgenes can lack the alternate promoters, enhancer elements, and 3' untranslated region (UTR) elements which play important roles in the control of gene expression in vivo 2 . For example, both the daf-12 and fah-1 genes have important enhancer elements which lie outside of the proximal promoter which were missed in promoter only constructs 3,4,5 . Further many transgene constructs use the unc-54 3'UTR which prevents regulation by the appropriate microRNA genes 6,7,8 .…”