Selenoprotein TRXR-1 and GSR-1 are essential for removal of old cuticle during molting in Caenorhabditis elegans

Abstract

Selenoproteins, in particular thioredoxin reductase, have been implicated in countering oxidative damage occurring during aging but the molecular functions of these proteins have not been extensively investigated in different animal models. Here we demonstrate that TRXR-1 thioredoxin reductase, the sole selenoprotein in Caenorhabditis elegans, does not protect against acute oxidative stress but functions instead together with GSR-1 glutathione reductase to promote the removal of old cuticle during molting. We show that the oxidation state of disulfide groups in the cuticle is tightly regulated during the molting cycle, and that when trxr-1 and gsr-1 function is reduced, disulfide groups in the cuticle remain oxidized. A selenocysteine-to-cysteine TRXR-1 mutant fails to rescue molting defects. Furthermore, worms lacking SELB-1, the C. elegans homolog of Escherichia coli SelB or mammalian EFsec, a translation elongation factor known to be specific for selenocysteine in E. coli, fail to incorporate selenocysteine, and display the same phenotype as those lacking trxr-1. Thus, TRXR-1 function in the reduction of old cuticle is strictly selenocysteine dependent in the nematode. Exogenously supplied reduced glutathione reduces disulfide groups in the cuticle and induces apolysis, the separation of old and new cuticle, strongly suggesting that molting involves the regulated reduction of cuticle components driven by TRXR-1 and GSR-1. Using dauer larvae, we demonstrate that aged worms have a decreased capacity to molt, and decreased expression of GSR-1. Together, our results establish a function for the selenoprotein TRXR-1 and GSR-1 in the removal of old cuticle from the surface of epidermal cells.

Fig. 1.

Disulfide groups in the cuticle are reduced during molting in C. elegans. Micrographs of worms stained with Alexa Fluor 488 C₅-maleimide viewed with DIC (D, G, and I) or fluorescence (A–C, E, F, and H) optics. The large and small arrows in A indicate the lumen of the pharynx and buccal cavity, respectively. The arrowhead in A and the arrow in B indicate one of a pair of cell or neuronal exensions lying adjacent to the buccal cavity stained by the dye. The large arrows in C and D indicate the lumen of the intestine. The small arrows in D indicate the cuticle, which is not appreciably stained by the dye in nonmolting worms under normal conditions. (E and F) Nonmolting worms that had been incubated with 5 mM DTT for 30 min before staining with the dye. (G and H) Worms at molt that had not been incubated with DTT. The arrows indicate cuticle. (I and J) Molted cuticle that had been incubated with N-ethylmaleimide before staining. (Scale bars, 10 μm.)

Fig. 2.

C. elegans TRXR-1 is required together with GSR-1 glutathione reductase for molting. (A Upper) The exon intron structure of the trxr-1 gene. Boxes represent exons, and lines represent introns. The sequence of the last four codons is shown, as is the position of the selenocysteine insertion sequence (SECIS) element in the 3′ untranslated region. The lines underneath denote the regions deleted in the sv43 and sv47 mutant alleles. (A Lower) Domain structure of the TRXR-1 protein. The sequence of the N-terminal redox active site is shown above, and the sequence of the C-terminal site is shown boxed below. (B) Western blot of C. elegans protein extracts probed with an antibody raised against TRXR-1. N2 indicates wild-type control. The upper bands result from nonspecific cross-reactivity of the antibody. (C) Micrographs of molting worms viewed with DIC optics. Old cuticle (indicated by arrows) associated with the buccal cavity (ii), midbody (iii), and rectum (v) is shown. The other panels show cuticle associated with either the head or tail regions. (i and iv) Wild-type control worm at molt. The complete genotype of the trxr-1 worms was trxr-1(sv47); rrf-3(pk1426); gsr-1(RNAi). (Scale bars, 10 μm.) (D) Graph showing percentage of larvae that arrest growth before becoming adults. All worms were homozygous for rrf-3(pk1426).

Fig. 3.

TRXR-1 and GSR-1 are required for the reduction of disulfide groups in the cuticle during molting, which is promoted by GSH. (A–I) Micrographs of worms stained with Alexa Fluor 488 C₅-maleimide viewed with DIC (B and C) or fluorescence (A and D–I) optics. The exposure time for the image in H was 1 s; for all other fluorescence images, exposure time was 100 ms. (A and D) Intermolt worms that had been incubated with 5 mM GSH for 30 min at room temperature before staining with the dye. (B, C, and E–H) Worms at molt that had not been incubated with GSH. The arrows indicate cuticle. (H) Note the punctate staining seen in some trxr-1; gsr-1(RNAi) worms at longer exposure times. (I) Cuticle from trxr-1; gsr-1(RNAi) worms that had been incubated with DTT before staining. (J–L) Partially detached cuticle in worms exposed to either 3 mM GSH (J) or that lacked maternal (m⁻) and zygotic (z⁻) activity of gcs-1 (K and L). (Scale bars, 10 μm.)

Fig. 4.

TRXR-1 is a selenoprotein; incorporation of selenium requires selb-1, and is necessary for function. (A Upper) Autoradiogram of a gel containing protein extracts of worms raised on ⁷⁵Se-labeled E. coli. (A Lower) The Coomassie-stained gel. (B) Nomarski micrograph of an arrested worm grown on selD mutant E. coli that expressed gsr-1 dsRNA. (Scale bars, 10 μm.) (C Upper) Autoradiogram of a gel containing protein extracts of worms raised on ⁷⁵Se-labeled E. coli. (C Lower) The Coomassie-stained gel. (D) Western blot of C. elegans protein extracts probed with an antibody raised against TRXR-1. (E) Micrograph of a selb-1 mutant worm grown on E. coli, wild type with respect to selD, that expressed gsr-1 dsRNA. Arrows indicate old cuticle. (F) Graph showing the activities of wild-type and mutant TRXR-1 proteins in catalyzing thioredoxin-mediated reduction of insulin in vitro (Materials and Methods). (G) Graph indicating the ability of wild-type or mutant trxr-1 transgenes to rescue the growth arrest defect displayed by trxr-1(sv47); gsr-1(RNAi) larvae. All strains were homozygous for rrf-3(pk1426).

Fig. 5.

Expression patterns of gsr-1::gfp, trxr-1::gfp and selb-1::gfp transgenes and focus for selb-1 activity. (A–D) Micrographs of worms with the indicated transgenes viewed with fluorescence (A and B) or confocal fluorescence (C and D) optics. The arrows in A, C, and D indicate GFP fluorescence in the hypodermis; arrows in B indicate GFP fluorescence in the pharynx. The worms in C and D were fixed and stained with an anti-GFP antibody. (Scale bars, 10 μm.) (E) Graph indicating the ability of seven-transgene arrays to rescue the growth arrest defect displayed by selb-1; gsr-1(RNAi) larvae. The arrays contain a selb-1 cDNA under the control of different tissue-specific promoters. The myo-2 promoter is active in the pharynx. The osm-6 and lon-3 promoters are active in ciliated sensory neurons and the hypodermis, respectively. All strains in E were homozygous for rrf-3(pk1426).