Genome-wide RNAi screening identifies protein damage as a regulator of osmoprotective gene expression

Abstract

The detection, stabilization, and repair of stress-induced damage are essential requirements for cellular life. All cells respond to osmotic stress-induced water loss with increased expression of genes that mediate accumulation of organic osmolytes, solutes that function as chemical chaperones and restore osmotic homeostasis. The signals and signaling mechanisms that regulate osmoprotective gene expression in animal cells are poorly understood. Here, we show that gpdh-1 and gpdh-2, genes that mediate the accumulation of the organic osmolyte glycerol, are essential for survival of the nematode Caenorhabditis elegans during osmotic stress. Expression of GFP driven by the gpdh-1 promoter (P gpdh-1::GFP) is detected only during hypertonic stress but is not induced by other stressors. Using Pgpdh-1::GFP expression as a phenotype, we screened Ϸ16,000 genes by RNAi feeding and identified 122 that cause constitutive activation of gpdh-1 expression and glycerol accumulation. Many of these genes function to regulate protein translation and cotranslational protein folding and to target and degrade denatured proteins, suggesting that the accumulation of misfolded proteins functions as a signal to activate osmoprotective gene expression and organic osmolyte accumulation in animal cells. Consistent with this hypothesis, 73% of these protein-homeostasis genes have been shown to slow age-dependent protein aggregation in C. elegans. Because diverse environmental stressors and numerous disease states result in protein misfolding, mechanisms must exist that discriminate between osmotically induced and other forms of stress-induced protein damage. Our findings provide a foundation for understanding how these damage-selectivity mechanisms function.Caenorhabditis elegans ͉ functional genomics ͉ organic osmolytes ͉ osmotic stress

Adaptation costs to constant and alternating polluted environments

“…In response to salt exposure, an extreme hypertonic stress, C. elegans regulates the rapid loss of water and solute content in its cells by synthesizing glycerol through transcriptional upregulation of an enzyme (gpdh‐1) in the intestine and hypodermis, two tissues that are both in direct contact with the external medium (Lamitina, Huang, & Strange, 2006; Lamitina et al., 2004). In our experiment, C. elegans populations responded to salt by reducing early and total fertility (Figure 2), thereby producing individuals with longer life cycles and lower fertility.…”

Physiological and molecular mechanisms of salt and water homeostasis in the nematodeCaenorhabditis elegans

“…Two to four hours after transfer, body volume and motility recover (Fig. 3) and the life cycle resumes, although with a reduced number of offspring and slower development (61).…”

“…In adult worms, 20 epithelial cells make up the intestine, which functions in digestion, nutrient absorption, energy storage, and detoxification. Genes responsible for organic osmolyte synthesis are induced in the intestine during acclimation to high osmolarity (61).…”

“…The functions of osmolytes like glycerol have long been assumed to include balancing high environmental osmolarity and stabilizing protein structure as a chemical chaperone (53,121). Glycerol synthesis is essential for long-term growth and reproduction of C. elegans on agar plates containing elevated NaCl (61).…”

“…GPDH-1 catalyzes the rate-limiting step of glycerol synthesis. The gpdh-1 gene is expressed at low levels in worms on standard agar but is induced several-fold within the first hour of exposure to nonlethal high osmolarity (59,(61)(62) (Fig. 3).…”