Keith P. Choe

The WD40 Repeat Protein WDR-23 Functions with the CUL4/DDB1 Ubiquitin Ligase To Regulate Nuclear Abundance and Activity of SKN-1 in Caenorhabditis elegans

ABSTRACT The transcription factor SKN-1 protects Caenorhabditis elegans from stress and promotes longevity. SKN-1 is regulated by diverse signals that control metabolism, development, and stress responses, but the mechanisms of regulation and signal integration are unknown. We screened the C. elegans genome for regulators of cytoprotective gene expression and identified a new SKN-1 regulatory pathway. SKN-1 protein levels, nuclear accumulation, and activity are repressed by the WD40 repeat protein WDR-23, which interacts with the CUL-4/DDB-1 ubiquitin ligase to presumably target the transcription factor for proteasomal degradation. WDR-23 regulates SKN-1 target genes downstream from p38 mitogen-activated protein kinase, glycogen synthase kinase 3, and insulin-like receptor pathways, suggesting that phosphorylation of SKN-1 may function to modify its interaction with WDR-23 and/or CUL-4/DDB-1. These findings define the mechanism of SKN-1 accumulation in the cell nucleus and provide a new mechanistic framework for understanding how phosphorylation signals are integrated to regulate stress resistance and longevity.

Genome-wide RNAi screen and in vivo protein aggregation reporters identify degradation of damaged proteins as an essential hypertonic stress response

The damaging effects of hypertonic stress on cellular proteins are poorly defined, and almost nothing is known about the pathways that detect and repair hypertonicity-induced protein damage. To begin addressing these problems, we screened approximately 19,000 Caenorhabditis elegans genes by RNA interference (RNAi) feeding and identified 40 that are essential for survival during acute hypertonic stress. Half (20 of 40) of these genes encode proteins that function to detect, transport, and degrade damaged proteins, including components of the ubiquitin-proteasome system, endosomal sorting complexes, and lysosomes. High-molecular-weight ubiquitin conjugates increase during hypertonic stress, suggesting a global change in the ubiquitinylation state of endogenous proteins. Using a polyglutamine-containing fluorescent reporter, we demonstrate that cell shrinkage induces rapid protein aggregation in vivo and that many of the genes that are essential for survival during hypertonic stress function to prevent accumulation of aggregated proteins. High levels of urea, a strong protein denaturant, do not cause aggregation, suggesting that factors such as macromolecular crowding also contribute to protein aggregate formation during cell shrinkage. Acclimation of C. elegans to mild hypertonicity dramatically increases the osmotic threshold for protein aggregation, demonstrating that protein aggregation-inhibiting pathways are activated by osmotic stress. Our studies demonstrate that hypertonic stress induces protein damage in vivo and that detection and degradation of damaged proteins are essential mechanisms for survival under hypertonic conditions.

Immunological detection of Na+/H+ exchangers in the gills of a hagfish, Myxine glutinosa, an elasmobranch, Raja erinacea, and a teleost, Fundulus heteroclitus

Na(+)/H(+) exchangers (NHE) are a family of ion exchangers with diverse functions that are well defined in mammals. NHE-1 is expressed in the plasma membrane of most mammalian cells where it regulates intracellular pH, and usually in the basolateral membrane of epithelial cells. It has also been detected in teleost gills where it may participate in systemic pH regulation. NHE-3 is usually expressed in the apical membrane of mammalian epithelial cells where it helps reabsorb Na(+) and HCO(3)(-); it has also been detected in teleost gills. We used Western blotting and heterologous antibodies to screen for expression of NHE-1 and NHE-3 in gills of an agnathan (Myxine glutinosa) and an elasmobranch (Raja erinacea), and NHE-3 in gills of a teleost (Fundulus heteroclitus). Positive NHE-1 bands were detected in gills from the agnathan and elasmobranch. Using the NHE-3 antibody, bands were detected in the gills of the elasmobranch and teleost. These data are some of the first direct evidence of NHEs in the gills of an agnathan and elasmobranch, and confirm the presence of NHEs in the gills of teleosts.

Hypertonic stress induces rapid and widespread protein damage in C. elegans

Proteostasis is defined as the homeostatic mechanisms that maintain the function of all cytoplasmic proteins. We recently demonstrated that the capacity of the proteostasis network is a critical factor that defines the limits of cellular and organismal survival in hypertonic environments. The current studies were performed to determine the extent of protein damage induced by cellular water loss. Using worm strains expressing fluorescently tagged foreign and endogenous proteins and proteins with temperature-sensitive point mutations, we demonstrate that hypertonic stress causes aggregation and misfolding of diverse proteins in multiple cell types. Protein damage is rapid. Aggregation of a polyglutamine yellow fluorescent protein reporter is observable with <1 h of hypertonic stress, and aggregate volume doubles approximately every 10 min. Aggregate formation is irreversible and occurs after as little as 10 min of exposure to hypertonic conditions. To determine whether endogenous proteins are aggregated by hypertonic stress, we quantified the relative amount of total cellular protein present in detergent-insoluble extracts. Exposure for 4 h to 400 mM or 500 mM NaCl induced a 55-120% increase in endogenous protein aggregation. Inhibition of insulin signaling or acclimation to mild hypertonic stress increased survival under extreme hypertonic conditions and prevented aggregation of endogenous proteins. Our results demonstrate that hypertonic stress causes widespread and dramatic protein damage and that cells have a significant capacity to remodel the network of proteins that function to maintain proteostasis. These findings have important implications for understanding how cells cope with hypertonic stress and other protein-damaging stressors.

COX2 in a euryhaline teleost, Fundulus heteroclitus: primary sequence, distribution, localization, and potential function in gills during salinity acclimation.

SUMMARY In the kidneys of mammals, cyclooxygenase type 2 (COX2) is expressed in medullary interstitial cells, the macula densa and epithelial cells of the cortical thick ascending limb where it generates prostaglandins that regulate hormone secretion, inhibit ion transport, and support cell survival during salt loading and dehydration. In teleosts, the gills are in direct contact with an aquatic environment and are the dominant site of osmoregulation. During transfers between salinities, specialized cells in the gills (chloride cells) rapidly regulate NaCl secretion for systemic osmoregulation while they simultaneously are exposed to acute osmotic shock. This study was conducted to determine if COX2 is expressed in the gills, and if so, to evaluate its function in cellular and systemic osmoregulation. Degenerate primers, reverse transcription–PCR and rapid amplification of cDNA ends were used to deduce the complete cDNA sequence of a putative COX2 enzyme from the gills of the euryhaline killifish (Fundulus heteroclitus). The 2738 base pair cDNA includes a coding region for a 610 amino acid protein that is over 70% identical to mammalian COX2. A purified antibody generated against a conserved region of mouse COX2 labeled chloride cells, suggesting that the enzyme may control NaCl secretion as an autocrine agent. Real-time PCR was then used to demonstrate that mRNA expression of the COX2 homologue was threefold greater in gills from chronic seawater killifish than in gills from chronic freshwater killifish. Expression of Na+/K+/2Cl– cotransporter and the cystic fibrosis transmembrane conductance regulator were also greater in seawater, suggesting that chronic COX2 expression in the gills is regulated in parallel to the key ion transporters that mediate NaCl secretion. Real-time PCR was also used to demonstrate that acute transfer from seawater to freshwater and from freshwater to seawater led to rapid, transient inductions of COX2 expression. Together with previous physiological evidence, the present molecular and immunological data suggest that constitutive branchial COX2 expression is enhanced in seawater, where prostaglandins can regulate NaCl secretion in chloride cells. Our data also suggest that branchial COX2 expression may play a role in cell survival during acute osmotic shock.

An Ultra High-Throughput, Whole-Animal Screen for Small Molecule Modulators of a Specific Genetic Pathway in Caenorhabditis elegans

High-throughput screening (HTS) is a powerful approach to drug discovery, but many lead compounds are found to be unsuitable for use in vivo after initial screening. Screening in small animals like C. elegans can help avoid these problems, but this system has been limited to screens with low-throughput or no specific molecular target. We report the first in vivo 1536-well plate assay for a specific genetic pathway in C. elegans. Our assay measures induction of a gene regulated by SKN-1, a master regulator of detoxification genes. SKN-1 inhibitors will be used to study and potentially reverse multidrug resistance in parasitic nematodes. Screens of two small commercial libraries and the full Molecular Libraries Small Molecule Repository (MLSMR) of ∼364,000 compounds validate our platform for ultra HTS. Our platform overcomes current limitations of many whole-animal screens and can be widely adopted for other inducible genetic pathways in nematodes and humans.

Unique structure and regulation of the nematode detoxification gene regulator, SKN-1: implications to understanding and controlling drug resistance

Nematodes parasitize an alarming number of people and agricultural animals globally and cause debilitating morbidity and mortality. Anthelmintics have been the primary tools used to control parasitic nematodes for the past several decades, but drug resistance is becoming a major obstacle. Xenobiotic detoxification pathways defend against drugs and other foreign chemicals in diverse organisms, and evidence is accumulating that they play a role in mediating resistance to anthelmintics in nematodes. Related antioxidation pathways may also provide filarial parasites with protection against host free-radical–mediated immune responses. Upstream regulatory pathways have received almost no attention in nematode parasites, despite their potential to coregulate multiple detoxification and antioxidation genes. The nuclear eurythroid 2–related factor 2 (NRF2) transcription factor mediates inducible detoxification and antioxidation defenses in mammals, and recent studies have demonstrated that it promotes multidrug resistance in some human tumors. Recent studies in the free-living model nematode, Caenorhabditis elegans, have defined the homologous transcription factor, SKN-1, as a master regulator of detoxification and antioxidation genes. Despite similar functions, SKN-1 and NRF2 have important differences in structure and regulatory pathways. Protein alignment and phylogenetic analyses indicate that these differences are shared among many nematodes, making SKN-1 a candidate for specifically targeting nematode detoxification and antioxidation.

Characterization of the Proteostasis Roles of Glycerol Accumulation, Protein Degradation and Protein Synthesis during Osmotic Stress in C. elegans

Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50–70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50–80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70–180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage brought about by an environmental stressor, demonstrate important differences in aging- versus stress-induced protein damage, and challenge the widely held view that chemical chaperones are accumulated during hypertonic stress to protect protein structure/function.

In vitro and in vivo characterization of a tunable dual-reactivity probe of the Nrf2-ARE pathway.

The cell utilizes the Keap1/Nrf2-ARE signaling pathway to detoxify harmful chemicals in order to protect itself from oxidative stress and to maintain its reducing environment. When exposed to oxidative stress and xenobiotic inducers, the redox sensitive Keap1 is covalently modified at specific cysteine residues. Consequently, the latent transcription factor Nrf2 is stabilized and translocates into the nucleus, where it transactivates the expression of detoxification genes through binding to the antioxidant response element (ARE). In the pursuit of potent and bioavailable activators of the ARE, we validated hits from a pathway-directed high-throughput screening campaign by testing them in cell culture and a reporter strain of a whole animal model, Caenorhabditis elegans. These studies allowed us to identify AI-3 as an ARE activator that induces cytoprotective genes in human cells and in worms, which also translated into in vivo activity in mice. AI-3 is an electrophilic ARE activator with two thiol sensitive sites toward a nucleophilic aromatic substitution, and SAR studies indicated the tunability of the system. Tandem LC-MS analysis revealed that AI-3 alkylates Keap1 primarily at Cys151, while AI-3 is reactive toward additional cysteine residues at higher doses in vitro and in vivo. The immediate effects of such alkylation included the disruption of Keap1-Cul3 (low [AI-3]) and/or Keap1-Nrf2 (high [AI-3]) interactions that both led to the stabilization of Nrf2. This further translated into the downstream Nrf2-ARE regulated cytoprotective gene activation. Collectively, AI-3 may become a valuable biological tool and may even provide therapeutic benefits in oxidative stress related diseases.

Physiological and molecular mechanisms of salt and water homeostasis in the nematode Caenorhabditis elegans

Intracellular salt and water homeostasis is essential for all cellular life. Extracellular salt and water homeostasis is also important for multicellular organisms. Many fundamental mechanisms of compensation for osmotic perturbations are well defined and conserved. Alternatively, molecular mechanisms of detecting salt and water imbalances and regulating compensatory responses are generally poorly defined for animals. Throughout the last century, researchers studying vertebrates and vertebrate cells made critical contributions to our understanding of osmoregulation, especially mechanisms of salt and water transport and organic osmolyte accumulation. Researchers have more recently started using invertebrate model organisms with defined genomes and well-established methods of genetic manipulation to begin defining the genes and integrated regulatory networks that respond to osmotic stress. The nematode Caenorhabditis elegans is well suited to these studies. Here, I introduce osmoregulatory mechanisms in this model, discuss experimental advantages and limitations, and review important findings. Key discoveries include defining genetic mechanisms of osmolarity sensing in neurons, identifying protein damage as a sensor and principle determinant of hypertonic stress resistance, and identification of a putative sensor for hypertonic stress associated with the extracellular matrix. Many of these processes and pathways are conserved and, therefore, provide new insights into salt and water homeostasis in other animals, including mammals.