Inactivation of the Caenorhabditis elegans RNF-5 E3 ligase promotes IRE-1-independent ER functions.

RNF5 is implicated in ERAD and in negative regulation of macroautophagy/autophagy. To better understand the function of RNF-5 under ER-stress conditions, we studied the ability of Caenorhabditis elegans rnf-5(tm794) mutant animals to cope with stress in the background of impaired UPR machinery. We demonstrate that downregulation of RNF-5 decreased sensitivity to tunicamycin both in wild type and in an ire-1 mutant. Double-mutant rnf-5;ire-1 animals showed increased starvation resistance and extended lifespan when compared to the ire-1 mutant. This partial rescue of ire-1 required functional autophagy. Downregulation of RNF-5 rescued ER maturation defects and protein secretion of a DAF-28::GFP intestinal reporter in the ire-1 background. Proteomics and functional studies revealed an increase in lysosomal protease levels, in the frequency of intestinal lysosomes, and in lysosomal protease activity in rnf-5(tm794) animals. Together, these data suggest that RNF-5 is a negative regulator of ER stress, and that inactivation of RNF-5 promotes IRE-1-independent elevation of ER capacity.

S-allylmercapto-N-acetylcysteine protects against oxidative stress and extends lifespan in Caenorhabditis elegans.

S-allylmercapto-N-acetylcysteine (ASSNAC) was shown in our previous study to activate Nrf2-mediated processes and increase glutathione level and resistance to oxidative stress in cultured endothelial cells. In this study, we explored the antioxidant protective effect of ASSNAC in Caenorhabditis elegans (C. elegans). Treatment of gst-4 reporter strain (CL2166) with increasing concentrations of ASSNAC (0.2 to 20 mM) for 24 hours and with ASSNAC (10 mM) for various time periods demonstrated a significant concentration- and time-dependent increase in Glutathione S-transferase (GST) gene expression (up to 60-fold at 20 mM after 24 hours). In addition, ASSNAC (2 mM; 24 hours) treatment of C. elegans strains N2 (wild type strain), gst-4 reporter (CL2166) and temperature sensitive sterile strain (CF512) significantly increased GST enzyme activity by 1.9-, 1.5- and 1.8-fold, respectively. ASSNAC (2.0 mM; 24 hours) increased the reduced glutathione content in N2 and CF512 strains by 5.9- and 4.9-fold, respectively. Exposure of C. elegans (N2 strain) to a lethal concentration of H2O2 (3.5 mM; 120 min) resulted in death of 88% of the nematodes while pretreatment with ASSNAC (24 hours) reduced nematodes death in a concentration-dependent manner down to 8% at 2.0 mM. C. elegans nematodes (strain CF512) cultured on agar plates containing ASSNAC (0.5 to 5.0 mM) demonstrated a significant increase in lifespan compared to control (mean lifespan 26.45 ± 0.64 versus 22.90 ± 0.59 days; log-rank p ≤ 0.001 at 2.0 mM) with a maximal lifespan of 40 versus 36 days. In conclusion, ASSNAC up-regulates the GST gene expression and enzyme activity as well as the glutathione content in C. elegans nematodes and thereby increases their resistance to oxidative stress and extends their lifespan.

ULP-2 SUMO Protease Regulates E-Cadherin Recruitment to Adherens Junctions.

Adherens junctions (AJs) are membrane-anchored structures composed of E-cadherin and associated proteins, including catenins and actin. The unique plasticity of AJs mediates both the rigidity and flexibility of cell-cell contacts essential for embryonic morphogenesis and adult tissue remodeling. We identified the SUMO protease ULP-2 as a regulator of AJ assembly and show that dysregulated ULP-2 activity impairs epidermal morphogenesis in Caenorhabditis elegans embryos. The conserved cytoplasmic tail of HMR-1/E-cadherin is sumoylated and is a target of ULP-2 desumoylation activity. Coupled sumoylation and desumoylation of HMR-1 are required for its recruitment to the subapical membrane during AJ assembly and the formation of the linkages between AJs and the apical actin cytoskeleton. Sumoylation weakens HMR-1 binding to HMP-2/ß-catenin. Our study provides a mechanistic link between the dynamic nature of the SUMO machinery and AJ plasticity and highlight sumoylation as a molecular switch that modulates the binding of E-cadherin to the actin cytoskeleton.

Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging.

Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin-proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies.

RNF-121 is an endoplasmic reticulum-membrane E3 ubiquitin ligase involved in the regulation of beta-integrin.

We report on the characterization of RNF-121, an evolutionarily conserved E3 ligase RING finger protein that is expressed in the endoplasmic reticulum (ER) of various cells and tissues in Caenorhabditis elegans. Inactivation of RNF-121 induced an elevation in BiP expression and increased the sensitivity of worms to ER stress. Genetic analysis placed RNF-121 downstream of the unfolded protein response (UPR) regulator protein kinase-like endoplasmic reticulum kinase (PERK). We identify PAT-3::GFP, the beta subunit of the heterodimeric integrin receptors, as an RNF-121 substrate; whereas induction of RNF-121 expression reduced the level of PAT-3::GFP in the gonad distal tip cells, inhibition of RNF-121 led to the accumulation of stably bound PAT-3::GFP inclusions. Correspondingly, overexpression of RNF-121 during early stages of gonad development led to aberrations in germline development and gonad migration that overlap with those observed after PAT-3 inactivation. The formation of these gonad abnormalities required functional ER-associated degradation (ERAD) machinery. Our findings identify RNF-121 as an ER-anchored ubiquitin ligase that plays a specific role in the ERAD pathway by linking it to the regulation of the cell adhesion integrin receptors.

SUMO regulates the assembly and function of a cytoplasmic intermediate filament protein in C. elegans.

Sumoylation is a reversible posttranslational modification that plays roles in many processes, including transcriptional regulation, cell division, chromosome integrity, and DNA damage response. Using a proteomics approach, we identified approximately 250 candidate targets of sumoylation in C. elegans. One such target is the cytoplasmic intermediate filament (cIF) protein named IFB-1, which is expressed in hemidesmosome-like structures in the worm epidermis and is essential for embryonic elongation and maintenance of muscle attachment to the cuticle. In the absence of SUMO, IFB-1 formed ectopic filaments and protein aggregates in the lateral epidermis. Moreover, depletion of SUMO or mutation of the SUMO acceptor site on IFB-1 resulted in a reduction of its cytoplasmic soluble pool, leading to a decrease in its exchange rate within epidermal attachment structures. These observations indicate that SUMO regulates cIF assembly by maintaining a cytoplasmic pool of nonpolymerized IFB-1, and that this is necessary for normal IFB-1 function.