Real-time redox measurements during endoplasmic reticulum stress reveal interlinked protein folding functions.

Disruption of protein folding in the endoplasmic reticulum (ER) causes unfolded proteins to accumulate, triggering the unfolded protein response (UPR). UPR outputs in turn decrease ER unfolded proteins to close a negative feedback loop. However, because it is infeasible to directly measure the concentration of unfolded proteins in vivo, cells are generically described as experiencing "ER stress" whenever the UPR is active. Because ER redox potential is optimized for oxidative protein folding, we reasoned that measureable redox changes should accompany unfolded protein accumulation. To test this concept, we employed fluorescent protein reporters to dynamically measure ER redox status and UPR activity in single cells. Using these tools, we show that diverse stressors, both experimental and physiological, compromise ER protein oxidation when UPR-imposed homeostatic control is lost. Using genetic analysis we uncovered redox heterogeneities in isogenic cell populations, and revealed functional interlinks between ER protein folding, modification, and quality control systems.

Chaperone-mediated reflux of secretory proteins to the cytosol during endoplasmic reticulum stress.

Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such "ER stress," we employed an ER-targeted, redox-responsive, green fluorescent protein-eroGFP-that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress regimes cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to "reflux" back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in Saccharomyces cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and cochaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.

Establishment of a system for monitoring endoplasmic reticulum redox state in mammalian cells.

The endoplasmic reticulum (ER) performs a critical role in the oxidative folding of nascent proteins, such that perturbations to ER homeostasis may lead to protein misfolding and subsequent pathological processes. Among the mechanisms for maintaining ER homeostasis is a redox regulation, which is a critical determinant of the fate of ER-stressed cells. Here, we report the establishment of a system for monitoring the ER redox state in mammalian cells. The new ER redox-sensing system was developed based on the previously described monitoring system in yeast. Our system could successfully monitor the dynamic ER redox state in mammalian cells. Using this system, we find that manipulation of ER oxidases changes the ER redox state. The mammalian ER redox-sensing system could be used to study the mechanisms of ER redox regulation and provide a foundation for an approach to develop novel therapeutic modalities for human diseases related to dysregulated ER homeostasis including diabetes, neurodegeneration, and Wolfram syndrome.

The sirtuins, oxidative stress and aging: an emerging link.

Reactive oxygen species (ROS) are a family of compounds that can oxidatively damage cellular macromolecules and may influence lifespan. Sirtuins are a conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases that regulate lifespan in many model organisms including yeast and mice. Recent work suggests that sirtuins can modulate ROS levels notably during a dietary regimen known as calorie restriction which enhances lifespan for several organisms. Although both sirtuins and ROS have been implicated in the aging process, their precise roles remain unknown. In this review, we summarize current thinking about the oxidative stress theory of aging, discuss some of the compelling data linking the sirtuins to ROS and aging, and propose a conceptual model placing the sirtuins into an ROS-driven mitochondria-mediated hormetic response.