Olli Matilainen

Specific SKN-1/Nrf Stress Responses to Perturbations in Translation Elongation and Proteasome Activity

SKN-1, the Caenorhabditis elegans Nrf1/2/3 ortholog, promotes both oxidative stress resistance and longevity. SKN-1 responds to oxidative stress by upregulating genes that detoxify and defend against free radicals and other reactive molecules, a SKN-1/Nrf function that is both well-known and conserved. Here we show that SKN-1 has a broader and more complex role in maintaining cellular stress defenses. SKN-1 sustains expression and activity of the ubiquitin-proteasome system (UPS) and coordinates specific protective responses to perturbations in protein synthesis or degradation through the UPS. If translation initiation or elongation is impaired, SKN-1 upregulates overlapping sets of cytoprotective genes and increases stress resistance. When proteasome gene expression and activity are blocked, SKN-1 activates multiple classes of proteasome subunit genes in a compensatory response. SKN-1 thereby maintains UPS activity in the intestine in vivo under normal conditions and promotes survival when the proteasome is inhibited. In contrast, when translation elongation is impaired, SKN-1 does not upregulate proteasome genes, and UPS activity is then reduced. This indicates that UPS activity depends upon presence of an intact translation elongation apparatus; and it supports a model, suggested by genetic and biochemical studies in yeast, that protein synthesis and degradation may be coupled processes. SKN-1 therefore has a critical tissue-specific function in increasing proteasome gene expression and UPS activity under normal conditions, as well as when the UPS system is stressed, but mounts distinct responses when protein synthesis is perturbed. The specificity of these SKN-1–mediated stress responses, along with the apparent coordination between UPS and translation elongation activity, may promote protein homeostasis under stress or disease conditions. The data suggest that SKN-1 may increase longevity, not only through its well-documented role in boosting stress resistance, but also through contributing to protein homeostasis.

A photoconvertible reporter of the ubiquitin-proteasome system in vivo.

The ubiquitin-proteasome system (UPS) orchestrates many cellular and tissue-specific processes by degrading damaged and key regulatory proteins. To enable investigation of UPS activity in different cell types in a living animal, we developed a photoconvertible fluorescent UPS reporter system for live imaging and quantification of protein degradation in Caenorhabditis elegans. Our reporter consists of the photoconvertible fluorescent protein Dendra2 targeted for proteasomal degradation by fusion to the UbG76V mutant form of ubiquitin. In contrast to previous reporters, this system permits quantification of UPS activity independently of protein synthesis. Our reporter revealed that UPS-mediated protein degradation varies in a cell type–specific and age-dependent manner in C. elegans.

Mitochondria and Epigenetics – Crosstalk in Homeostasis and Stress

Through epigenetic mechanisms cells integrate environmental stimuli to fine-tune gene expression levels. Mitochondrial function is essential to provide the intermediate metabolites necessary to generate and modify epigenetic marks in the nucleus, which in turn can regulate the expression of mitochondrial proteins. In this review we summarize the function of mitochondria in the regulation of epigenetic mechanisms as a new aspect of mitonuclear communication. We focus in particular on the most common epigenetic modifications - histone acetylation and histone and DNA methylation. We also discuss the emerging field of mitochondrial DNA (mtDNA) methylation, whose physiological role remains unknown. Finally, we describe the essential role of some histone modifications in regulating the mitochondrial unfolded protein response (UPRmt) and the mitochondrial stress-dependent lifespan extension.

Proteasome Activity Influences UV-Mediated Subnuclear Localization Changes of NPM

UV damage activates cellular stress signaling pathways, causes DNA helix distortions and inhibits transcription by RNA polymerases I and II. In particular, the nucleolus, which is the site of RNA polymerase I transcription and ribosome biogenesis, disintegrates following UV damage. The disintegration is characterized by reorganization of the subnucleolar structures and change of localization of many nucleolar proteins. Here we have queried the basis of localization change of nucleophosmin (NPM), a nucleolar granular component protein, which is increasingly detected in the nucleoplasm following UV radiation. Using photobleaching experiments of NPM-fluorescent fusion protein in live human cells we show that NPM mobility increases after UV damage. However, we show that the increase in NPM nucleoplasmic abundance after UV is independent of UV-activated cellular stress and DNA damage signaling pathways. Unexpectedly, we find that proteasome activity affects NPM redistribution. NPM nucleolar expression was maintained when the UV-treated cells were exposed to proteasome inhibitors or when the expression of proteasome subunits was inhibited using RNAi. However, there was no evidence of increased NPM turnover in the UV damaged cells, or that ubiquitin or ubiquitin recycling affected NPM localization. These findings suggest that proteasome activity couples to nucleolar protein localizations in UV damage stress.

The chromatin remodeling factor ISW-1 integrates organismal responses against nuclear and mitochondrial stress

Age-associated changes in chromatin structure have a major impact on organismal longevity. Despite being a central part of the ageing process, the organismal responses to the changes in chromatin organization remain unclear. Here we show that moderate disturbance of histone balance during C. elegans development alters histone levels and triggers a stress response associated with increased expression of cytosolic small heat-shock proteins. This stress response is dependent on the transcription factor, HSF-1, and the chromatin remodeling factor, ISW-1. In addition, we show that mitochondrial stress during developmental stages also modulates histone levels, thereby activating a cytosolic stress response similar to that caused by changes in histone balance. These data indicate that histone and mitochondrial perturbations are both monitored through chromatin remodeling and involve the activation of a cytosolic response that affects organismal longevity. HSF-1 and ISW-1 hence emerge as a central mediator of this multi-compartment proteostatic response regulating longevity.Changes in chromatin structure have been linked to organismal ageing. Here the authors show that altered histone expression and mitochondrial stress during C. elegans development result in chromatin changes and a cytosolic stress response that affects organismal longevity, and depends on HSF-1 and the chromatin remodeller, ISW-1.

De novo NAD+ synthesis enhances mitochondrial function and improves health.

Nicotinamide adenine dinucleotide (NAD+) is a co-substrate for several enzymes, including the sirtuin family of NAD+-dependent protein deacylases. Beneficial effects of increased NAD+ levels and sirtuin activation on mitochondrial homeostasis, organismal metabolism and lifespan have been established across species. Here we show that α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), the enzyme that limits spontaneous cyclization of α-amino-β-carboxymuconate-ε-semialdehyde in the de novo NAD+ synthesis pathway, controls cellular NAD+ levels via an evolutionarily conserved mechanism in Caenorhabditis elegans and mouse. Genetic and pharmacological inhibition of ACMSD boosts de novo NAD+ synthesis and sirtuin 1 activity, ultimately enhancing mitochondrial function. We also characterize two potent and selective inhibitors of ACMSD. Because expression of ACMSD is largely restricted to kidney and liver, these inhibitors may have therapeutic potential for protection of these tissues from injury. In summary, we identify ACMSD as a key modulator of cellular NAD+ levels, sirtuin activity and mitochondrial homeostasis in kidney and liver.Genetic or pharmacological inhibition of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase increases NAD+ and improves mitochondrial function in nematodes and mice, and may have therapeutic potential in kidney and liver disease.

Fluorescent Tools for In Vivo Studies on the Ubiquitin-Proteasome System

The ubiquitin-proteasome system (UPS) plays a key role in maintaining proteostasis by degrading most of the cellular proteins. Traditionally, UPS activity is studied in vitro, in yeast, or in mammalian cell cultures by using short-lived GFP-based UPS reporters. Here, we present protocols for two fluorescent tools facilitating real-time imaging of UPS activity in living animals. We have generated transgenic Caenorhabditis elegans (C. elegans) expressing a photoconvertible UbG76V-Dendra2 UPS reporter, which permits measurement of reporter degradation by the proteasome independently of reporter protein synthesis, and a fluorescent polyubiquitin-binding reporter for detection of the endogenous pool of Lys48-linked polyubiquitinated proteasomal substrates. These reporter systems facilitate cell- and tissue-specific analysis of UPS activity especially in young adult animals, but can also be used for studies during development, aging, and for example stress conditions.