Reactive oxygen species (ROS) are highly reactive, oxygen-containing molecules that can cause molecular damage within the cell. While the accumulation of ROS-mediated damage is widely believed to be one of the main causes of aging, ROS also act in signaling pathways. Recent work has demonstrated that increasing levels of superoxide, one form of ROS, through treatment with paraquat, results in increased lifespan. Interestingly, treatment with paraquat robustly increases the already long lifespan of the clk-1 mitochondrial mutant, but not other long-lived mitochondrial mutants such as isp-1 or nuo-6. To genetically dissect the subcellular compartment in which elevated ROS act to increase lifespan, we deleted individual superoxide dismutase (sod) genes in clk-1 mutants, which are sensitized to ROS. We find that only deletion of the primary mitochondrial sod gene, sod-2 results in increased lifespan in clk-1 worms. In contrast, deletion of either of the two cytoplasmic sod genes, sod-1 or sod-5, significantly decreases the lifespan of clk-1 worms. Further, we show that increasing mitochondrial superoxide levels through deletion of sod-2 or treatment with paraquat can still increase lifespan in clk-1;sod-1 double mutants, which live shorter than clk-1 worms. The fact that mitochondrial superoxide can increase lifespan in worms with a detrimental level of cytoplasmic superoxide demonstrates that ROS have a compartment specific effect on lifespan – elevated ROS in the mitochondria acts to increase lifespan, while elevated ROS in the cytoplasm decreases lifespan. This work also suggests that both ROS-dependent and ROS-independent mechanisms contribute to the longevity of clk-1 worms.

BackgroundThe mitochondrial unfolded protein response (mitoUPR) is a stress response pathway activated by disruption of proteostasis in the mitochondria. This pathway has been proposed to influence lifespan, with studies suggesting that mitoUPR activation has complex effects on longevity.ResultsHere, we examined the contribution of the mitoUPR to the survival and lifespan of three long-lived mitochondrial mutants in Caenorhabditis elegans by modulating the levels of ATFS-1, the central transcription factor that mediates the mitoUPR. We found that clk-1, isp-1, and nuo-6 worms all exhibit an ATFS-1-dependent activation of the mitoUPR. While loss of atfs-1 during adulthood does not affect lifespan in any of these strains, absence of atfs-1 during development prevents clk-1 and isp-1 worms from reaching adulthood and reduces the lifespan of nuo-6 mutants. Examining the mechanism by which deletion of atfs-1 reverts nuo-6 lifespan to wild-type, we find that many of the transcriptional changes present in nuo-6 worms are mediated by ATFS-1. Genes exhibiting an ATFS-1-dependent upregulation in nuo-6 worms are enriched for transcripts that function in stress response and metabolism. Consistent, with this finding, loss of atfs-1 abolishes the enhanced stress resistance observed in nuo-6 mutants and prevents upregulation of multiple stress response pathways including the HIF-1-mediated hypoxia response, SKN-1-mediated oxidative stress response and DAF-16-mediated stress response.ConclusionsOur results suggest that in the long-lived mitochondrial mutant nuo-6 activation of the mitoUPR causes atfs-1-dependent changes in the expression of genes involved in stress response and metabolism, which contributes to the extended longevity observed in this mutant. This work demonstrates that the mitoUPR can modulate multiple stress response pathways and suggests that it is crucial for the development and lifespan of long-lived mitochondrial mutants.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0615-3) contains supplementary material, which is available to authorized users.

Mild deficits in mitochondrial function have been shown to increase lifespan in multiple species including worms, flies and mice. Here, we study three C. elegans mitochondrial mutants (clk-1, isp-1 and nuo-6) to identify overlapping genetic pathways that contribute to their longevity. We find that genes regulated by the FOXO transcription factor DAF-16 are upregulated in all three strains, and that the transcriptional changes present in these worms overlap significantly with the long-lived insulin-IGF1 signaling pathway mutant daf-2. We show that DAF-16 and multiple DAF-16 interacting proteins (MATH-33, IMB-2, CST-1/2, BAR-1) are required for the full longevity of all three mitochondrial mutants. Our results suggest that the activation of DAF-16 in these mutants results from elevated levels of reactive oxygen species. Overall, this work reveals an overlapping genetic pathway required for longevity in three mitochondrial mutants, and, combined with previous work, demonstrates that DAF-16 is a downstream mediator of lifespan extension in multiple pathways of longevity.

In this work, we examine the relationship between stress resistance and aging. We find that resistance to multiple types of stress peaks during early adulthood and then declines with age. To dissect the underlying mechanisms, we use C. elegans transcriptional reporter strains that measure the activation of different stress responses including: the heat shock response, mitochondrial unfolded protein response, endoplasmic reticulum unfolded protein response, hypoxia response, SKN-1-mediated oxidative stress response, and the DAF-16-mediated stress response. We find that the decline in stress resistance with age is at least partially due to a decreased ability to activate protective mechanisms in response to stress. In contrast, we find that any baseline increase in stress caused by the advancing age is too mild to detectably upregulate any of the stress response pathways. Further exploration of how worms respond to stress with increasing age revealed that the ability to mount a hormetic response to heat stress is also lost with increasing age. Overall, this work demonstrates that resistance to all types of stress declines with age. Based on our data, we speculate that the decrease in stress resistance with advancing age results from a genetically-programmed inactivation of stress response pathways, not accumulation of damage.

Mutations affecting components of the mitochondrial electron transport chain have been shown to increase lifespan in multiple species including the worm Caenorhabditis elegans. While it was originally proposed that decreased generation of reactive oxygen species (ROS) resulting from lower rates of electron transport could account for the observed increase in lifespan, recent evidence indicates that ROS levels are increased in at least some of these long-lived mitochondrial mutants. Here, we show that the long-lived mitochondrial mutant isp-1 worms have increased resistance to oxidative stress. Our results suggest that elevated ROS levels in isp-1 worms cause the activation of multiple stress-response pathways including the mitochondrial unfolded protein response, the SKN-1-mediated stress response, and the hypoxia response. In addition, these worms have increased expression of specific antioxidant enzymes, including a marked upregulation of the inducible superoxide dismutase genes sod-3 and sod-5. Examining the contribution of sod-3 and sod-5 to the oxidative stress resistance in isp-1 worms revealed that loss of either of these genes increased resistance to oxidative stress, but not other forms of stress. Deletion of sod-3 or sod-5 decreased the lifespan of isp-1 worms and further exacerbated their slow physiologic rates. Thus, while deletion of sod-3 and sod-5 genes has little impact on stress resistance, physiologic rates or lifespan in wild-type worms, these genes are required for the longevity of isp-1 worms. Overall, this work shows that the increased resistance to oxidative stress in isp-1 worms does not account for their longevity, and that resistance to oxidative stress can be experimentally dissociated from lifespan.

In 2000, the National Institutes of Health initiated the Protein Structure Initiative as a multi‐center structural biology program with “an initial goal to make the three‐dimensional, atomic‐level structures of most proteins easily obtainable from knowledge of their corresponding DNA sequences.” The third and final phase of this program concluded in 2015 with the publication and distribution of more than 5000 previously uncharacterized proteins.The work described here leverages the availability of high‐quality structures and pre‐cloned expression plasmids to combine forces of undergraduate biochemistry teaching lab courses across a diverse range of participating institutions. This consortium of undergraduate biochemistry faculty and students seeks to identify functional properties of a subset of these uncharacterized proteins, seeking to unify structure□ and □function relationships. Through structural and bioinformatics tools, an number of these proteins were characterized as putative hydrolases due to similarity to known hydrolase enzymes. Some appeared, by sequence homology, to possess features indicative of more specifically identifiable activity, such as halogenases and nucleases. Students in the current biochemistry laboratory class at Hope College have expressed and purified seven of these proteins, finding that structural information can guide, although not predict entirely, functional predictions regarding substrate specificity.Support or Funding InformationThis work was funded in part with support from the National Science Foundation IUSE program.