Progress in Understanding Calorie Restricted Life Extension

The enzyme WWP-1, shown in green, is a key player in the signaling cascade that links dietary restriction to longevity in roundworms. Sensory neurons are shown in red. Image: Courtesy of Dr. Andrea C. Carrano, Salk Institute for Biological Studies

Researchers at the Salk Institute for Biological Studies have identified a pivotal role for two enzymes that work together to determine the health benefits of diet restriction.

– enzyme wwp-1 is required and specific for the extension of lifespan by dietary restriction.
– WWP-1 ubiquitin ligase activity is essential for diet-restriction-induced longevity.
– WWP-1 exhibits ubiquitin ligase activity in a UBC-18 dependent manner in vitro.
– WWP-1 and UBC-18 function together to regulate diet-restriction-induced longevity.

a, Lifespan analysis of eat-2(ad1116) mutant worms fed bacteria expressing ubc-18 dsRNA or control vector initiated after hatching of eggs (L1) or day 1 adults (D1). b, c, Lifespan analysis of isp-1(qm150) (b) and daf-2(e1368) (c) fed bacteria expressing ubc-18 dsRNA or control vector. d, Lifespan analysis of eat-2(ad1116) mutant animals fed bacteria expressing wwp-1 dsRNA and vector (wwp-1 RNAi), ubc-18 dsRNA and vector (ubc-18 RNAi), wwp-1 and ubc-18 dsRNA (wwp-1 + ubc-18 RNAi), or control vector. e, Lifespan analysis of wwp-1 overexpressing worms (GFP::WWP-1) or control worms (GFP) fed bacteria expressing ubc-18 dsRNA or control vector.

There are only three known genetic networks that ensure youthfulness when manipulated. One centers on the insulin/insulin growth factor-1, which regulates metabolism and growth; the second is driven by mitochondria, the cell’s power plants; and the third is linked to diet restriction.

Author Andrea C. Carrano, Ph.D., a postdoctoral researcher in American Cancer Society Professor Tony Hunter’s laboratory, hadn’t set out to unravel the molecular connection between dietary restriction and increased lifespan when she started to investigate the role of the mammalian enzyme WWP-1. “I only knew that WWP-1 was a ubiquitin ligase and that mammalian cells contain three copies, which would make it difficult to study its function.”

Ubiquitin ligases work in tandem with so called ubiquitin-conjugating enzymes to attach a chain of ubiquitin molecules to other proteins. This process, called ubiquitination, flags protein substrates for destruction but can also serve as a regulatory signal.

Since the laboratory roundworm Caenorhabditis elegans only contains one copy, Carrano teamed up with Salk researcher Dillin, who studies aging and longevity in C. elegans. Initial experiments revealed that worms without the WWP-1 gene seemed normal but were more susceptible to various forms of stress. “This finding was the first hint that WWP-1 might play a role in the aging process since mutations that affect stress very often correlate with longevity,” she says.

Prompted by the findings, Carrano’s next set of experiments focused on WWP-1’s potential role in the regulation of lifespan. When she genetically engineered worms to overexpress WWP-1, well-fed worms lived on average 20 percent longer. Deleting PHA-4, which was discovered in Dillin’s lab and so far is the only gene known to be essential for lifespan extension in response to diet restriction, abolished the life-extending effects of additional WWP-1 placing the ubiquitin ligase as a central rung on the same genetic ladder as PHA-4. Without WWP-1, cutting down on calories no longer staved off death.

When a study by others found that UBC-18 interacts with WWP-1, Carrano wondered whether it could play a role in diet-restriction-induced longevity as well. She first confirmed that the UBC-18 functions as an ubiquitin-conjugating enzyme and gives WWP-1 a hand. She then tested whether it played a role in lifespan regulation. “Overexpression of UBC-18 was not enough to extend the lifespan of worms but depleting it negated the effects of caloric restriction,” says Carrano, who is busy looking for potential substrates of the UBC-18-WWP-1 ubiquitination complex.

Journal Nature: A conserved ubiquitination pathway determines longevity in response to diet restriction

Dietary restriction extends longevity in diverse species, suggesting that there is a conserved mechanism for nutrient regulation and prosurvival responses. Here we show a role for the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase WWP-1 as a positive regulator of lifespan in Caenorhabditis elegans in response to dietary restriction. We find that overexpression of wwp-1 in worms extends lifespan by up to 20% under conditions of ad libitum feeding. This extension is dependent on the FOXA transcription factor pha-4, and independent of the FOXO transcription factor daf-16. Reduction of wwp-1 completely suppresses the extended longevity of diet-restricted animals. However, the loss of wwp-1 does not affect the long lifespan of animals with compromised mitochondrial function or reduced insulin/IGF-1 signalling. Overexpression of a mutant form of WWP-1 lacking catalytic activity suppresses the increased lifespan of diet-restricted animals, indicating that WWP-1 ubiquitin ligase activity is essential for longevity. Furthermore, we find that the E2 ubiquitin conjugating enzyme, UBC-18, is essential and specific for diet-restriction-induced longevity. UBC-18 interacts with WWP-1 and is required for the ubiquitin ligase activity of WWP-1 and the extended longevity of worms overexpressing wwp-1. Taken together, our results indicate that WWP-1 and UBC-18 function to ubiquitinate substrates that regulate diet-restriction-induced longevity.

12 megabyte pdf with supplemental information.