Extensive remodeling of the ubiquitination landscape during aging in C. elegans

Abstract

In previous work, we investigated ubiquitination changes during aging in C. elegans. We identified 2,163 peptides undergoing age-related ubiquitination changes in wild-type animals, corresponding to 1,050 proteins. While many lysine sites had increased ubiquitination with age, a larger number exhibited decreased ubiquitination. Longevity pathways, such as reduced insulin signaling and dietary restriction, prevented ubiquitination changes. Treatment with a broad-spectrum inhibitor of deubiquitinating enzymes (DUBs) or knockdown of specific DUBs ameliorated ubiquitination loss in old worms. Moreover, we identified proteins that accumulate with aging due to reduced ubiquitination and subsequent proteasomal degradation. Our conclusions were supported by multiple approaches, including ubiquitin and total proteomics, western blot, and ubiquitin-less mutations. Concerns were raised by a laboratory regarding the possibility of our protocol omitting insoluble proteins due to a centrifugation step after protein extraction and solubilization. To address these concerns, we have focused on proteins previously reported to become insoluble with aging in C. elegans. Our proteomics experiments successfully detected and quantified all these proteins in old worms. In many cases, the proteins that become insoluble with aging did not change or even increased at the total levels. However, they often exhibited ubiquitination changes, primarily a loss of ubiquitination. Independent work combining analysis of conformational changes with our datasets demonstrated that 92% of the age-dependent metastable proteins exhibit differential ubiquitination during aging. In addition, we performed experiments to confirm that the buffers used for proteomics and western blot efficiently solubilize most of the proteome. Importantly, the analysis of total homogenates combining clear lysates and the remaining debris after protein extraction also revealed decreased ubiquitination during aging, whereas DUB inhibitor treatment (4 h) in old worms restored ubiquitination levels. These data further support our previous conclusions regarding extensive ubiquitination changes during aging in C. elegans.