TitleIn vivo study of the ubiquitin-proteasome system in C.elegans
NameLiu, Gang (author), Barr, Maureen (chair), Rongo, Chris (internal member), Wadsworth, William (internal member), Grant, Barth (outside member), Rutgers University, Graduate School - New Brunswick,
DescriptionThe Ubiquitin-Proteasome System (UPS) is an important regulatory mechanism used to control acute protein turnover and scavenge misfolded proteins in numerous cellular processes. (CM Pickart etc 2001). Indeed, the failure of the UPS to remove misfolded proteins is believed to contribute to aging as well as several neurodegenerative disorders. Although recent studies have explored the biochemical function and protein-protein interactions of the UPS, regulated changes in UPS activity have been difficult to observe in vivo in specific tissues as animals develop and age. Here we have employed the GFP reporter UbG76V-GFP, which was developed for monitoring UPS activity (N. P. Dantuma, K etc, 2000), to study the tissue- and age-specific regulation of proteolysis in C. elegans. The UbG76V-GFP reporter contains a mutated ubiquitin fused to GFP, and is a substrate for polyubiquitination and degradation by the 26S proteasome. Here we employ this reporter as a substrate for degradation by UPS activity to study the regulation of proteolysis in intact C. elegans animals. Mutations in the ubiquitin moiety that prevent K48 and K63 polyubiquitin chain addition block turnover of the reporter. Mutations and RNAi treatment that reduce 26S Proteasome activity also block reporter turnover, confirming the in vivo functionality of this UPS reporter. With this reporter we identify several different regulators of the UPS activity. The UFD pathway, including the E3 ligase CHN-1 and the E4 enzyme UFD-2, as well as a new E3 ligase, HECD-1, are required for maintaining UPS activity iii in maturing adults. Mutations that reduce UFD gene activity do not appear to grossly affect larval health, but greatly shorten the lifespan of adult nematodes, suggesting that the UPS activity is critical for long-term viability. We also find that EGF signaling through the Ras-MAPK pathway and the transcription factors EOR-1 and EOR-2 is critical for maintaining UPS activity in adult worms. Animals that fail to maintain their UPS activity at midlife via EGF signaling have a shortened lifespan, whereas animals with over-activated EGF signaling have an increased lifespan. Taken together, our results suggest that the UFD pathway and EGF signaling maintain long-term protein homeostasis by regulating UPS activity in anticipation of aging. We also apply microarray analysis to identify genes that are regulated by the EGF signaling and we find that several F-box proteins and a Skp1 ortholog, which are E3 components, are up-regulated by EGF signaling. Finally, we performed an RNAi library screen for modulators of UPS activity, and we also found that knocking down F-box proteins can suppress the UPS activity. We also tested whether worms had activated their oxidative stress response using the gcs-1::GFP reporter, and we observed accumulation of GFP in the tail and intestine of both hecd-1 and EFG signaling mutant worms. Thus we reach the conclusion the UPS activity might affect life span by regulating oxidative response in worms.
NoteIncludes bibliographical references
Noteby Gang Liu
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.