ABSTRACT
Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCF(Cyclin F) substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.
Subject(s)
Genome, Human , Proteome/analysis , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Cyclopentanes/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Pyrimidines/pharmacology , Ubiquitin-Protein Ligases/geneticsABSTRACT
The abundance of cellular proteins is determined largely by the rate of transcription and translation coupled with the stability of individual proteins. Although we know a great deal about global transcript abundance, little is known about global protein stability. We present a highly parallel multiplexing strategy to monitor protein turnover on a global scale by coupling flow cytometry with microarray technology to track the stability of individual proteins within a complex mixture. We demonstrated the feasibility of this approach by measuring the stability of approximately 8000 human proteins and identifying proteasome substrates. The technology provides a general platform for proteome-scale analysis of protein turnover under various physiological and disease conditions.
Subject(s)
Proteasome Endopeptidase Complex/metabolism , Protein Stability , Proteins/metabolism , Amino Acids/analysis , Cell Cycle , Cell Line , DNA, Complementary , Flow Cytometry , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/metabolism , Half-Life , Humans , Luminescent Proteins/analysis , Luminescent Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Open Reading Frames , Protein Biosynthesis , Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , Transcription, Genetic , Red Fluorescent ProteinABSTRACT
Ubiquitin-mediated proteolysis regulates all aspects of cellular function, and defects in this process are associated with human diseases. The limited number of identified ubiquitin ligase-substrate pairs is a major bottleneck in the ubiquitin field. We established and applied genetic technologies that combine global protein stability (GPS) profiling and genetic perturbation of E3 activity to screen for substrates of the Skp1-cullin-F-box (SCF) ubiquitin ligase in mammalian cells. Among the >350 potential substrates identified, we found most known SCF targets and many previously unknown substrates involved in cell cycle, apoptosis, and signaling pathways. Exploring cell cycle-stage stability, we found that several substrates used the SCF and other E3s in different cell cycle stages. Our results demonstrate the potential of these technologies as general platforms for the global discovery of E3-substrate regulatory networks.
