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Closer scrutiny of Nedd8, an accessory to protein murder
A QSTAR XL instrument, which has high mass accuracy and resolution, and Ubiquitin is the small but powerful regan LTQ mass spectrometer, which offers ulatory protein that all other proteins high sensitivity, were used. Huang says, fear to meet in the dark alleyways of a “The use of both instruments provided cell. Once a protein has been marked us with complementary as well as for death by the covalent attachment of overlapping results, which allowed us ubiquitin, it usually isn’t long before the to identify a larger number of proteins condemned protein is looking down the with high confidence.” barrel of a proteasome. The researchers identified a total of Though similar in some ways, ubiq496 Nedd8-modified or -associated prouitin-like (Ubl) proteins such as SUMO teins, including all eight cullin family and Nedd8 have not been studied as members. The neddylation sites in five extensively as their more notorious of the cullins were determined by pepcousin. To identify novel tide sequencing. Other Nedd8-modified and enzymes and regulators -associated proteins, Lan of the Nedd8 and ubiq76 RGG K* (a) NH2 Signature peptide Huang and co-workers at uitin pathways also were Trypsin GG K* Nedd8 COOH the University of Califordetected by MS/MS. PreNH2 Substrate nia Irvine and the Mount viously unknown Nedd8GG GG Sinai School of Medicine associated proteins were TLTG(K11)*EIEIDIEPTDK EIEIDIEPTD(K22)*VER RGG76 (b) NH2 Lys11 chain Lys22 chain Nedd8 76 conducted a proteomics discovered in the analyNH2 K* RGG Trypsin GG GG Nedd8 analysis of affinity-purisis, including proteins LIYSG(K48)*QMNDEK TAADY(K60)*ILGGSVLHLVLALR fied Nedd8 substrates and involved in the cell cycle Lys48 chain Lys60 chain interacting proteins. The and signal transduction. work is described in JPR Some of the Nedd8 interDetermination of neddylation sites. (a) Identification of neddylation sites in (2008, 7, 1274−1287). actions were confirmed Nedd8-modified substrates. GG (blue) is the remnant of neddylation left after The ~9 kDa Nedd8 by immunoblotting. tryptic digestion. (b) Identification of neddylation sites in Nedd8 itself (polynedprotein, which is 80% Unexpectedly, Huang dylation). and co-workers deter homologous to ubiquitin, is attached to substrates mined that Nedd8 in a process called neddylation. As with lines by affinity purification of Nedd8 can form chains in vivo, primarily by ubiquitination, neddylation requires a followed by MS/MS. “After many failed covalent attachment of the carboxyl Nedd8-specific E1 activating enzyme, attempts to purify Nedd8-modified proterminus of one Nedd8 molecule to the an E2 conjugating enzyme, and an E3 teins from cultured cells, we realized K48 residue of another Nedd8. Although ligase to link a lysine residue of the subthat the study of Nedd8 modification ubiquitin is known to form chains strate protein to the carboxyl terminus presents distinct challenges compared that function differently from single (Gly76) of Nedd8. Unlike most instances with studies of ubiquitination or sumoubiquitin conjugates, polyneddylation of ubiquitination, neddylation does not ylation,” says Huang. “This is mainly had never before been demonstrated directly target substrates for proteadue to the low abundance of neddylated in cells. The biological significance of somal degradation but instead modusubstrates in cells.” The researchers these chains remains to be determined. lates their biochemical activities. overcame the difficulties in sample In future studies, Huang hopes to Highly conserved among eukaryotes, preparation by expressing a glutathione identify differences in Nedd8-modified Nedd8 is essential for fission-yeast viS-transferase (GST)-tagged precursor proteins between normal and cancerability and embryonic development in form of Nedd8 (GST-proNedd8), which ous prostate cells. “We want to underanimals. Interestingly, Nedd8 is downis processed in a stable HEK293 cell line stand how the Nedd8 pathway plays a regulated in prostate cancer. According to yield mature GST-Nedd8. role in cell proliferation and tumorito Huang, “Despite the importance of After large-scale affinity purification genesis in prostate cancer,” she says. “I the Nedd8 pathway in the regulation of GST-Nedd8 with a glutathione–Sephthink our research in that direction will of the cell cycle, embryogenesis, and arose column, Huang and co-workers help reveal new insights not only into possibly tumorigenesis, the number separated the purified proteins by 1DE. neddylation-associated proteolysis but of known Nedd8 substrates was very To isolate low-abundance proteins, the also neddylation-associated transcriplimited.” Until recently, the cullin famresearchers excised individual protein tional regulation.” ily of proteins, which are components bands for in-gel digestion with trypsin. —Laura Cassiday LAN HUANG
of SCF or SCF-like E3 ubiquitin ligases, were the only known Nedd8 substrates. Although Nedd8 does not directly target proteins for degradation, it can act as an “accessory to murder” via the neddylation of cullins; this stimulates the activity of the cullin-containing E3 ubiquitin ligases. Nedd8 also modulates the activities of proteins that are not involved in the ubiquitin pathway. To gain a better understanding of Nedd8 function, Huang and co-workers identified Nedd8-modified and -associated proteins in mammalian cell
838 Journal of Proteome Research • Vol. 7, No. 3, 2008
© 2008 American Chemical Society
