The tumor suppressor protein p16(INK4a) and the human papillomavirus oncoprotein-58 E7 are naturally occurring lysine-less proteins that are degraded by the ubiquitin system. Direct evidence for ubiquitination at the N-terminal residue.
Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16(INK4a). For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16(INK4a) is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes.
Ben-Saadon
Ronen
R
Cancer and Vascular Biology Research Center, The Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Efron Street, Bat Galim, P. O. Box 9649, Haifa 31096, Israel.
Fajerman
Ifat
I
Ziv
Tamar
T
Hellman
Ulf
U
Schwartz
Alan L
AL
Ciechanover
Aaron
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D002452:Cell Count; D002455:Cell Division; D002460:Cell Line; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008239:Lysine; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000378:metabolism*; D027383:Papillomaviridae; D050725:Papillomavirus E7 Proteins; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
D019941:Cyclin-Dependent Kinase Inhibitor p16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D025801:Ubiquitin; C059731:oncogene protein E7, Human papillomavirus type 16; D008239:Lysine