Proteome-wide Identification of Ubiquitylation Sites by Conjugation of Engineered Lysine-less Ubiquitin

Ubiquitin conjugation (ubiquitylation) plays important roles not only in protein degradation but also in many other cellular functions. However, the sites of proteins that are targeted for such modification have remained poorly characterized at the proteomic level. We have now developed a method for the efficient identification of ubiquitylation sites in target proteins with the use of an engineered form of ubiquitin (K0-Ub), in which all seven lysine residues are replaced with arginine. K0-Ub is covalently attached to lysine residues of target proteins via an isopeptide bond, but further formation of a polyubiquitin chain does not occur on K0-Ub. We identified a total of 1392 ubiquitylation sites of 794 proteins from HEK293T cells. Profiling of ubiquitylation sites indicated that the sequences surrounding lysine residues targeted for ubiquitin conjugation do not share a common motif or structural feature. Furthermore, we identified a critical ubiquitylation site of the cyclin-dependent kinase inhibitor p27^<i>Kip1</i>. Mutation of this site thus inhibited ubiquitylation of and stabilized p27^<i>Kip1</i>, suggesting that this lysine residue is the target site of p27^<i>Kip1</i> for ubiquitin conjugation in vivo. In conclusion, our method based on K0-Ub is a powerful tool for proteome-wide identification of ubiquitylation sites of target proteins

Proteome-wide Identification of Ubiquitylation Sites by Conjugation of Engineered Lysine-less Ubiquitin

Ubiquitin conjugation (ubiquitylation) plays important roles not only in protein degradation but also in many other cellular functions. However, the sites of proteins that are targeted for such modification have remained poorly characterized at the proteomic level. We have now developed a method for the efficient identification of ubiquitylation sites in target proteins with the use of an engineered form of ubiquitin (K0-Ub), in which all seven lysine residues are replaced with arginine. K0-Ub is covalently attached to lysine residues of target proteins via an isopeptide bond, but further formation of a polyubiquitin chain does not occur on K0-Ub. We identified a total of 1392 ubiquitylation sites of 794 proteins from HEK293T cells. Profiling of ubiquitylation sites indicated that the sequences surrounding lysine residues targeted for ubiquitin conjugation do not share a common motif or structural feature. Furthermore, we identified a critical ubiquitylation site of the cyclin-dependent kinase inhibitor p27^<i>Kip1</i>. Mutation of this site thus inhibited ubiquitylation of and stabilized p27^<i>Kip1</i>, suggesting that this lysine residue is the target site of p27^<i>Kip1</i> for ubiquitin conjugation in vivo. In conclusion, our method based on K0-Ub is a powerful tool for proteome-wide identification of ubiquitylation sites of target proteins

Comprehensive Identification of Substrates for F-box Proteins by Differential Proteomics Analysis

Although elucidation of enzyme–substrate relations is fundamental to the advancement of biology, universal approaches to the identification of substrates for a given enzyme have not been established. It is especially difficult to identify substrates for ubiquitin ligases, given that most such substrates are immediately ubiquitylated and degraded as a result of their association with the enzyme. We here describe the development of a new approach, DiPIUS (differential proteomics-based identification of ubiquitylation substrates), to the discovery of substrates for ubiquitin ligases. We applied DiPIUS to Fbxw7α, Skp2, and Fbxl5, three of the most well-characterized F-box proteins, and identified candidate substrates including previously known targets. DiPIUS is thus a powerful tool for unbiased and comprehensive screening for substrates of ubiquitin ligases

Comprehensive Identification of Substrates for F-box Proteins by Differential Proteomics Analysis

Although elucidation of enzyme–substrate relations is fundamental to the advancement of biology, universal approaches to the identification of substrates for a given enzyme have not been established. It is especially difficult to identify substrates for ubiquitin ligases, given that most such substrates are immediately ubiquitylated and degraded as a result of their association with the enzyme. We here describe the development of a new approach, DiPIUS (differential proteomics-based identification of ubiquitylation substrates), to the discovery of substrates for ubiquitin ligases. We applied DiPIUS to Fbxw7α, Skp2, and Fbxl5, three of the most well-characterized F-box proteins, and identified candidate substrates including previously known targets. DiPIUS is thus a powerful tool for unbiased and comprehensive screening for substrates of ubiquitin ligases

Comprehensive Identification of Substrates for F-box Proteins by Differential Proteomics Analysis

Although elucidation of enzyme–substrate relations is fundamental to the advancement of biology, universal approaches to the identification of substrates for a given enzyme have not been established. It is especially difficult to identify substrates for ubiquitin ligases, given that most such substrates are immediately ubiquitylated and degraded as a result of their association with the enzyme. We here describe the development of a new approach, DiPIUS (differential proteomics-based identification of ubiquitylation substrates), to the discovery of substrates for ubiquitin ligases. We applied DiPIUS to Fbxw7α, Skp2, and Fbxl5, three of the most well-characterized F-box proteins, and identified candidate substrates including previously known targets. DiPIUS is thus a powerful tool for unbiased and comprehensive screening for substrates of ubiquitin ligases

Numbers of TUNEL-positive apoptotic tubular epithelial and interstitial cells in UUO kidneys.

<p>(<b>A–E</b>) Sections from CLK kidneys in WT mice (<b>A</b>) and UUO kidneys in WT (<b>B</b>), Skp2^−/− (<b>C</b>), Skp2^−/−p27^−/− (<b>D</b>) and p27^−/− (<b>E</b>) mice were subjected to TUNEL staining, scale bars: 50 µm. (<b>F, G</b>) The numbers of TUNEL-positive tubular epithelial cells (<b>F</b>) and TUNEL-positive interstitial cells (<b>G</b>) in the UUO kidneys were counted in the mice of each genotype. The CLK kidneys in WT mice were evaluated as controls. *<i>P</i><0.05, **<i>P</i><0.01 versus WT CLK kidneys, ^#<i>P</i><0.05, ^##<i>P</i><0.01 versus WT UUO kidneys and ^†<i>P</i><0.01 versus Skp2^−/− UUO kidneys.</p

Immunoblot analyses of the previously reported Skp2 target proteins in UUO kidneys.

<p>The levels of the previously reported Skp2 target proteins were detected by western blot analysis in the obstructed kidneys in WT (Skp2^+/+) and Skp2^−/− mice at 7 days after UUO. α-tubulin was evaluated as an internal control.</p

Levels of UUO renal injury.

<p>(<b>A</b>) Representative microscopic images of CLK and UUO kidneys in WT, Skp2^−/−, Skp2^−/−p27^−/− and p27^−/− mice (Masson's trichrome staining; scale bars: 50 µm). Increases in the interstitial area, tubular dilatation and atrophy, and interstitial cell infiltration are observed in the UUO kidneys in WT mice. However, the severities of these lesions are markedly less in the UUO kidneys in Skp2<b>^−/−</b> mice. In contrast, aggravation of the UUO renal injury is noted in Skp2^−/−p27^−/− and p27^−/− mice. (<b>B</b>) The severity of fibrotic tubulointerstitial lesions was graded semiquantitatively as follows: 0, absent (0%); 1, weak (≤10%); 2, mild (>10 to ≤30%); 3, moderate (>30 to ≤50%); 4, strong (>50%) in WT, Skp2^−/−, Skp2^−/−p27^−/− and p27^−/− mice at 7 days after UUO. (<b>C–E</b>) The numbers of dilated tubules (<b>C</b>), renal tubular epithelial cells in a tubule (<b>D</b>), and tubular interstitial cells (<b>E</b>) were counted and evaluated statistically in the UUO kidneys in WT, Skp2^−/−, Skp2^−/−p27^−/− and p27^−/− mice at 7 days after UUO. The CLK kidneys in WT mice were evaluated as controls. *<i>P</i><0.05, **<i>P</i><0.01 versus WT CLK kidneys, ^#<i>P</i><0.05, ^##<i>P</i><0.01 versus WT UUO kidneys and ^†<i>P</i><0.05, ^††<i>P</i><0.01 versus Skp2^−/− UUO kidneys.</p

Levels of tubulointerstitial fibrotic lesions.

<p>(<b>A</b>) Representative images of UUO kidney sections in WT, Skp2^−/−, Skp2^−/−p27^−/− and p27^−/− mice immunostained for type I collagen (top panels), α-SMA (middle panels) and F4/80 (bottom panels) at 7 days after UUO, scale bars: 50 µm. (<b>B</b>) The ratios of the interstitial area in the UUO kidneys were quantified using the sections stained for type I collagen. (<b>C, D</b>) The intensities of the α-SMA (<b>C</b>) and F4/80 (<b>D</b>) immunostaining were quantified using Scion Image software. The CLK kidneys in WT mice were evaluated as controls. *<i>P</i><0.01 versus WT CLK kidneys, ^#<i>P</i><0.01 versus WT UUO kidneys and ^†<i>P</i><0.05, ^††<i>P</i><0.01 versus Skp2^−/− UUO kidneys.</p

Numbers of Ki67-positive proliferative tubular epithelial and interstitial cells in UUO kidneys.

<p>(<b>A–E</b>) Sections of CLK kidneys in WT mice (<b>A</b>) and UUO kidneys in WT (<b>B</b>), Skp2^−/− (<b>C</b>), Skp2^−/−p27^−/− (<b>D</b>) and p27^−/− (<b>E</b>) mice were subjected to immunostaining with an anti-Ki67 antibody, scale bars: 50 µm (<b>F, G</b>) The numbers of Ki67-positive tubular epithelial cells (<b>F</b>) and Ki67-positive interstitial cells (<b>G</b>) in the UUO kidneys were counted in the mice of each genotype. The CLK kidneys in WT mice were evaluated as controls. *<i>P</i><0.05, **<i>P</i><0.01 versus WT CLK kidneys, ^#<i>P</i><0.05 versus WT UUO kidneys and ^†<i>P</i><0.05, ^††<i>P</i><0.01 versus Skp2^−/− UUO kidneys.</p