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Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast.

Gupta R, Kus B, Fladd C, Wasmuth J, Tonikian R, Sidhu S, Krogan NJ, Parkinson J, Rotin D - Mol. Syst. Biol. (2007)

Bottom Line: Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase.Our enzymatic approach was complemented by a parallel protein microarray protein interaction study.Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets.

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Phage display logos. Peptides identified as substrates of Rsp5, using the phage display system, were aligned and are graphically displayed as logos, as shown in Figure 3 above.
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f4: Phage display logos. Peptides identified as substrates of Rsp5, using the phage display system, were aligned and are graphically displayed as logos, as shown in Figure 3 above.

Mentions: To further confirm these findings, we performed a modified phage display screen to explore substrate specificity of each of the three WW domains of Rsp5. All peptides identified through this screen (over 300) were found to contain a PY motif. Consistent with data presented above, Ser and Ala were both found to be preferred at the third position (Figure 4). More interestingly, the most common amino-acid residue associated with the third position was Pro, suggesting an important biological role for this residue.


Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast.

Gupta R, Kus B, Fladd C, Wasmuth J, Tonikian R, Sidhu S, Krogan NJ, Parkinson J, Rotin D - Mol. Syst. Biol. (2007)

Phage display logos. Peptides identified as substrates of Rsp5, using the phage display system, were aligned and are graphically displayed as logos, as shown in Figure 3 above.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1911201&req=5

f4: Phage display logos. Peptides identified as substrates of Rsp5, using the phage display system, were aligned and are graphically displayed as logos, as shown in Figure 3 above.
Mentions: To further confirm these findings, we performed a modified phage display screen to explore substrate specificity of each of the three WW domains of Rsp5. All peptides identified through this screen (over 300) were found to contain a PY motif. Consistent with data presented above, Ser and Ala were both found to be preferred at the third position (Figure 4). More interestingly, the most common amino-acid residue associated with the third position was Pro, suggesting an important biological role for this residue.

Bottom Line: Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase.Our enzymatic approach was complemented by a parallel protein microarray protein interaction study.Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets.

Show MeSH