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Quantitative proteomics identifies the Myb-binding protein p160 as a novel target of the von Hippel-Lindau tumor suppressor.

Lai Y, Qiao M, Song M, Weintraub ST, Shiio Y - PLoS ONE (2011)

Bottom Line: Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL.A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates.The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases.

View Article: PubMed Central - PubMed

Affiliation: Greehey Children's Cancer Research Institute, San Antonio, Texas, United States of America.

ABSTRACT

Background: The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex, which is best understood as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the α subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. However, several lines of evidence suggest that there are unidentified substrates or targets for VHL that play important roles in tumor suppression.

Methodology/principal findings: Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL.

Conclusions/significance: A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates. The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases.

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Related in: MedlinePlus

Outline of proteomic screen for VHL substrates.In cells with functional VHL, VHL(+), VHL substrates are ubiquitinated and degraded by the proteasome. In cells without functional VHL, VHL(-), VHL substrates accumulate. By comparing the global protein expression of VHL(+) and VHL(-) cells by quantitative proteomics, candidate VHL substrates can be identified.
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pone-0016975-g001: Outline of proteomic screen for VHL substrates.In cells with functional VHL, VHL(+), VHL substrates are ubiquitinated and degraded by the proteasome. In cells without functional VHL, VHL(-), VHL substrates accumulate. By comparing the global protein expression of VHL(+) and VHL(-) cells by quantitative proteomics, candidate VHL substrates can be identified.

Mentions: To identify novel ubiquitination substrates of VHL, we undertook a proteomic screening using ICAT (isotope-coded affinity tag) quantitative proteomics technology [16], [25]. Unlike other isotope-labeling proteomics approaches such as SILAC and iTRAQ, the ICAT procedure selects only cysteine-containing peptides (Note that 96% of all human proteins contain at least one cysteine) and thus effectively reduces the complexity of peptide mixtures, allowing sensitive detection of low-abundance proteins. Since the VHL ubiquitin ligase catalyzes the formation of lysine-48-linked poly-ubiquitin chains which target proteins for proteasomal degradation, we reasoned that the VHL substrates would accumulate in cells that do not have functional VHL, which can be detected by comparing the global protein expression in cells with and without functional VHL (Figure 1). Because the ubiquitination and degradation of HIF by VHL can be inhibited by iron chelation, we used iron chelation to inhibit protein ubiquitination by VHL and analyzed the resulting protein expression changes.


Quantitative proteomics identifies the Myb-binding protein p160 as a novel target of the von Hippel-Lindau tumor suppressor.

Lai Y, Qiao M, Song M, Weintraub ST, Shiio Y - PLoS ONE (2011)

Outline of proteomic screen for VHL substrates.In cells with functional VHL, VHL(+), VHL substrates are ubiquitinated and degraded by the proteasome. In cells without functional VHL, VHL(-), VHL substrates accumulate. By comparing the global protein expression of VHL(+) and VHL(-) cells by quantitative proteomics, candidate VHL substrates can be identified.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046137&req=5

pone-0016975-g001: Outline of proteomic screen for VHL substrates.In cells with functional VHL, VHL(+), VHL substrates are ubiquitinated and degraded by the proteasome. In cells without functional VHL, VHL(-), VHL substrates accumulate. By comparing the global protein expression of VHL(+) and VHL(-) cells by quantitative proteomics, candidate VHL substrates can be identified.
Mentions: To identify novel ubiquitination substrates of VHL, we undertook a proteomic screening using ICAT (isotope-coded affinity tag) quantitative proteomics technology [16], [25]. Unlike other isotope-labeling proteomics approaches such as SILAC and iTRAQ, the ICAT procedure selects only cysteine-containing peptides (Note that 96% of all human proteins contain at least one cysteine) and thus effectively reduces the complexity of peptide mixtures, allowing sensitive detection of low-abundance proteins. Since the VHL ubiquitin ligase catalyzes the formation of lysine-48-linked poly-ubiquitin chains which target proteins for proteasomal degradation, we reasoned that the VHL substrates would accumulate in cells that do not have functional VHL, which can be detected by comparing the global protein expression in cells with and without functional VHL (Figure 1). Because the ubiquitination and degradation of HIF by VHL can be inhibited by iron chelation, we used iron chelation to inhibit protein ubiquitination by VHL and analyzed the resulting protein expression changes.

Bottom Line: Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL.A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates.The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases.

View Article: PubMed Central - PubMed

Affiliation: Greehey Children's Cancer Research Institute, San Antonio, Texas, United States of America.

ABSTRACT

Background: The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex, which is best understood as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the α subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. However, several lines of evidence suggest that there are unidentified substrates or targets for VHL that play important roles in tumor suppression.

Methodology/principal findings: Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL.

Conclusions/significance: A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates. The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases.

Show MeSH
Related in: MedlinePlus