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Large-scale detection of ubiquitination substrates using cell extracts and protein microarrays.

ABSTRACT: Identification of protein targets of post-translational modification is an important analytical problem in biology. Protein microarrays exposed to cellular extracts could offer a rapid and convenient means of identifying modified proteins, but this kind of biochemical assay, unlike DNA microarrays, depends on a faithful reconstruction of in vivo conditions. Over several years, concentrated cellular extracts have been developed, principally for cell cycle studies that reproduce very complex cellular states. We have used extracts that replicate the mitotic checkpoint and anaphase release to identify differentially regulated poyubiquitination. Protein microarrays were exposed to these complex extracts, and the polyubiquitinated products were detected by specific antibodies. We expected that among the substrates revealed by the microarray should be substrates of the anaphase promoting complex (APC). Among 8,000 proteins on the chip, 10% were polyubiquitinated. Among those, we found 11 known APC substrates (out of 16 present on the chip) to be polyubiquitinated. Interestingly, only 1.5% of the proteins were differentially ubiquitinated on exit from the checkpoint. When we arbitrarily chose 6 proteins thought to be involved in mitosis from the group of differentially modified proteins, all registered as putative substrates of the APC, and among 4 arbitrarily chosen non-mitotic proteins picked from the same list, 2 were ubiquitinated in an APC-dependent manner. The striking yield of potential APC substrates from a simple assay with concentrated cell extracts suggests that combining microarray analysis of the products of post-translational modifications with extracts that preserve the physiological state of the cell can yield information on protein modification under various in vivo conditions.

SUBMITTER: Merbl Y

PROVIDER: S-EPMC2633216 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia coli S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to E. coli, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize in vitro translation for protein microarray assembly. RESULTS:Self-assembling autofluorescent protein microarrays were produced by in situ transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when in vitro translation was performed with E. coli S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid in vitro translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested. CONCLUSION:The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the in vitro system by providing eukaryotic ribosomes and chaperones and, at the same time, the E. coli S30 extract, which includes an ATP regeneration system, translates the polypeptides at high rates. This hybrid cell-free expression system allows the facile production of high-yield protein arrays suitable for downstream assays.

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Large-scale detection of ubiquitination substrates using cell extracts and protein microarrays.

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