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Research Themes Cell biology

iTRAQing the Ubiquitinome

SBKB [doi:10.1038/sbkb.2014.211]
Technical Highlight - July 2014
Short description: An inflammatory signal triggers broad changes to protein ubiquitination and chromatin regulation in immune cells.

Immunofluorescence reveals DBC1 protein degradation in immune cells after inflammatory stimulus, (left) DBC1 in green in the control and (right) following LPS stimulation. Figure courtesy of Joshua Adkins.

Immune cells must respond quickly to invading pathogens. One strategy for rapid regulatory change is to alter post-translational modifications such as ubiquitination across the proteome. Adkins and colleagues (PSI MCSG in partnership with PCSEP) have taken a quantitative proteomics approach to study ubiquitination dynamics in macrophage-like murine cells upon stimulation by bacterial lipopolysaccharide (LPS), a pro-inflammatory elicitor of innate immunity.

The authors used the ubiquitin-binding domain Dsk2-UBA to enrich for modified proteins at four time points within four hours of LPS treatment, corresponding to a window of dynamic regulation assessed by western blot. Proteins were labeled using 8-plex isobaric tags for relative and absolute quantitation (iTRAQ), followed by fractionation and liquid chromatography–tandem mass spectrometry.

They detected 1,057 proteins enriched for pathways in which ubiquitinated proteins are overrepresented. Seventy-eight of those were differentially abundant between time points, among which was the deubiquitinase ataxin-3. Since ataxin-3 activity is known to be regulated by ubiquitination, the authors examined whether its ubiquitination affected the overall deubiquitinase activity of the cell: ataxin-3 activity increased slightly at four hours, linked to an increase in its own ubiquitination level and a decrease in global protein ubiquitination levels. Treating cells with proteasome inhibitor confirmed that deubiquitinases play an important role alongside protein degradation in regulating ubiquitination levels.

Leveraging prior proteomic and microarray time course data from LPS-treated cells, the researchers found proteins that appeared to be degraded after treatment, identified by a drop in protein but not in gene expression levels. An interesting member of this set is deleted in breast cancer 1 (DBC1), a histone deacetylase inhibitor that has been implicated in inflammation response in mice. Immunofluorescence and western blotting revealed that levels of a nuclear form of DBC1 are reduced after LPS treatment, while a truncated cytoplasm version stays constant. Based on proteasome and caspase inhibitor experiments, the researchers suggest a model linking inflammation to chromatin remodeling: LPS causes caspase processing and ubiquitin-mediated degradation of DBC1, which allows for the deacetylation of an acetylated form of histone 4 (H4K12ac).

This work shows that integrating data from large-scale approaches enables the study of both global regulatory patterns and the specific mechanisms of response to an inflammatory signal.

Tal Nawy

References

  1. E.S. Nakayasu et al. Multi-omic data integration links Deleted in Breast Cancer 1 (DBC1) degradation to chromatin remodeling in inflammatory response.
    Mol. Cell. Proteomics. 12, 2136-47 (2013). doi:10.1074/mcp.M112.026138

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