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Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling.

Nature methods | Mar 29, 2012

http://www.ncbi.nlm.nih.gov/pubmed/22306808

Polyubiquitin chain topology is thought to direct modified substrates to specific fates, but this function-topology relationship is poorly understood, as are the dynamics and subcellular locations of specific polyubiquitin signals. Experimental access to these questions has been limited because linkage-specific inhibitors and in vivo sensors have been unavailable. Here we present a general strategy to track linkage-specific polyubiquitin signals in yeast and mammalian cells, and to probe their functions. We designed several high-affinity Lys63 polyubiquitin-binding proteins and demonstrate their specificity in vitro and in cells. We apply these tools as competitive inhibitors to dissect the polyubiquitin-linkage dependence of NF-κB activation in several cell types, inferring the essential role of Lys63 polyubiquitin for signaling via the IL-1β and TNF-related weak inducer of apoptosis (TWEAK) but not TNF-α receptors. We anticipate live-cell imaging, proteomic and biochemical applications for these tools and extension of the design strategy to other polymeric ubiquitin-like protein modifications.

Pubmed ID: 22306808 RIS Download

Mesh terms: Animals | Binding Sites | Humans | Molecular Probe Techniques | Protein Binding | Protein Interaction Mapping | Signal Transduction | Ubiquitin

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Associated grants

  • Agency: NIGMS NIH HHS, Id: 1R01 GM097452
  • Agency: NCI NIH HHS, Id: P01 CA139980
  • Agency: NIGMS NIH HHS, Id: R01 GM097452
  • Agency: NIGMS NIH HHS, Id: R01 GM097452-01
  • Agency: NIGMS NIH HHS, Id: RC1 GM091424
  • Agency: NIGMS NIH HHS, Id: RC1 GM091424
  • Agency: NIGMS NIH HHS, Id: RC1 GM091424-01
  • Agency: NIGMS NIH HHS, Id: RC1 GM091424-02
  • Agency: NCRR NIH HHS, Id: RR020839
  • Agency: Intramural NIH HHS, Id:

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