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Reference: Sims JJ, et al. (2012) Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling.LID - 10.1038/nmeth.1888 [doi] Nat Methods

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Abstract


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-kappaB activation in several cell types, inferring the essential role of Lys63 polyubiquitin for signaling via the IL-1beta and TNF-related weak inducer of apoptosis (TWEAK) but not TNF-alpha 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.

Reference Type
Journal Article
Authors
Sims JJ, Scavone F, Cooper EM, Kane LA, Youle RJ, Boeke JD, Cohen RE
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