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Flexible regions within I{kappa}B{alpha} create the ubiquitin-independent degradation signal.

ABSTRACT: Homeostatic regulation of NF-?B requires the continuous synthesis of I?B? and its rapid degradation by the proteasome through a ubiquitin-independent pathway. We previously showed that the ubiquitin-independent degradation signal of unbound I?B? was located in the C-terminal PEST region, and we have now identified a single tyrosine, Tyr-289, and determined that the hydrophobic character of the tyrosine is important for the rapid turnover of I?B?. The sequence composition of the PEST peptide surrounding this Tyr-289 imposes a distinct polyproline II conformation. Enhancing the polyproline II helix formation correlates with slower degradation rates of unbound I?B?. We have further identified a degradation signal located within the 5th ankyrin repeat that is functional once the C terminus is removed. Both the C-terminal and 5th ankyrin repeat degradation signals have inherent flexibility and specific hydrophobic residue(s), which together constitute the ubiquitin-independent degradation signal for I?B?.

SUBMITTER: Mathes E

PROVIDER: S-EPMC2963393 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Flexible regions within I{kappa}B{alpha} create the ubiquitin-independent degradation signal.

Mathes Erika E Wang Lily L Komives Elizabeth E Ghosh Gourisankar G

The Journal of biological chemistry 20100803 43

Homeostatic regulation of NF-κB requires the continuous synthesis of IκBα and its rapid degradation by the proteasome through a ubiquitin-independent pathway. We previously showed that the ubiquitin-independent degradation signal of unbound IκBα was located in the C-terminal PEST region, and we have now identified a single tyrosine, Tyr-289, and determined that the hydrophobic character of the tyrosine is important for the rapid turnover of IκBα. The sequence composition of the PEST peptide surr ...[more]

PMID: 20682784

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Project description:Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases. Apomyoglobin emerges as a new model substrate to further explore the role of ATPases and protein structure in proteasomal degradation.

Dataset Information

Flexible regions within I{kappa}B{alpha} create the ubiquitin-independent degradation signal.

Publications

Flexible regions within I{kappa}B{alpha} create the ubiquitin-independent degradation signal.

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