A Regulated, Ubiquitin-Independent Degron in IκBα

Abstract

Whereas ubiquitin-dependent degrons have been characterized in some detail, how proteins may be targeted to ubiquitin-independent proteasomal degradation remains unclear. Here we show that IκBα contains a ubiquitin-independent degron whose activity is portable to heterologous proteins such as the globular protein GFP via a proteasome-dependent, ubiquitin-independent, non-lysosomal pathway. The ubiquitin-independent degradation signal resides in a 11 amino acid sequence, which is not only sufficient but also required for IκBα’s short half-life. Finally, we show that this degron’s activity is regulated by the interaction with NFκB, which controls its solvent exposure, and we demonstrate that this regulation of the degron’s activity is critical for IκBα’s signaling functions.

An NFκB Activity Calculator to Delineate Signaling Crosstalk: Type I and II Interferons Enhance NFκB via Distinct Mechanisms

“…As two out of the three reactions represented in SiMoN were found unaffected by IFNγ we tested the third, the degradation rate of unbound IκBα. Whereas, NFκB-bound IκBα is degraded through IKK-mediated phosphorylation and the ubiquitin-proteasome system, free IκBα is degraded independently of IKK activity through a ubiquitin-independent, but 20S proteasome-dependent mechanism (65, 66). To determine whether IFNγ affects the stability of free IκBα, we employed MEFs deficient in the NFκB proteins RelA, cRel, and p50 (termed “ nfk b −/− ”) in which all IκBα is in fact free, a previously established assay system for free IκBα turnover (51): nfk b −/− cells were treated with IFNγ, and IκBα levels were measured by Western blotting.…”

“…SiMoN predicts the molecular mechanism need not be poly(I:C) specific as selective amplification of weak NFκB activators can emerge through the kinetics of non-specific increased degradation of free IκBα. Whereas, ubiquitinated proteins are recognized and degraded by the 26S proteasome, which consists of the 20S barrel-shaped core and a 19S regulatory cap, free IκBα was shown to be degraded in a ubiquitin-independent manner (65). An alternative 11S regulatory cap, consisting of oligomers of the PA28α and PA28β proteins allows for ubiquitin-independent entry into the proteasome and has been implicated in antigen processing in antigen-presenting cells (66, 67).…”

The Atypical Ubiquitin E2 Conjugase UBE2L3 Is an Indirect Caspase-1 Target and Controls IL-1β Secretion by Inflammasomes

“…Further, the homeostatic turnover of non-ubiquitylatable UBE2L3 (UBE2L3 18R variant) was also proteasome-dependent. Interestingly, ubiquitin-independent degradation has been reported for several common proteins such as IκBε ( Xu et al., 2016 ), IκBα ( Fortmann et al., 2015 ), p21 ( Erales and Coffino, 2014 ), p53 ( Tsvetkov et al., 2010 ), and myelin basic protein ( Belogurov et al., 2015 ), among others ( Ben-Nissan and Sharon, 2014 , Inobe and Matouschek, 2014 ). Local disordered regions can target proteins for degradation by 20S proteasomes independently of their ubiquitylation and this can rely on mechanisms such as alternative proteasome subunits or regulatory particles such as the PA28α/β ( Ben-Nissan and Sharon, 2014 ).…”

Functional importance of stripping in NFκB signaling revealed by a stripping-impaired IκBα mutant

“…1B). The similarity of the IκBα PEST sequence to other negatively charged PEST sequences first suggested its involvement in degradation (14); however, more recent studies have not supported these claims and instead revealed degradation signals in the AR domain (15,16). NMR studies showed that the PEST sequence interacts with positively charged residues in the DNA-binding pocket of NFκB (17), and simulations using a coarse-grained model showed that the IκBα PEST sequence electrostatically repels DNA from NFκB (8).…”