NBE5

Amino Acid Sequence

3D Structure (/structures/AF-Q14145-F1-model_v6.cif)

Function

Substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase complex that regulates the response to oxidative stress by targeting NFE2L2/NRF2 for ubiquitination. KEAP1 acts as a key sensor of oxidative and electrophilic stress: in normal conditions, the BCR(KEAP1) complex mediates ubiquitination and degradation of NFE2L2/NRF2, a transcription factor regulating expression of many cytoprotective genes. In response to oxidative stress, different electrophile metabolites trigger non-enzymatic covalent modifications of highly reactive cysteine residues in KEAP1, leading to inactivate the ubiquitin ligase activity of the BCR(KEAP1) complex, promoting NFE2L2/NRF2 nuclear accumulation and expression of phase II detoxifying enzymes. In response to selective autophagy, KEAP1 is sequestered in inclusion bodies following its interaction with SQSTM1/p62, leading to inactivation of the BCR(KEAP1) complex and activation of NFE2L2/NRF2. The BCR(KEAP1) complex also mediates ubiquitination of SQSTM1/p62, increasing SQSTM1/p62 sequestering activity and degradation. The BCR(KEAP1) complex also targets BPTF and PGAM5 for ubiquitination and degradation by the proteasome

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

To investigate the degradation mechanism mediated by NBE5, a series of rescue experiments were carried out with the UPS inhibitors MG132 (proteasome inhibitor), MLN4924 (NEDD8-activating enzyme inhibitor), lysosomal inhibitor chloroquine (CQ), and CDDO (competing with NBE5). As shown in Fig. 4A-B, the Keap1 degradation was significantly suppressed by pre-treatment with UPS inhibitors MG132, MLN4924, and CDDO, which demonstrated that the Keap1 degradation mediated by NBE5 was dependent on the UPS and the ability of binding to Keap1. It is well-known that Hsp90 plays a critical role in the degradation of misfolded proteins via the ubiquitin-proteasome system (UPS) and chaperone-mediated autophagy (CMA) [25,26]. Therefore, co-immunoprecipitation (co-IP) was per formed to examine whether the chaperonin Hsp90 was involved in the Keap1 degradation mediated by NBE5. As expected, an enhanced interaction between Keap1 and Hsp90 was detected after NBE5 treat ment in HCT116 cells (Fig. 4F).

Mechanism

The autophagy-lysosomal pathway (ALP) is capable of degrading a broader range of substrates, including longer-lived and larger misfolded proteins and damaged organelles. ALP is classified into three subtypes: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, substrates are directly sequestered and degraded by lysosomes and late endosomes via membrane protrusion and invagination. In CMA, HSPA8/HSC70 recognizes specific substrates containing KFERQ-like sequences; these substrates subsequently bind to LAMP2A on the lysosomal surface and are delivered into lysosomes for degradation. In macroautophagy, substrates are encapsulated through the formation of double-membrane autophagosomes, which subsequently fuse with lysosomes to degrade the substrates.

Description

In addition, CQ could also block the Keap1 degradation, which suggested that the degradation of Keap1 by NBE5 may also partly rely on the autophagy-lysosome system. To further demonstrate the relationship between NBE5-mediated Keap1 degradation and the autophagy-lysosome system, the v-ATPase inhibitor bafilomycin A1 inactivating lysosomal hydrolases and the mTOR inhibitor torin 1 as an autophagy inducer were employed [24]. As shown in Fig. 4C-D, the inhibition of lysosomal activity could also decrease the degradation of Keap1 induced by NBE5. It is well-known that Hsp90 plays a critical role in the degradation of misfolded proteins via the ubiquitin-proteasome system (UPS) and chaperone-mediated autophagy (CMA) [25,26]. Therefore, co-immunoprecipitation (co-IP) was per formed to examine whether the chaperonin Hsp90 was involved in the Keap1 degradation mediated by NBE5. As expected, an enhanced interaction between Keap1 and Hsp90 was detected after NBE5 treatment in HCT116 cells (Fig. 4F).

Description

To further evaluate the Keap1 degradation efficacy of NBE5, HCT116 and RKO cells were treated with NBE5 at a wider range of concentrations from 0.1 to 10 μМ for 12 h. DC50 was described as the concentration for inducing half degradation and Dmax was the maximal degradation ratio of Keap1. The DC50 of NBE5 mediated Keap1 degradation was 0.82 μМ and 1.72 μМ in HCT116 and RKO cells, respectively, while the Dmax at 5.0 μМ was 82.7% and 72.2% in the cells.

Description

To further evaluate the Keap1 degradation efficacy of NBE5, HCT116 and RKO cells were treated with NBE5 at a wider range of concentrations from 0.1 to 10 μМ for 12 h. DC50 was described as the concentration for inducing half degradation and Dmax was the maximal degradation ratio of Keap1. The DC50 of NBE5 mediated Keap1 degradation was 0.82 μМ and 1.72 μМ in HCT116 and RKO cells, respectively, while the Dmax at 5.0 μМ was 82.7% and 72.2% in the cells.

Description

To further evaluate the Keap1 degradation efficacy of NBE5, HCT116 and RKO cells were treated with NBE5 at a wider range of concentrations from 0.1 to 10 μМ for 12 h. DC50 was described as the concentration for inducing half degradation and Dmax was the maximal degradation ratio of Keap1. The DC50 of NBE5 mediated Keap1 degradation was 0.82 μМ and 1.72 μМ in HCT116 and RKO cells, respectively, while the Dmax at 5.0 μМ was 82.7% and 72.2% in the cells.

Description

To further evaluate the Keap1 degradation efficacy of NBE5, HCT116 and RKO cells were treated with NBE5 at a wider range of concentrations from 0.1 to 10 μМ for 12 h. DC50 was described as the concentration for inducing half degradation and Dmax was the maximal degradation ratio of Keap1. The DC50 of NBE5 mediated Keap1 degradation was 0.82 μМ and 1.72 μМ in HCT116 and RKO cells, respectively, while the Dmax at 5.0 μМ was 82.7% and 72.2% in the cells.