NFE2L2 binds KEAP1:NEDD8-CUL3:RBX1

Under the basal resting conditions, cytosolic Nuclear factor erythroid 2-related NFE2L2 (NRF2) is maintained at low basal levels by constitutive proteasomal degradation. Kelch-like ECH associated protein 1 (KEAP1), which is a substrate adaptor protein for the Cullin 3 (CUL3)-dependent E3 ubiquitin ligase complex binds with and represses NFE2L2 by promoting its ubiquitination and subsequent proteasomal degradation (Itoh et al. 1999, Cullinan et al. 2004, Kobayashi et al. 2004, Zhang et al. 2004, Furukawa & Xiong 2005). Therefore, the KEAP1–CUL3–E3 ubiquitin ligase complex tightly regulates NFE2L2 protein to maintain it at a low level. NFE2L2 contains seven functional domains, known as Neh1-Neh7. Neh2 domain contains two motifs termed ETGE and DLG that are involved in interacting with KEAP1. The ETGE and the DLG motifs have overlapping binding sites on KEAP1, with the ETGE motif mediating a higher affinity interaction with KEAP1 than the DLG motif. One molecule of NFE2L2 interacts simultaneously with two KEAP1 molecules, with the DLG motif and the ETGE motif on NFE2L2 contacting similar sites on each member of the KEAP1 dimer (Tong et al, 2006; McMahon et al, 2006; Baird et al, 2013; Fukutomi et al, 2014). This complex assembly positions NFE2L2 appropriately to be ubiquitinated by the CUL3/RBX1 ubiquitin ligase, targeting it for degradation. In the presence of electrophiles or other NFE2L2 inducers, conformational changes within KEAP1 occur as inducers interact with KEAP1 'sensor cysteines'. These conformational changes disrupt the KEAP1-DLG motif interaction, repositioning NFE2L2 within the KEAP1 complex in such a way as to prevent its ubiquitination. In this 'hinge and latch model', saturation of the KEAP1:CUL3:RBX1 complex with mal-positioned and thus not degradable NFE2L2 allows newly translated NFE2L2 to accumulate and translocate into the nucleus to stimulate transcription (Tong et al, 2006; Tong et al, 2007; reviewed in Baird and Yamamoto, 2020). This NFE2L2-KEAP1 interaction is also known to be regulated negatively by multiple proteins like PALB2, DPP3 and AMER(WTX1) which competitively bind with NFE2L2/KEAP1 and allow NFE2L2 nuclear translocation. They play an important role in NFE2L2 regulation to other pathways (Mizukami et al, 2012; Lu et al, 2017; Camp et al, 2012). In addition to that, this NFE2L2-KEAP1 binding is also regulated by succinylation of KEAP1 as cysteine residue and inhibits KEAP1 binding with NRF2 in FH mutant (LOF) condition (Ooi et al, 2011). <br>KEAP1 and NFE2L2 mutations occur in several tumor types and KEAP1 and NFE2L2 mutations occur at a frequency of around 25% in lung cancer. The NFE2L2 pathway has multiple pro-tumorigenic functions, and NFE2L2 levels are increased in head and neck squamous cell carcinoma (HNSCC). KEAP1 somatic mutant C23Y is observed in tumors from approximately 15% of patients with lung cancer (Hayes & McMahon 2009).

在基础静息状态下，细胞质中的核因子E2相关因子2（NRF2）通过组成性蛋白酶体降解维持在低基础水平。Kelch样ECH相关蛋白1（KEAP1），作为Cullin 3（CUL3）依赖性E3泛素连接酶复合物的底物适配蛋白，通过与NFE2L2结合并抑制其表达，通过促进其泛素化及其后续蛋白酶体降解来抑制NFE2L2（Itoh等，1999，Cullinan等，2004，Kobayashi等，2004，Zhang等，2004，Furukawa & Xiong，2005）。因此，KEAP1-CUL3-E3泛素连接酶复合物严格调控NFE2L2蛋白，以维持其低水平。NFE2L2包含七个功能结构域，称为Neh1-Neh7。Neh2结构域包含两个称为ETGE和DLG的基序，这些基序参与与KEAP1的相互作用。ETGE和DLG基序在KEAP1上具有重叠的结合位点，其中ETGE基序与KEAP1的相互作用亲和力高于DLG基序。一个NFE2L2分子同时与两个KEAP1分子相互作用，NFE2L2上的DLG和ETGE基序与KEAP1二聚体每个成员的相似位点接触（Tong等，2006；McMahon等，2006；Baird等，2013；Fukutomi等，2014）。这种复合物组装使NFE2L2适当地定位，以便被CUL3/RBX1泛素连接酶泛素化，从而将其降解。在有亲电剂或其他NFE2L2诱导剂存在的情况下，KEAP1内的构象变化发生，因为诱导剂与KEAP1的“传感器半胱氨酸”相互作用。这些构象变化破坏了KEAP1-DLG基序的相互作用，以防止NFE2L2泛素化，从而将NFE2L2在KEAP1复合物中的位置重新定位。在此“铰链和闩锁模型”中，KEAP1:CUL3:RBX1复合物与错位且不可降解的NFE2L2饱和，允许新合成的NFE2L2积累并转位至细胞核，从而刺激转录（Tong等，2006；Tong等，2007；参见Baird和Yamamoto，2020年的综述）。这种NFE2L2-KEAP1相互作用还已知受到多个蛋白质如PALB2、DPP3和AMER(WTX1)的负调控，这些蛋白质通过与NFE2L2/KEAP1竞争性结合，允许NFE2L2的核转位。它们在NFE2L2调控其他通路中起着重要作用（Mizukami等，2012；Lu等，2017；Camp等，2012）。此外，这种NFE2L2-KEAP1结合还受到KEAP1琥珀酰化（半胱氨酸残基）的调控，并抑制KEAP1在FH突变（LOF）条件下与NRF2的结合（Ooi等，2011）。KEAP1和NFE2L2突变见于多种肿瘤类型，KEAP1和NFE2L2突变在肺癌中的发生频率约为25%。NFE2L2通路具有多种促肿瘤发生功能，NFE2L2水平在头颈鳞状细胞癌（HNSCC）中升高。在约15%的肺癌患者肿瘤中发现KEAP1体细胞突变C23Y（Hayes & McMahon，2009）。