A Small Peptide Modeled after the NRAGE Repeat Domain Inhibits XIAP-TAB1-TAK1 Signaling for NF-κB Activation and Apoptosis in P19 Cells

In normal growth and development, apoptosis is necessary to shape the central nervous system and to eliminate excess neurons which are not required for innervation. In some diseases, however, apoptosis can be either overactive as in some neurodegenerative disorders or severely attenuated as in the spread of certain cancers. Bone morphogenetic proteins (BMPs) transmit signals for regulating cell growth, differentiation, and apoptosis. Responding to BMP receptors stimulated from BMP ligands, neurotrophin receptor-mediated MAGE homolog (NRAGE) binds and functions with the XIAP-TAK1-TAB1 complex to activate p38MAPK and induces apoptosis in cortical neural progenitors. NRAGE contains a unique repeat domain that is only found in human, mouse, and rat homologs that we theorize is pivotal in its BMP MAPK role. Previously, we showed that deletion of the repeat domain inhibits apoptosis, p38MAPK phosphorylation, and caspase-3 cleavage in P19 neural progenitor cells. We also showed that the XIAP-TAB1-TAK1 complex is dependent on NRAGE for IKK-α/β phosphorylation and NF-κB activation. XIAP is a major inhibitor of caspases, the main executioners of apoptosis. Although it has been shown previously that NRAGE binds to the RING domain of XIAP, it has not been determined which NRAGE domain binds to XIAP. Here, we used fluorescence resonance energy transfer (FRET) to determine that there is a strong likelihood of a direct interaction between NRAGE and XIAP occurring at NRAGE's unique repeat domain which we also attribute to be the domain responsible for downstream signaling of NF-κB and activating IKK subunits. From these results, we designed a small peptide modeled after the NRAGE repeat domain which we have determined inhibits NF-κB activation and apoptosis in P19 cells. These intriguing results illustrate that the paradigm of the NRAGE repeat domain may hold promising therapeutic strategies in developing pharmaceutical solutions for combating harmful diseases involving excessive downstream BMP signaling, including apoptosis.在正常生长发育过程中，细胞凋亡（apoptosis）对于塑造中枢神经系统以及清除无需参与神经支配的多余神经元至关重要。然而在部分疾病中，细胞凋亡或表现为过度激活（如某些神经退行性疾病），或严重受抑（如部分癌症的扩散）。 骨形态发生蛋白（Bone morphogenetic proteins, BMPs）可传递调控细胞增殖、分化与凋亡的信号。当骨形态发生蛋白配体刺激骨形态发生蛋白受体后，神经营养因子受体介导的黑素瘤抗原同源物（neurotrophin receptor-mediated MAGE homolog, NRAGE）会与X连锁凋亡抑制蛋白-TAK1-TAB1（XIAP-TAK1-TAB1）复合物结合并发挥功能，激活p38丝裂原活化蛋白激酶（p38MAPK），进而诱导皮层神经前体细胞发生凋亡。 NRAGE含有一个独特的重复结构域，该结构域仅在人类、小鼠和大鼠的同源蛋白中存在，我们推测其在NRAGE介导骨形态发生蛋白信号通路调控丝裂原活化蛋白激酶（BMP MAPK）的过程中发挥关键作用。 此前我们的研究证实，在P19神经前体细胞中，删除该重复结构域会抑制细胞凋亡、p38MAPK磷酸化以及半胱天冬酶-3（caspase-3）的剪切活化。我们还发现，XIAP-TAB1-TAK1复合物依赖NRAGE实现IκB激酶-α/β（IKK-α/β）磷酸化与核因子-κB（NF-κB）的激活。 XIAP是半胱天冬酶的主要抑制剂，而半胱天冬酶是细胞凋亡的核心执行因子。尽管已有研究证实NRAGE可结合XIAP的环指结构域（RING domain），但目前尚未明确NRAGE的哪个结构域与XIAP发生相互作用。 本研究中，我们利用荧光共振能量转移（fluorescence resonance energy transfer, FRET）技术证实，NRAGE与XIAP极有可能发生直接相互作用，且相互作用位点位于NRAGE的独特重复结构域；同时我们证实，该结构域也是介导NF-κB下游信号通路激活以及IKK亚基活化的关键结构域。 基于上述结果，我们设计了一段以NRAGE重复结构域为模板的小肽，经实验证实该小肽可抑制P19细胞中的NF-κB激活与细胞凋亡。这些引人关注的研究结果表明，靶向NRAGE重复结构域的策略，有望为开发针对过度激活下游骨形态发生蛋白信号通路（包括细胞凋亡异常）的有害疾病的药物治疗方案提供极具前景的治疗思路。