Functional characterization of the Bax-interacting factor-1 interactome in neurons

Bax相互作用因子-1(Bif-1)最初被鉴定为与促凋亡蛋白Bax相互作用的蛋白质 自那以后，它与线粒体动力学、自噬和非神经细胞的凋亡有关 它的总体活性是促凋亡的。我们最近报道了新的神经元特异性剪接的鉴定 形式，Bif-1b和Bif-1c，具有神经保护作用。相比之下，Bif-1a，唯一表达的剪接形式 非神经细胞，通过与促凋亡蛋白Bax相互作用促进细胞凋亡。我们报道了 Bif-1b/c降低，主要在受AD病理影响的中枢神经系统区域，在邻近的脑区 中风导致的脑梗塞，以及老化的大脑。有趣的是，仅Bif-1的丢失就足以导致衰老 小鼠海马区的依赖行为缺陷和突触变性。这些发现表明 Bif-1b/c的丢失可能有助于神经元对与以下相关的多种应激反应敏感 脑部损伤和疾病。在鉴定和鉴定Bif-1相互作用的初步研究过程中 在原代培养的皮质神经元中的蛋白质我们观察到蛋白质交联之后 Bif-1a、Bif-1b和Bif-1c的免疫共沉淀下调了BIF-2家族中支持生存的成员BIF-XL 和线粒体分裂因子(MFF)。BCL-XL是成人表达的主要抗细胞凋亡蛋白 大脑。重要的是，Bclxl维持线粒体功能，并通过增强 线粒体产生三磷酸腺苷的效率。Mff是线粒体表面促进分裂的受体 GTP酶Drp1。在对初级皮质神经元的新的初步研究中，与我们之前的研究不同 SH-SY5Y细胞，只有神经元特异性形式的Bif-1与Bcl-xL和MFF强烈相互作用，这与我们的 先前的研究表明，只有Bif-1b和Bif-1c显示出神经保护活性和促进线粒体 伸长率。这些发现表明，不同的Bif-1亚型可能具有不同的功能 拥有独特的结合伙伴。由于对BIF-1促进BIF-1的机制知之甚少 神经元活性，Bif-1相互作用体的特征可以为解剖Bif-1‘S提供更多线索 神经元中新的功能和作用机制。这里我们建议：i)使用集成的交联剂和 用质谱学方法表征和比较神经元特异性和普遍存在的相互作用组 BIF-1在正常和应激条件下呈异构体。这种方法将允许我们提取特定于异构体的 Bif-1相互作用组的差异以确定可能导致Bif-1相互作用的选择性相互作用 神经元特异性Bif-1亚型的神经保护作用和II)证实了Bif-1亚型的生物学意义 Bif-1与Bclxl和MFf的相互作用这些研究将有助于阐明一些具体的机制。 通过Bif-1蛋白的改变影响神经元的功能和生存能力。我们的长期目标是确定 调节Bif-1和Bcl-xl功能相互作用的抑制肽和激动肽/模拟肽 或MFF，用于增强损伤后和中枢神经系统疾病的脑功能。

Bax-interacting factor-1 (Bif-1) was initially identified as a protein that interacts with the pro-apoptotic protein Bax and has since been implicated in mitochondrial dynamics, autophagy and apoptosis in non-neuronal cells with its overall activity being pro-apoptotic. We recently reported the identification of novel neuron specific splice forms, Bif-1b and Bif-1c that have neuroprotective action. In contrast, Bif-1a, the only splice form expressed by non-neuronal cells, promotes apoptosis through interactions with the pro-apoptotic protein Bax. We reported reduced Bif-1b/c specifically in CNS regions predominantly affected by AD pathology, in brain regions adjacent to stroke-induced infarcts and also in aging brain. Interestingly, loss of Bif-1 alone is sufficient to cause age dependent behavioral deficits and synaptic degeneration in the mouse hippocampus. These findings suggest that the loss of Bif-1b/c may be instrumental in sensitizing neurons to a multitude of stresses associated with brain injury and disease. During the course of preliminary studies to identify and characterize Bif-1 interacting proteins in primary cortical neurons in culture we observed that protein crosslinking followed by immunoprecipitation of Bif-1a, Bif-1b and Bif-1c brought down a pro-survival member of the Bcl-2 family, Bcl-xL and mitochondrial fission factor (Mff). Bcl-xL represents the principal anti-apoptotic protein expressed in adult brain. Importantly, Bcl-xL maintains mitochondrial function and regulates neuronal metabolism by enhancing the efficiency of ATP production in mitochondria. Mff is a mitochondrial surface receptor for the fission promoting GTPase Drp1. In new preliminary studies with primary cortical neurons, in contrast to our previous studies with SH-SY5Y cells, only neuron-specific forms of Bif-1 interacted strongly with Bcl-xL and Mff, consistent with our previous studies showing that only Bif-1b and Bif-1c exhibit neuroprotective activity and promote mitochondrial elongation. These findings suggest that different Bif-1 isoforms may be endowed with distinct functions by virtue of having unique binding partners. Since little is known regarding the mechanism by which Bif-1 promotes neuronal viability, characterization of the Bif-1 interactome could provide additional clues to dissecting Bif-1’s novel function and mechanism of action in neurons. Here we propose to: i) use an integrated crosslinking and mass spectrometry approach to characterize and compare the interactome for neuron-specific and ubiquitous Bif-1 isoforms under normal and stressful conditions. This approach will allow us to extract isoform-specific differences in the Bif-1 interactome to identify selective interactions that are potentially responsible for the neuroprotective potency of neuron-specific Bif-1 isoforms and ii) validate the biological significance of interactions between Bif-1 and Bcl-xL and Mff. These studies will help elucidate some of the specific mechanisms by which alterations in the Bif-1 protein impact neuronal function and viability. Our long term goal is to identify inhibitor peptides and agonist/mimetic peptides for modulating functional interactions between Bif-1 and Bcl-xL or Mff to enhance brain function after injury and in CNS disease.