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

神经元中 Bax 相互作用因子 1 相互作用组的功能表征

• 批准号: 9475333
• 负责人: RICHARD S MORRISON
• 金额: $ 23.59万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2019-12-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9475333
• 关键词: Adult; Affect; Agonist; Alternative Splicing; Alzheimer' s Disease; Alzheimer' s disease model; Amyloid beta-Protein; Animal Model; Apoptosis; Apoptotic; Attenuated; Autophagocytosis; BCL2 gene; Bax protein; Behavioral; Binding; Biological; Brain; Brain Diseases; Brain Injuries; Brain Mass; Brain region; Cell Survival; Cells; Central Nervous System Diseases; Disease; Enterobacteria phage P1 Cre recombinase; Exhibits; Family; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); Homeostasis; Immunoprecipitation; Impaired cognition; In Vitro; Infarction; Injury; Knock-out; Length; Mass Spectrum Analysis; Membrane Potentials; Messenger RNA; Mitochondria; Mus; Neuroglia; Neurons; Pathology; Peptides; Performance; Perfusion; Production; Protein Isoforms; Proteins; RNA Splicing; Reporting; Stress; Stroke; Surface; Synapses; Transgenic Mice; age related; aging brain; astrogliosis; bcl-xlong protein; crosslink; cytochrome c; in vivo; inhibitor/antagonist; knock-down; member; mitochondrial membrane; mouse model; neuronal metabolism; neuronal survival; neuroprotection; novel; overexpression; peptidomimetics; prevent; pro-apoptotic protein; protein crosslink; receptor; time use

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.

促凋亡蛋白相互作用因子-1（Bif-1）最初被鉴定为与促凋亡蛋白Bax相互作用的蛋白质 并且已经涉及非神经元细胞中的线粒体动力学、自噬和凋亡， 其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。与此相反，Bif-1a，表达的唯一剪接形式， 非神经元细胞通过与促凋亡蛋白Bax相互作用促进凋亡。我们报道 降低Bif-1b/c，尤其是在主要受AD病理影响的CNS区域，在邻近的脑区域 中风引起的梗塞和老化的大脑。有趣的是，仅BIF-1的缺失就足以导致衰老。 依赖性行为缺陷和小鼠海马中的突触变性。这些发现表明 Bif-1b/c的缺失可能有助于使神经元对多种应激敏感， 脑损伤和疾病。在鉴定和表征Bif-1相互作用的初步研究过程中， 在培养的原代皮层神经元中，我们观察到蛋白质交联， Bif-1a、Bif-1b和Bif-1c的免疫沉淀使Bcl-2家族的促存活成员Bcl-xL下降 和线粒体分裂因子（Mff）。Bcl-xL代表了在成人中表达的主要抗凋亡蛋白。 个脑袋重要的是，Bcl-xL通过增强线粒体功能和调节神经元代谢来维持线粒体功能。 线粒体ATP生成效率。Mff是线粒体表面的一种促分裂受体， GTADRP Drp1.在对初级皮质神经元的新的初步研究中，与我们以前的研究相反， SH-SY 5 Y细胞中，只有神经元特异性形式的Bif-1与Bcl-xL和Mff强烈相互作用，与我们的研究结果一致。 以前的研究表明，只有Bif-1b和Bif-1c表现出神经保护活性，并促进线粒体 伸长率这些发现表明，不同的Bif-1亚型可能被赋予不同的功能， 拥有独特的结合伴侣。由于对Bif-1促进细胞凋亡的机制知之甚少， 神经元活力，Bif-1相互作用组的表征可以为解剖Bif-1提供额外的线索。 神经元中的新功能和作用机制。在这里，我们提出：i）使用一体化交联， 质谱法表征和比较神经元特异性和普遍存在的相互作用组 在正常和应激条件下的Bif-1亚型。这种方法将使我们能够提取亚型特异性 Bif-1相互作用组中的差异，以识别可能导致疾病的选择性相互作用 神经元特异性Bif-1同种型的神经保护效力和ii）验证Bif-1同种型的生物学意义， Bif-1与Bcl-xL和Mff之间的相互作用。这些研究将有助于阐明一些具体的机制 Bif-1蛋白的改变通过其影响神经元功能和存活力。我们的长期目标是确定 用于调节Bif-1和Bcl-xL之间功能性相互作用的抑制肽和激动剂/模拟肽 或Mff以增强损伤后和CNS疾病中的脑功能。