Identifying binding partners and molecular functions of the Bcl-2 family member Bok

鉴定 Bcl-2 家族成员 Bok 的结合伴侣和分子功能

• 批准号: 245708129
• 负责人: Professor Dr. Thomas Kaufmann
• 金额: --
• 依托单位: Institut für Pharmakologie
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/245708129?language=en
• 关键词: Identifying; binding; partners; molecular; functions

Despite its discovery 15 years ago, Bok remains a poorly characterised member of the Bcl-2 family. Initially reported to be expressed selectively in reproductive tissues it has meanwhile become clear that Bok is much more widely expressed and detectable in most tissue. Based on its amino acid sequence, Bok resembles the pro-apoptotic multi BH domain members Bax and Bak and, accordingly, enforced expression of Bok leads to apoptosis in many cell types. Intriguingly, a recent report identified the Bok gene to be deleted in human cancers with high frequency. We could recently show that Bok-induced apoptosis relies on a mechanism largely dependent on Bax or Bak. Furthermore, we could demonstrate that major portions of Bok are localised at non-mitochondrial sites, in particular at the membranes of the Golgi apparatus and the endoplasmic reticulum (ER). Consistent with its subcellular localisation we found that Bok-deficient cells display with an aberrant unfolded protein response upon treatment with ER stressors. With the here-proposed project, which involves multiple collaborations with principal investigators within our Research Group, we aim to better understand the molecular functions of Bok. We have recently generated a Bok-deficient mouse model and acquired a large arsenal of molecular tools facilitating analysis of Bok. We first want to analyse Bokcontaining multiprotein complexes isolated from various intracellular membranes of healthy and dying cells. We will then isolate and identify Bok binding proteins by non-biased approaches using mass spectrometry and subsequently test the biological relevance of newly identified protein interactions. We will characterise the oligomerisation and poreforming potential of recombinant Bok in cell free systems and assist in the quantitative measurement of Bok interactions to other Bcl-2 family members. Another major aim consists in the elucidation of the molecular mechanisms that regulate Bok-induced apoptosis and the potential of Bok to sensitise cancer cells towards classical chemotherapeutics. Finally, we would like to clarify the underlying mechanisms that lead to a disturbed ER stress response in the absence of Bok, using both in vitro and in vivo approaches in the mouse.

尽管Bok在15年前就被发现了，但它仍然是Bcl-2家族中特征不明显的成员。最初报道Bok在生殖组织中选择性表达，同时已经清楚Bok在大多数组织中更广泛地表达和可检测。基于其氨基酸序列，Bok类似于促细胞凋亡多BH结构域成员Bax和巴克，因此，Bok的增强表达导致许多细胞类型中的细胞凋亡。有趣的是，最近的一份报告发现，Bok基因在人类癌症中被删除的频率很高。我们最近发现Bok诱导的细胞凋亡依赖于一种主要依赖于Bax或巴克的机制。此外，我们可以证明，博克的主要部分位于非线粒体位点，特别是在高尔基体和内质网（ER）的膜。与其亚细胞定位一致，我们发现Bok缺陷细胞在用ER应激物处理后显示异常的未折叠蛋白反应。通过这里提出的项目，涉及与我们研究小组内的主要研究人员的多次合作，我们的目标是更好地了解Bok的分子功能。我们最近产生了一个博克缺陷小鼠模型，并获得了大量的分子工具，促进博克分析。我们首先要分析从健康和垂死细胞的各种细胞内膜中分离的Bokcontaining多蛋白复合物。然后，我们将分离和确定博克结合蛋白的非偏见的方法，使用质谱，随后测试新发现的蛋白质相互作用的生物相关性。我们将研究重组博克在无细胞系统中的寡聚化和穿孔潜力，并协助定量测量博克与其他Bcl-2家族成员的相互作用。另一个主要目的在于阐明调控博克诱导的细胞凋亡的分子机制和博克使癌细胞对经典化疗药物敏感的潜力。最后，我们想澄清的潜在机制，导致干扰ER应激反应的情况下，博克，在体外和体内的方法在小鼠。