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

尽管Bok在15年前被发现，但它仍然是Bcl-2家族中一个特征不明确的成员。最初报道Bok在生殖组织中有选择性地表达，但同时也清楚地表明Bok在大多数组织中更广泛地表达和可检测到。根据其氨基酸序列，Bok类似于促凋亡的多BH结构域成员Bax和Bak，因此，Bok的强制表达导致许多细胞类型的凋亡。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。我们最近可以证明，bok诱导的细胞凋亡依赖于一种主要依赖于Bax或Bak的机制。此外，我们可以证明Bok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）的膜上。与其亚细胞定位一致，我们发现bok缺陷细胞在内质网应激源治疗时表现出异常的未折叠蛋白反应。在此提议的项目中，我们与研究小组的主要研究人员进行了多次合作，我们的目标是更好地了解Bok的分子功能。我们最近建立了一个Bok缺陷小鼠模型，并获得了大量的分子工具来促进Bok的分析。我们首先要分析从健康细胞和垂死细胞的各种细胞膜中分离出的含有bok的多蛋白复合物。然后，我们将使用质谱法分离和鉴定Bok结合蛋白，并随后测试新鉴定的蛋白相互作用的生物学相关性。我们将描述重组Bok在无细胞系统中的寡聚化和预重整潜力，并协助定量测量Bok与其他Bcl-2家族成员的相互作用。另一个主要目的是阐明Bok诱导细胞凋亡的分子机制，以及Bok对经典化疗药物敏感的潜力。最后，我们希望通过小鼠体内和体外实验，阐明在缺乏Bok的情况下导致内质网应激反应紊乱的潜在机制。

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.