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家族中一个特征不佳的成员。最初的报道是选择性地在生殖组织中表达，但现在已经很清楚，BOK在大多数组织中得到了更广泛的表达和检测。根据其氨基酸序列，BOK类似于促凋亡的多个BH结构域成员Bax和Bak，因此，Bok的强制表达导致许多类型的细胞发生凋亡。有趣的是，最近的一份报告发现，在人类癌症中，BOK基因被高频率缺失。我们最近可以证明，BOK诱导的细胞凋亡依赖于一种在很大程度上依赖于Bax或Bak的机制。此外，我们可以证明BOK的主要部分定位在非线粒体位置，特别是高尔基体和内质网(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处理时表现出异常的未折叠蛋白反应。通过这里提出的项目，包括与我们研究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。我们最近建立了一个BOK缺陷的小鼠模型，并获得了大量有助于分析BOK的分子工具。我们首先想要分析从健康和濒死细胞的各种细胞膜中分离出来的含有Bokin的多蛋白复合体。然后，我们将使用质谱学无偏见的方法分离和鉴定BOK结合蛋白，并随后测试新发现的蛋白质相互作用的生物学相关性。我们将表征重组BOK在无细胞系统中的寡聚和孔道重整潜力，并协助定量测量BOK与其他Bcl-2家族成员的相互作用。另一个主要目的在于阐明调控BOK诱导的细胞凋亡的分子机制，以及BOK使癌细胞对经典化疗药物敏感的可能性。最后，我们想要阐明在没有BOK的情况下，通过体外和体内方法在小鼠中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.