Bcl-2 家族通过促凋亡蛋白和抗凋亡蛋白的直接相互作用来控制细胞凋亡。其主要机制是促凋亡蛋白的 BH3 结构域与抗凋亡蛋白的疏水沟结合，已被批准的 BH3 模拟抗癌药物在治疗上利用了这一机制。有证据表明，Bcl-2 蛋白的跨膜结构域 (TMD) 也可以介导 Bcl-2 相互作用。我们开发了一种高度特异性的裂解荧光素酶测定，能够分析活细胞中成孔凋亡效应器 BAX、BAK 和 BOK 与抗凋亡 Bcl-2 蛋白的 TMD 相互作用。我们证实了 BAX-TMD 的同型相互作用，而且还新鉴定了抗凋亡 BCL-2 的 TMD 与 BOK（一种特殊的促凋亡 Bcl-2 蛋白）的 TMD 的相互作用。 BOK-TMD 和 BCL-2-TMD 在内质网相互作用。分子动力学模拟证实了动态 BOK-TMD 和 BCL-2-TMD 二聚体和稳定的异四聚体。 BCL-2-TMD 在预测的关键残基处的突变会消除与 BOK-TMD 的相互作用。此外，BCL-2 对 BOK 诱导的细胞凋亡的抑制具体取决于其 TMD。因此，Bcl-2 蛋白的 TMD 是细胞凋亡调节的相关相互作用界面，并提供了新的潜在药物靶点。© 2024。作者。

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target.© 2024. The Author(s).