Vervliet Tim, Lemmens Irma, Vandermarliere Elien, Decrock Elke, Ivanova Hristina, Monaco Giovanni, Sorrentino Vincenzo, Nadif Kasri Nael, Missiaen Ludwig, Martens Lennart, De Smedt Humbert, Leybaert Luc, Parys Jan B, Tavernier Jan, Bultynck Geert
KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, B-3000 Leuven, Belgium.
University of Gent, Cytokine Receptor Lab, VIB Department of Medical Protein Research, B-9000 Gent, Belgium.
Sci Rep. 2015 Apr 15;5:9641. doi: 10.1038/srep09641.
Anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members target several intracellular Ca(2+)-transport systems. Bcl-2, via its N-terminal Bcl-2 homology (BH) 4 domain, inhibits both inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), while Bcl-XL, likely independently of its BH4 domain, sensitizes IP3Rs. It remains elusive whether Bcl-XL can also target and modulate RyRs. Here, Bcl-XL co-immunoprecipitated with RyR3 expressed in HEK293 cells. Mammalian protein-protein interaction trap (MAPPIT) and surface plasmon resonance (SPR) showed that Bcl-XL bound to the central domain of RyR3 via its BH4 domain, although to a lesser extent compared to the BH4 domain of Bcl-2. Consistent with the ability of the BH4 domain of Bcl-XL to bind to RyRs, loading the BH4-Bcl-XL peptide into RyR3-overexpressing HEK293 cells or in rat hippocampal neurons suppressed RyR-mediated Ca(2+) release. In silico superposition of the 3D-structures of Bcl-2 and Bcl-XL indicated that Lys87 of the BH3 domain of Bcl-XL could be important for interacting with RyRs. In contrast to Bcl-XL, the Bcl-XL(K87D) mutant displayed lower binding affinity for RyR3 and a reduced inhibition of RyR-mediated Ca(2+) release. These data suggest that Bcl-XL binds to RyR channels via its BH4 domain, but also its BH3 domain, more specific Lys87, contributes to the interaction.
抗凋亡B细胞淋巴瘤2（Bcl-2）家族成员作用于多个细胞内Ca(2+)转运系统。Bcl-2通过其N端Bcl-2同源（BH）4结构域抑制肌醇1,4,5-三磷酸受体（IP3Rs）和兰尼碱受体（RyRs），而Bcl-XL可能独立于其BH4结构域使IP3Rs敏感化。Bcl-XL是否也能作用并调节RyRs仍不清楚。在此，Bcl-XL与在HEK293细胞中表达的RyR3进行了共免疫沉淀。哺乳动物蛋白质-蛋白质相互作用捕获（MAPPIT）和表面等离子体共振（SPR）表明，Bcl-XL通过其BH4结构域与RyR3的中央结构域结合，尽管与Bcl-2的BH4结构域相比程度较小。与Bcl-XL的BH4结构域结合RyRs的能力一致，将BH4-Bcl-XL肽加载到过表达RyR3的HEK293细胞或大鼠海马神经元中可抑制RyR介导的Ca(2+)释放。Bcl-2和Bcl-XL的3D结构的计算机叠加表明，Bcl-XL的BH3结构域的Lys87可能对与RyRs相互作用很重要。与Bcl-XL相反，Bcl-XL(K87D)突变体对RyR3的结合亲和力较低，对RyR介导的Ca(2+)释放的抑制作用减弱。这些数据表明，Bcl-XL通过其BH4结构域与RyR通道结合，但其BH3结构域，更具体地说是Lys87，也有助于这种相互作用。
