Laboratoire de Chimie et Biologie des Métaux, UMR5249 Université Grenoble Alpes, CNRS, CEA, 17 rue des Martyrs, CEDEX 9, 38054 Grenoble, France.
Département d'immunologie et biologie cellulaire, Pavillon de recherche appliquée sur le cancer, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada.
Int J Mol Sci. 2020 Oct 15;21(20):7625. doi: 10.3390/ijms21207625.
Bcl-xL, a member of the Bcl-2 family, is a pro-survival protein involved in apoptosis regulation. We have previously reported the ability of Bcl-xL to form various types of fibers, from native to amyloid conformations. Here, we have mimicked the effect of apoptosis-induced caspase activity on Bcl-xL by limited proteolysis using trypsin. We show that cleaved Bcl-xL (ΔN-Bcl-xL) forms fibers that exhibit the features of amyloid structures (BclxLcf37). Moreover, three monoclonal antibodies (mAbs), produced by mouse immunization and directed against ΔN-Bcl-xL or Bcl-xL fibers, were selected and characterized. Our results show that these mAbs specifically target ΔN-Bcl-xL in amyloid fibers in vitro. Upon metal-stress-induced apoptosis, these mAbs are able to detect the presence of Bcl-xL in amyloid aggregates in neuroblastoma SH-SY5Y cell lines. In conclusion, these specific mAbs directed against amyloidogenic conformations of Bcl-xL constitute promising tools for studying, in vitro and in cellulo, the contribution of Bcl-xL in apoptosis. These mAbs may further help in developing new diagnostics and therapies, considering Bcl-xL as a strategic target for treating brain lesions relevant to stroke and neurodegenerative diseases.
Bcl-xL 是 Bcl-2 家族的一员，是一种参与细胞凋亡调控的抗凋亡蛋白。我们之前曾报道过 Bcl-xL 能够形成各种类型的纤维，从天然构象到淀粉样构象。在这里，我们通过使用胰蛋白酶进行有限水解模拟了细胞凋亡诱导的半胱天冬酶活性对 Bcl-xL 的影响。我们发现，切割的 Bcl-xL（ΔN-Bcl-xL）形成的纤维表现出淀粉样结构的特征（BclxLcf37）。此外，我们还筛选和鉴定了三种单克隆抗体（mAbs），这些 mAbs 由小鼠免疫产生，针对的是 ΔN-Bcl-xL 或 Bcl-xL 纤维。我们的结果表明，这些 mAbs 特异性靶向体外淀粉样纤维中的 ΔN-Bcl-xL。在金属应激诱导的细胞凋亡过程中，这些 mAbs 能够检测到神经母细胞瘤 SH-SY5Y 细胞系中淀粉样聚集物中 Bcl-xL 的存在。总之，这些针对 Bcl-xL 淀粉样构象的特异性 mAbs 为研究细胞凋亡中 Bcl-xL 的作用提供了有前途的工具，无论是在体外还是在细胞内。这些 mAbs 可能会进一步有助于开发新的诊断和治疗方法，因为 Bcl-xL 是治疗与中风和神经退行性疾病相关的脑损伤的一个重要治疗靶点。
