Department of Cell and Molecular Physiology, Loyola University Stritch School of Medicine, Maywood, Illinois, USA.
J Cell Biochem. 2022 Jan;123(1):128-141. doi: 10.1002/jcb.30140. Epub 2021 Sep 6.
The co-chaperone Bcl2-associated athanogene-3 (BAG3) maintains cellular protein quality control through the regulation of heat shock protein 70 (HSP70). Cancer cells manipulate BAG3-HSP70-regulated pathways for tumor initiation and proliferation, which has led to the development of promising small molecule therapies, such as JG-98, which inhibit the BAG3-HSP70 interaction and mitigate tumor growth. However, it is not known how these broad therapies impact cardiomyocytes, where the BAG3-HSP70 complex is a key regulator of protein turnover and contractility. Here, we show that JG-98 exposure is toxic in neonatal rat ventricular myocytes (NRVMs). Using immunofluorescence microscopy to assess cell death, we found that apoptosis increased in NRVMs treated with JG-98 doses as low as 10 nM. JG-98 treatment also reduced autophagy flux and altered expression of BAG3 and several binding partners involved in BAG3-dependent autophagy, including SYNPO2 and HSPB8. We next assessed protein half-life with disruption of the BAG3-HSP70 complex by treating with JG-98 in the presence of cycloheximide and found BAG3, HSPB5, and HSPB8 half-lives were reduced, indicating that complex formation with HSP70 is important for their stability. Next, we assessed sarcomere structure using super-resolution microscopy and found that disrupting the interaction with HSP70 leads to sarcomere structural disintegration. To determine whether the effects of JG-98 could be mitigated by pharmacological autophagy induction, we cotreated NRVMs with rapamycin, which partially reduced the extent of apoptosis and sarcomere disarray. Finally, we investigated whether the effects of JG-98 extended to skeletal myocytes using C2C12 myotubes and found again increased apoptosis and reduced autophagic flux. Together, our data suggest that nonspecific targeting of the BAG3-HSP70 complex to treat cancer may be detrimental for cardiac and skeletal myocytes.
伴侣蛋白 Bcl2 相关抗凋亡基因 3（BAG3）通过调节热休克蛋白 70（HSP70）来维持细胞的蛋白质质量控制。癌细胞操纵 BAG3-HSP70 调节的途径来启动和增殖肿瘤，这导致了有前途的小分子治疗方法的发展，例如 JG-98，它抑制 BAG3-HSP70 相互作用并减轻肿瘤生长。然而，目前尚不清楚这些广泛的治疗方法如何影响心肌细胞，在心肌细胞中，BAG3-HSP70 复合物是蛋白质周转和收缩性的关键调节剂。在这里，我们表明 JG-98 暴露对新生大鼠心室肌细胞（NRVM）有毒性。通过免疫荧光显微镜评估细胞死亡，我们发现 JG-98 处理剂量低至 10 nM 时 NRVM 中的细胞凋亡增加。JG-98 处理还降低了自噬通量，并改变了 BAG3 和几个参与 BAG3 依赖性自噬的结合伴侣的表达，包括 SYNPO2 和 HSPB8。接下来，我们通过用 JG-98 在环己酰亚胺存在下处理来评估 BAG3-HSP70 复合物的破坏对蛋白质半衰期的影响，发现 BAG3、HSPB5 和 HSPB8 的半衰期缩短，表明与 HSP70 的复合物形成对其稳定性很重要。接下来，我们使用超分辨率显微镜评估肌节结构，发现破坏与 HSP70 的相互作用导致肌节结构解体。为了确定 JG-98 的作用是否可以通过药理学自噬诱导来减轻，我们用雷帕霉素共同处理 NRVM，雷帕霉素部分减少了细胞凋亡和肌节紊乱的程度。最后，我们使用 C2C12 肌管再次研究了 JG-98 的作用是否扩展到骨骼肌细胞，发现细胞凋亡增加和自噬通量降低。总之，我们的数据表明，用 BAG3-HSP70 复合物的非特异性靶向治疗癌症可能对心脏和骨骼肌细胞有害。
