Cell Death & Biomedicine Laboratory, Centro de Biomedicina, Universidad Mayor, Santiago, Chile.
VIB Center for Inflammation Research, Universidad Mayor, Ghent, Belgium.
Autophagy. 2024 Nov;20(11):2444-2459. doi: 10.1080/15548627.2024.2367923. Epub 2024 Jul 3.
Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs. : BAF: bafilomycin A; CASP3: caspase 3; Caspases: cysteine-aspartic proteases; cCASP3: cleaved CASP3; CQ: chloroquine; CHX: cycloheximide; cPARP: cleaved poly (ADP-ribose) polymerase; DEPs: differential expressed proteins; ETO: etoposide; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain-like; MSC: mesenchymal stem cell; MTORC1: mechanistic target of rapamycin kinase complex 1; Nec1s: necrostatin 1s; NFKB/NF-kB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PLA: proximity ligation assay; RCD: regulated cell death; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RUBCNL/PACER: RUN and cysteine rich domain containing beclin 1 interacting protein like; si: small interfering RNA nonsense; siRNA: small interfering RNA; TdT: terminal deoxynucleotidyl transferase; Tm: tunicamycin; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a.
间充质干细胞 (MSCs) 被用于细胞治疗；然而，它们的应用受到其在促炎微环境中移植后存活能力差的限制。MSCs 中的巨自噬/自噬激活构成了应激适应途径，促进细胞内稳态。我们的蛋白质组学数据表明，RUBCNL/PACER（RUN 和富含半胱氨酸的域包含与 beclin 1 相互作用的蛋白样），一种自噬的正调节剂，也参与细胞死亡。因此，我们在 RUBCNL 缺失或功能获得的情况下筛选了各种细胞死亡刺激物对 MSC 存活的影响。当 RUBCNL 表达时，MSCs 可免受 TNF（肿瘤坏死因子）诱导的调节性细胞死亡的影响。TNF 通过诱导 RIPK1 激酶依赖性凋亡或坏死性凋亡来促进炎症。我们确定，当半胱天冬酶失活时，MSCs 在感知 TNF 时会发生 RIPK1 激酶依赖性凋亡和坏死性凋亡。我们表明，RUBCNL 是 RIPK1 依赖性凋亡和坏死性凋亡的负调节剂。此外，失去调节自噬能力的 RUBCNL 突变体保留了其负调控细胞死亡的功能。我们还发现，RUBCNL 与 RIPK1 形成复合物，该复合物响应 TNF 而解体。这一发现表明，RUBCNL 表达限制了 RIPK1-TNFRSF1A/TNFR1 复合物 I 的组装，表明 RUBCNL 与 RIPK1 之间的复合物形成抑制了 TNF 信号转导。这些结果为 RIPK1 介导的细胞死亡和自噬机制之间的串扰提供了新的见解，并表明由于其在自噬和凋亡/坏死性凋亡中的功能双重性，RUBCNL 可以作为靶点，以提高 MSC 的治疗效果。：BAF：巴弗洛霉素 A；CASP3：半胱天冬酶 3；Caspases：半胱天冬酶；cCASP3：裂解的 CASP3；CQ：氯喹；CHX：环己酰亚胺；cPARP：裂解的多聚（ADP-核糖）聚合酶；DEP：差异表达蛋白；ETO：依托泊苷；MEF：鼠胚胎成纤维细胞；MLKL：混合谱系激酶结构域样；MSC：间充质干细胞；MTORC1：雷帕霉素靶蛋白激酶复合物 1； Nec1s：坏死性凋亡抑制剂 1s；NFKB/NF-kB：B 细胞中κ轻肽基因增强子核因子；PLA：邻近连接测定；RCD：调节性细胞死亡；RIPK1：受体（TNFRSF）-相互作用丝氨酸-苏氨酸激酶 1；RIPK3：受体相互作用丝氨酸-苏氨酸激酶 3；RUBCNL/PACER：RUN 和富含半胱氨酸的域包含与 beclin 1 相互作用的蛋白样；si：无意义的小干扰 RNA；siRNA：小干扰 RNA；TdT：末端脱氧核苷酸转移酶；Tm：衣霉素；TNF：肿瘤坏死因子；TNFRSF1A/TNFR1：肿瘤坏死因子受体超家族成员 1a。
