Regulator of G-Protein Signaling 19 (RGS19) and Its Partner Gα-Inhibiting Activity Polypeptide 3 (GNAI3) Are Required for zVAD-Induced Autophagy and Cell Death in L929 Cells

Autophagy has diverse biological functions and is involved in many biological processes. The L929 cell death induced by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD) was shown to be an autophagy-mediated death for which RIP1 and RIP3 were both required. It was also reported that zVAD can induce a small amount of TNF production, which was shown to be required for zVAD-induced L929 cell death, arguing for the contribution of autophagy in the zVAD-induced L929 cell death. In an effort to study RIP3 mediated cell death, we identified regulator of G-protein signaling 19 (RGS19) as a RIP3 interacting protein. We showed that RGS19 and its partner Gα-inhibiting activity polypeptide 3 (GNAI3) are involved in zVAD-, but not TNF-, induced cell death. The role of RGS19 and GNAI3 in zVAD-induced cell death is that they are involved in zVAD-induced autophagy. By the use of small hairpin RNAs and chemical inhibitors, we further demonstrated that zVAD-induced autophagy requires not only RIP1, RIP3, PI3KC3 and Beclin-1, but also RGS19 and GNAI3, and this autophagy is required for zVAD-induced TNF production. Collectively, our data suggest that zVAD-induced L929 cell death is a synergistic result of autophagy, caspase inhibition and autocrine effect of TNF.

细胞自噬（autophagy）具有多样的生物学功能，参与诸多生理过程。泛半胱天冬酶抑制剂苄氧羰基-Val-Ala-Asp-(OMe)-氟甲基酮（benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone, zVAD）诱导的L929细胞死亡，被证实为一种依赖细胞自噬的死亡方式，且该过程同时需要受体相互作用蛋白1（RIP1）与受体相互作用蛋白3（RIP3）的参与。另有研究表明，zVAD可诱导少量肿瘤坏死因子（TNF）的产生，而该TNF的产生对于zVAD诱导的L929细胞死亡至关重要，这进一步佐证了细胞自噬在zVAD诱导的L929细胞死亡中的作用。本研究团队为探究RIP3介导的细胞死亡机制，筛选得到G蛋白信号调节因子19（RGS19）作为RIP3的互作蛋白。实验证实，RGS19及其结合蛋白G蛋白抑制活性多肽3（GNAI3）参与zVAD诱导的细胞死亡，但不参与TNF诱导的细胞死亡。RGS19与GNAI3在zVAD诱导细胞死亡中的作用机制，在于其参与了zVAD诱导的细胞自噬过程。通过短发夹RNA（small hairpin RNAs, shRNA）与化学抑制剂联用实验，本团队进一步证实，zVAD诱导的细胞自噬不仅需要RIP1、RIP3、磷脂酰肌醇3-激酶催化亚基3（PI3KC3）与自噬相关蛋白Beclin-1，同时也依赖RGS19与GNAI3；且该细胞自噬过程对于zVAD诱导的TNF产生不可或缺。综上，本研究数据表明，zVAD诱导的L929细胞死亡是细胞自噬、半胱天冬酶抑制以及TNF自分泌效应共同协同作用的结果。