Necroptosis and ferroptosis are two distinct necrotic cell death modalities with no known common molecular mechanisms. Necroptosis is activated by ligands of death receptors such as tumor necrosis factor-α (TNF-α) under caspase-deficient conditions, whereas ferroptosis is mediated by the accumulation of lipid peroxides upon the depletion/or inhibition of glutathione peroxidase 4 (GPX4). The molecular mechanism that mediates the execution of ferroptosis remains unclear. In this study, we identified 2-amino-5-chloro-N,3-dimethylbenzamide (CDDO), a compound known to inhibit heat shock protein 90 (HSP90), as an inhibitor of necroptosis that could also inhibit ferroptosis. We found that HSP90 defined a common regulatory nodal between necroptosis and ferroptosis. We showed that inhibition of HSP90 by CDDO blocked necroptosis by inhibiting the activation of RIPK1 kinase. Furthermore, we showed that the activation of ferroptosis by erastin increased the levels of lysosome-associated membrane protein 2a to promote chaperone-mediated autophagy (CMA), which, in turn, promoted the degradation of GPX4. Importantly, inhibition of CMA stabilized GPX4 and reduced ferroptosis. Our results suggest that activation of CMA is involved in the execution of ferroptosis.

Necroptosis, a form of regulated necrotic cell death, is mediated by receptor interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL). However, the mechanism by which necroptosis promotes inflammation is still unclear. Here we report that the expression of cytokines is robustly upregulated in a cell-autonomous manner during necroptosis induced by tumor necrosis factor alpha (TNFα). We demonstrate that TNFα-induced necroptosis leads to two waves of cytokine production. The first wave, more transient and weaker than the second, is in response to TNFα alone; whereas the second wave depends upon the necroptotic signaling. We show that necroptosis promotes the transcription of TNFα-target genes in a cell-intrinsic manner. The activation of both NF-κB and p38 by the necroptotic machinery, RIPK1, RIPK3, and MLKL, is involved in mediating the robust induction of cytokine expression in the second wave. In contrast, necroptosis induced by direct oligomerization of MLKL promotes cytokine production at much lower levels than that of necroptosis induced with TNFα. Thus, we conclude that TNFα-induced necroptosis signaling events mediated by RIPK1 and RIPK3 activation, in addition to the MLKL oligomerization, promotes the expression of cytokines involving multiple intracellular signaling mechanisms including NF-κB pathway and p38. These findings reveal that the necroptotic cell death machinery mounts an immune response by promoting cell-autonomous production of cytokines. Our study provides insights into the mechanism by which necroptosis promotes inflammation in human diseases.

The ubiquitin-proteasome system (UPS) and autophagy are two major intracellular degradative mechanisms that mediate the turnover of complementary repertoires of intracellular proteomes. Simultaneously activating both UPS and autophagy might provide a powerful strategy for the clearance of misfolded proteins. However, it is not clear whether UPS and autophagy can be controlled by a common regulatory mechanism. K48 deubiquitination by USP14 is known to inhibit UPS. Here we show that USP14 regulates autophagy by negatively controlling K63 ubiquitination of Beclin 1. Furthermore, we show that activation of USP14 by Akt-mediated phosphorylation provides a mechanism for Akt to negatively regulate autophagy by promoting K63 deubiquitination. Our study suggests that Akt-regulated USP14 activity modulates both proteasomal degradation and autophagy through controlling K48 and K63 ubiquitination, respectively. Therefore, regulation of USP14 provides a mechanism for Akt to control both proteasomal and autophagic degradation. We propose that inhibition of USP14 may provide a strategy to promote both UPS and autophagy for developing novel therapeutics targeting neurodegenerative diseases.[Keywords: USP14; autophagy; Beclin 1; Akt] Supplemental material is available for this article. Autophagy and the ubiquitin-proteasome system (UPS) are two major intracellular degradative mechanisms that function in a complementary manner. UPS mediates the degradation of short-lived proteins conjugated with K48 ubiquitin chains (Komander and Rape 2012). On the other hand, autophagy mediates the turnover of long-lived proteins and intracellular organelles encapsulated in autophagosomes that eventually fuse with lysosomes to allow degradation by lysosomal proteases. Ubiquitination is also involved as a signaling mechanism in targeting both protein substrates and organelles such as depolarized mitochondria for degradation by autophagy (Sarraf et al. 2013;Ordureau et al. 2014). Ubiquitination of protein substrates is a reversible process, as ubiquitin chains can be removed by deubiquitinating enzymes (DUBs). Deubiquitination is an important negative regulatory mechanism for reducing the levels of protein ubiquitination. Ubiquitin-specific protease-14 (USP14), a DUB reversibly associated with the proteasome, has been shown to negatively regulate the activity of proteasomes by trimming K48 ubiquitin chains on proteasome-bound substrates (Borodovsky et al. 2001;Koulich et al. 2008;Lee et al. 2010). USP14 can also be activated by Akt-mediated phosphorylation, which promotes its deubiquitinating activity for both K48 and K63 ubiquitin linkages (Xu et al. 2015a). The activity of USP14 in deubiquitinating K63 ubiquitin linkages is likely to be physiologically relevant, as inhibition of USP14 in vivo leads to increases in the levels of K63-linked ubiquitin conjugates in both spinal cords and neurons (Vaden et al. 2015). However, the mechanism by which USP14 regulates K63 ubiquitination in control of cellular processes and its functional significance are...