Abstract
Proteasome inhibitors have achieved clinical success because they trigger intrinsic and extrinsic cell death to eliminate susceptible human cancers. The ubiquitin-proteasome protein degradation system regulates signaling pathways by controlling levels of components such as cellular inhibitor of apoptosis (cIAP)1 and cIAP2 in TNF-mediated cell death. Here, we sought to evaluate the contribution of necroptosis to the cell death pattern induced by the specific proteasome inhibitor Carfilzomib (Cf). Proteasome inhibitor-sensitive multiple myeloma cell lines die in response to Cf by apoptosis in combination with serine protease-dependent death, without any contribution of RIPK3-dependent necroptosis. Proteasome inhibition leads to the induction of apoptotic markers such as activated caspase-3 rather than necroptotic markers such as phosphorylated-MLKL in all cell lines tested. In HT-29 cells, Cf attenuates the late RIPK1 interaction with TNFR1 during TNF-induced necroptosis without altering the sensitivity of cIAP antagonists. Cf treatment results in decreased translocation of death signaling components RIPK1, FADD, caspase-8, cFLIP, and RIPK3 to detergent insoluble fractions. Our results show that proteasome inhibition with Cf impairs necroptosis and favors apoptosis even in cells with intact necroptotic machinery. Following the induction of TNFR1-mediated necroptosis, proteasome activity stabilizes effective aggregation and activation of ripoptosome/necrosome complexes.
Highlights
The ubiquitin (Ub)-proteasome degradation system regulates the levels of proteins involved in receptor signaling pathways, such as those controlling cell death and cell cycle[1,2,3]
Cell death proteins RIPK1, RIPK3, Casp[8], and mixed lineage kinase domain-like (MLKL) all translocated to the pellet fraction of RPMI8226 cells following T/CH/ V14,33,34, treatment with Cf alone or Cf/V did not drive pMLKL into the pellet fractions (Fig. 1c)
Fully active P18-C8 translocated to the pellet in levels comparable to those occurring in T/CH-induced apoptosis (Fig. 1c)
Summary
The ubiquitin (Ub)-proteasome degradation system regulates the levels of proteins involved in receptor signaling pathways, such as those controlling cell death and cell cycle[1,2,3]. Necroptosis is a form of regulated lytic cell death characterized by swelling of intracellular organelles and leakage through the plasma membrane[7] triggered by TNF family death ligands[8], pathogen recognition[9], T cell activation[10] interferon[11] or virus infection[12,13] when caspase activation is compromised This pathway contributes to host defense during infection[14,15,16] as well as to inflammatory tissue injury[12,17,18]. TNFR1 activation leads to the recruitment of an Ub ligation complex that includes the TNFR-associated factor (TRAF)[2] and the cellular inhibitor of apoptosis (cIAP)[1] and cIAP2 This complex adds K63-linked Ub chains to TNFR1 associated signaling components including receptor interacting protein (RIPK)[17], favoring the Official journal of the Cell Death Differentiation Association
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