Suppression of RIP3-dependent Necroptosis by Human Cytomegalovirus

Necroptosis is an alternate programmed cell death pathway that is unleashed by caspase-8 compromise and mediated by receptor-interacting protein kinase 3 (RIP3). Murine cytomegalovirus (CMV) and herpes simplex virus (HSV) encode caspase-8 inhibitors that prevent apoptosis together with competitors of RIP homotypic interaction motif (RHIM)-dependent signal transduction to interrupt the necroptosis. Here, we show that pro-necrotic murine CMV M45 mutant virus drives virus-induced necroptosis during nonproductive infection of RIP3-expressing human fibroblasts, whereas WT virus does not. Thus, M45-encoded RHIM competitor, viral inhibitor of RIP activation, sustains viability of human cells like it is known to function in infected mouse cells. Importantly, human CMV is shown to block necroptosis induced by either TNF or M45 mutant murine CMV in RIP3-expressing human cells. Human CMV blocks TNF-induced necroptosis after RIP3 activation and phosphorylation of the mixed lineage kinase domain-like (MLKL) pseudokinase. An early, IE1-regulated viral gene product acts on a necroptosis step that follows MLKL phosphorylation prior to membrane leakage. This suppression strategy is distinct from RHIM signaling competition by murine CMV or HSV and interrupts an execution process that has not yet been fully elaborated.     

RIP3-mediated necroptosis is regulated by inter-filament assembly of RIP homotypic interaction motif

Necroptosis is mediated by signaling complexes called necrosomes, which contain receptor-interacting protein 3 (RIP3) and upstream effectors, such as RIP1. In necrosomes, the RIP homotypic interaction motif (RHIM) of RIP3 and RIP1 forms amyloidal complex. But how the amyloidal necrosomes control RIP3 activation and cell necroptosis has not been determined. Here, we showed that RIP3 amyloid fibrils could further assemble into large fibrillar networks which presents as cellular puncta during necroptosis. A viral RHIM-containing necroptosis inhibitor M45 could form heteroamyloid with RIP3 in cells and prevent RIP3 puncta formation and cell necroptosis. We characterized mutual antagonism between RIP3–RHIM and M45–RHIM in necroptosis regulation, which was caused by distinct inter-filament interactions in RIP3, M45 amyloids revealed with atomic force microscopy. Moreover, double mutations Asn464 and Met468 in RIP3–RHIM to Asp disrupted RIP3 kinase-dependent necroptosis. While the mutant RIP3(N464D/M468D) could form amyloid as wild type upon necroptosis induction. Based on these results, we propose that RIP3 amyloid formation is required but not sufficient in necroptosis signaling, the ordered inter-filament assembly of RIP3 is critical in RIP3 amyloid mediated kinase activation and cell necroptosis.Subject terms: Kinases, Cell biology, Protein aggregation     

DAI/ZBP1/DLM-1 complexes with RIP3 to mediate virus-induced programmed necrosis that is targeted by murine cytomegalovirus vIRA

Programmed necrosis, like apoptosis, eliminates pathogen-infected cells as a component of host defense. Receptor-interacting protein kinase (RIP) 3 (also called RIPK3) mediates RIP homotypic interaction motif (RHIM)-dependent programmed necrosis induced by murine cytomegalovirus (MCMV) infection or death receptor activation and suppressed by the MCMV-encoded viral inhibitor of RIP activation (vIRA). We find that interferon-independent expression of DNA-dependent activator of interferon regulatory factors (DAI, also known as ZBP1 or DLM-1) sensitizes cells to virus-induced necrosis and that DAI knockdown or knockout cells are resistant to this death pathway. Importantly, as with RIP3(-/-) mice, vIRA mutant MCMV pathogenesis is restored in DAI(-/-) mice, consistent with a DAI-RIP3 complex being the natural target of vIRA. Thus, DAI interacts with RIP3 to mediate virus-induced necrosis analogous to the RIP1-RIP3 complex controlling death receptor-induced necroptosis. These studies unveil a role for DAI as the RIP3 partner mediating virus-induced necrosis.     

Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3.

Receptor-interacting protein (RIP), a Ser/Thr kinase component of the tumor necrosis factor (TNF) receptor-1 signaling complex, mediates activation of the nuclear factor kappaB (NF-kappaB) pathway. RIP2 and RIP3 are related kinases that share extensive sequence homology with the kinase domain of RIP. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 possesses a unique C terminus. RIP3 binds RIP through this unique C-terminal segment to inhibit RIP- and TNF receptor-1-mediated NF-kappaB activation. We have identified a unique homotypic interaction motif at the C terminus of both RIP and RIP3 that is required for their association. Sixty-four amino acids within RIP3 and 88 residues within RIP are sufficient for interaction of the two proteins. This interaction is a prerequisite for RIP3-mediated phosphorylation of RIP and subsequent attenuation of TNF-induced NF-kappaB activation.     

Toll-like Receptor 3-mediated Necrosis via TRIF,RIP3, and MLKL

Toll-like receptor (TLR) signaling is triggered by pathogen-associated molecular patterns that mediate well established cytokine-driven pathways, activating NF-κB together with IRF3/IRF7. In addition, TLR3 drives caspase 8-regulated programmed cell death pathways reminiscent of TNF family death receptor signaling. We find that inhibition or elimination of caspase 8 during stimulation of TLR2, TLR3, TLR4, TLR5, or TLR9 results in receptor interacting protein (RIP) 3 kinase-dependent programmed necrosis that occurs through either TIR domain-containing adapter-inducing interferon-β (TRIF) or MyD88 signal transduction. TLR3 or TLR4 directly activates programmed necrosis through a RIP homotypic interaction motif-dependent association of TRIF with RIP3 kinase (also called RIPK3). In fibroblasts, this pathway proceeds independent of RIP1 or its kinase activity, but it remains dependent on mixed lineage kinase domain-like protein (MLKL) downstream of RIP3 kinase. Here, we describe two small molecule RIP3 kinase inhibitors and employ them to demonstrate the common requirement for RIP3 kinase in programmed necrosis induced by RIP1-RIP3, DAI-RIP3, and TRIF-RIP3 complexes. Cell fate decisions following TLR signaling parallel death receptor signaling and rely on caspase 8 to suppress RIP3-dependent programmed necrosis whether initiated directly by a TRIF-RIP3-MLKL pathway or indirectly via TNF activation and the RIP1-RIP3-MLKL necroptosis pathway.     

Receptor-interacting protein homotypic interaction motif-dependent control of NF-kappa B activation via the DNA-dependent activator of IFN regulatory factors

DNA-dependent activator of IFN regulatory factors (IRF; DAI, also known as ZBP1 or DLM-1) is a cytosolic DNA sensor that initiates IRF3 and NF-kappaB pathways leading to activation of type I IFNs (IFNalpha, IFNbeta) and other cytokines. In this study, induction of NF-kappaB is shown to depend on the adaptor receptor-interacting protein kinase (RIP)1, acting via a RIP homotypic interaction motif (RHIM)-dependent interaction with DAI. DAI binds to and colocalizes with endogenous RIP1 at characteristic cytoplasmic granules. Suppression of RIP1 expression by RNAi abrogates NF-kappaB activation as well as IFNbeta induction by immunostimulatory DNA. DAI also interacts with RIP3 and this interaction potentiates DAI-mediated activation of NF-kappaB, implicating RIP3 in regulating this RHIM-dependent pathway. The role of DAI in activation of NF-kappaB in response to immunostimulatory DNA appears to be analogous to sensing of dsRNA by TLR3 in that both pathways involve RHIM-dependent signaling that is mediated via RIP1, reinforcing a central role for this adaptor in innate sensing of intracellular microbes.     

Multiple roles for nuclear localization signal (NLS, aa 442-472) of receptor interacting protein 3 (RIP3)

RIP3, a Ser/Thr kinase of RIP (Receptor Interacting Protein) family, is recruited to the TNFR1 signaling complex through RIP and has been shown to mediate apoptosis induction and NF-κB activation. RIP3 is a nucleocytoplasmic shuttling protein and its unconventional nuclear localization signal (NLS, 442-472 aa) is sufficient to trigger apoptosis in the nucleus. In this study, we demonstrate that this NLS exhibits several other roles besides apoptotic function. Firstly, this NLS was found to be required for both RIP3-induced apoptosis and RIP3-mediated NF-κB activation. Next, similar to RHIM motif (RIP homotypic interaction motif), NLS of RIP3 was found to be involved in RIP3-RIP interaction. Furthermore, this NLS was found to be both sufficient and necessary for RIP3 self-association. Our primary data also showed that RIP3 might form a homodimer within cells, and its apoptotic activity may not be required for this dimerization, rather the intactness of NLS determines RIP3-induced apoptosis, since a point mutation at amino acid residue 452 (Ile to Ala) within NLS greatly reduced its apoptotic ability, despite that RIP3 point mutant RIP3/I452A is able to dimerize with wild type RIP3 or itself.     

Apoptosis induced by the toll-like receptor adaptor TRIF is dependent on its receptor interacting protein homotypic interaction motif

TLRs detect specific molecular features of microorganisms and subsequently engage distinct signaling networks through the differential use of Toll/IL-1R (TIR)-domain-containing adapter proteins. In this study, we investigated the control of apoptosis by the TIR domain-containing adapter proteins MyD88, TIR-domain containing adapter protein (TIRAP), TIR-domain-containing adapter-inducing IFN-beta (TRIF), TRIF-related adapter molecule (TRAM), and sterile alpha motifs and beta-catenin/armadillo repeats (SARM). Upon overexpression, TRIF was the sole TIR-adapter to potently engage mammalian cell death signaling pathways. TRIF-induced cell death required caspase activity initiated by the Fas/Apo-1-associated DD protein-caspase-8 axis and was unaffected by inhibitors of the intrinsic apoptotic machinery. The proapoptotic potential of TRIF mapped to the C-terminal region that was found to harbor a receptor interacting protein (RIP) homotypic interaction motif (RHIM). TRIF physically interacted with the RHIM-containing proteins RIP1 and RIP3, and deletion and mutational analyses revealed that the RHIM in TRIF was essential for TRIF-induced apoptosis and contributed to TRIF-induced NF-kappa B activation. The domain that was required for induction of apoptosis could activate NF-kappa B but not IFN regulatory factor-3, yet the activation of NF-kappa B could be blocked by superrepressor I kappa B alpha without blocking apoptosis. Thus, the ability of TRIF to induce apoptosis was not dependent on its ability to activate either IFN regulatory factor-3 or NF-kappa B but was dependent on the presence of an intact RHIM. TRIF serves as an adaptor for both TLR3 and TLR4, receptors that are activated by dsRNA and LPS, respectively. These molecular motifs are encountered during viral and bacterial infection, and the apoptosis that occurs when TRIF is engaged represents an important host defense to limit the spread of infection.     

RIP1 kinase activity promotes steatohepatitis through mediating cell death and inflammation in macrophages

Hepatocyte cell death and liver inflammation have been well recognized as central characteristics of nonalcoholic steatohepatitis (NASH), however, the underlying molecular basis remains elusive. The kinase receptor-interacting protein 1 (RIP1) is a multitasking molecule with distinct functions in regulating apoptosis, necroptosis, and inflammation. Dissecting the role of RIP1 distinct functions in different pathophysiology has absorbed huge research enthusiasm. Wild-type and RIP1 kinase-dead (Rip1^K45A/K45A) mice were fed with high-fat diet (HFD) to investigate the role of RIP1 kinase activity in the pathogenesis of NASH. Rip1^K45A/K45A mice exhibited significantly alleviated NASH phenotype of hepatic steatosis, liver damage, fibrosis as well as reduced hepatic cell death and inflammation compared to WT mice. Our results also indicated that both in vivo lipotoxicity and in vitro saturated fatty acids (palmitic acid) treatment were able to induce the kinase activation of RIP1 in liver macrophages. RIP1 kinase was required for mediating inflammasome activation, apoptotic and necrotic cell death induced by palmitic acid in both bone marrow-derived macrophage and mouse primary Kupffer cells. Results from chimeric mice established through lethal irradiation and bone marrow transplantation further confirmed that the RIP1 kinase in hematopoietic-derived macrophages contributed mostly to the disease progression in NASH. Consistent with murine models, we also found that RIP1 kinase was markedly activated in human NASH, and the kinase activation mainly occurred in liver macrophages as indicated by immunofluorescence double staining. In summary, our study indicated that RIP1 kinase was phosphorylated and activated mainly in liver macrophages in both experimental and clinical NASH. We provided direct genetic evidence that the kinase activity of RIP1 especially in hematopoietic-derived macrophages contributes to the pathogenesis of NASH, through mediating inflammasome activation and cell death induction. Macrophage RIP1 kinase represents a specific and potential therapeutic target for NASH.Subject terms: Cell death and immune response, Chronic inflammation     

Functional conservation and innovation of amphioxus RIP1-mediated signaling in cell fate determination

Recently, receptor interacting protein (RIP)-1 has been recognized as an intracellular sensor at the crossroads of apoptosis, necroptosis, and cell survival. To reveal when this crucial molecule originated and how its function in integrating stress signals evolved, in this study we report on two RIP1 homologs in Chinese amphioxus (Branchiostoma belcheri tsingtauense), designated B. belcheri tsingtauense RIP1a and B. belcheri tsingtauense RIP1b. Phylogenetic analysis indicates that they are generated by domain recombination and lineage-specific duplication. Similar to human RIP1, both B. belcheri tsingtauense RIP1a and B. belcheri tsingtauense RIP1b activate NF-κB in a kinase activity-independent manner and induce apoptosis through the Fas-associated death domain protein-caspase cascade. Moreover, we found that the natural point mutation of Q to I in the RIP homotypic interaction motif of B. belcheri tsingtauense RIP1a provides negative feedback for amphioxus RIP1-mediated signaling. Thus, our study not only suggests that RIP1 has emerged as a molecular switch in triggering cell death or survival in a basal chordate, but also adds new insights into the regulation mechanisms of RIP1-related signaling, providing a novel perspective on human diseases mediated by RIP1.     

RIP1-dependent and independent effects of necrostatin-1 in necrosis and T cell activation

Background

Programmed necrosis/necroptosis is an emerging form of cell death that plays important roles in mammalian development and the immune system. The pro-necrotic kinases in the receptor interacting protein (RIP) family are crucial mediators of programmed necrosis. Recent advances in necrosis research have been greatly aided by the identification of chemical inhibitors that block programmed necrosis. Necrostatin-1 (Nec-1) and its derivatives were previously shown to target the pro-necrotic kinase RIP1/RIPK1. The protective effect conferred by Nec-1 and its derivatives in many experimental model systems was often attributed to the inhibition of RIP1 function.

Methodology/Principal Findings

We compared the effect of Nec-1 and siRNA-mediated silencing of RIP1 in the murine fibrosarcoma cell line L929. Treatment of L929 cells with the pan-caspase inhibitor zVAD-fmk or exogenous TNF induces necrosis. Strikingly, we found that siRNA-mediated silencing of RIP1 inhibited zVAD-fmk induced necrosis, but not TNF-induced necrosis. TNF-induced cell death in RIP1 knocked down L929 cells was inhibited by Nec-1, but not the caspase inhibitor zVAD-fmk. We found that PKA-C§ expression, but not Jnk or Erk activation, was moderately inhibited by Nec-1. Moreover, we found that Nec-1 inhibits proximal T cell receptor signaling independent of RIP1, leading to inhibition of T cell proliferation.

Conclusions/Significance

Our results reveal that besides RIP1, Nec-1 also targets other factors crucial for necrosis induction in L929 cells. In addition, high doses of Nec-1 inhibit other signal transduction pathways such as that for T cell receptor activation. These results highlight the importance to independently validate results obtained using Nec-1 with other approaches such as siRNA-mediated gene silencing. We propose that some of the previous published results obtained using Nec-1 should be re-evaluated in light of our findings.     

NMR resonance assignments of caspase recruitment domain of RIP2 kinase

Receptor interacting protein-2, RIP2, is a serine/threonine kinase and has sequence homology to RIP. It functions as an adaptor molecule for some members from the tumor necrosis factor receptor family and mediates divergent signaling pathways including NF-κB activation and cell death. RIP2 contains an N-terminal kinases domain and a C-terminal caspase activation and recruitment domain (CARD). The apoptotic activity of RIP2 is restricted to its C-terminal CARD domain while NF-κB activation requires the intact RIP2 for binding. RIP2 CARD involved homotypic or heterotypic interactions with members of the death domains superfamily. Here I report backbone and sidechain ¹H, ¹³C and ¹⁵N resonance assignments of soluble RIP2 CARD as a basis for further structural and functional studies.     

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis

RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that?the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked?by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in?vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.     

The serine threonine kinase RIP3: lost and found

Receptor-interacting protein kinase-3 (RIP3, or RIPK3) is an essential protein in the “programmed”, or “regulated” necrosis cell death pathway that is activated in response to death receptor ligands and other types of cellular stress. Programmed necrotic cell death is distinguished from its apoptotic counterpart in that it is not characterized by the activation of caspases; unlike apoptosis, programmed necrosis results in plasma membrane rupture, thus spilling the contents of the cell and triggering the activation of the immune system and inflammation. Here we discuss findings, including our own recent data, which show that RIP3 protein expression is absent in many cancer cell lines. The recent data suggests that the lack of RIP3 expression in a majority of these deficient cell lines is due to methylation-dependent silencing, which limits the responses of these cells to pro-necrotic stimuli. Importantly, RIP3 expression may be restored in many cancer cells through the use of hypomethylating agents, such as decitabine. The potential implications of loss of RIP3 expression in cancer are explored, along with possible consequences for chemotherapeutic response. [BMB Reports 2015; 48(6): 303-312]     

RIP3, a novel apoptosis-inducing kinase.

RIP3 is a novel gene product containing a N-terminal kinase domain that shares extensive homology with the corresponding domain in RIP (receptor-interacting protein) and RIP2. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 has a unique C terminus. RIP3 binds RIP through its unique C-terminal segment and by virtue of this interaction is recruited to the tumor necrosis factor (TNF) receptor-1 signaling complex. Previous studies have shown that RIP mediates TNF-induced activation of the anti-apoptotic NF-kappaB pathway. RIP3, however, attenuates both RIP and TNF receptor-1-induced NF-kappaB activation. Overexpression studies revealed RIP3 to be a potent inducer of apoptosis, capable of selectively binding to large prodomain initiator caspases.     

RIP kinases: key decision makers in cell death and innate immunity

Innate immunity represents the first line of defence against invading pathogens. It consists of an initial inflammatory response that recruits white blood cells to the site of infection in an effort to destroy and eliminate the pathogen. Some pathogens replicate within host cells, and cell death by apoptosis is an important effector mechanism to remove the replication niche for such microbes. However, some microbes have evolved evasive strategies to block apoptosis, and in these cases host cells may employ further countermeasures, including an inflammatory form of cell death know as necroptosis. This review aims to highlight the importance of the RIP kinase family in controlling these various defence strategies. RIP1 is initially discussed as a key component of death receptor signalling and in the context of dictating whether a cell triggers a pathway of pro-inflammatory gene expression or cell death by apoptosis. The molecular and functional interplay of RIP1 and RIP3 is described, especially with respect to mediating necroptosis and as key mediators of inflammation. The function of RIP2, with particular emphasis on its role in NOD signalling, is also explored. Special attention is given to emphasizing the physiological and pathophysiological contexts for these various functions of RIP kinases.     

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.