SUMMARY 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.

SummaryFADD is a common adaptor shared by several death-receptors (DRs) for signaling apoptosis through recruitment and activation of caspase 81-3. DRs are essential for immune homeostasis, but dispensable during embryogenesis. Surprisingly, FADD−/− mice die in utero4-5 and conditional deletion of FADD leads to impaired lymphocyte proliferation6-7. How FADD regulates embryogenesis and lymphocyte responses has been a long standing enigma. FADD could directly bind to RIP1, a serine/threonine kinase which mediates both necrosis and NF-κB activation. Here we show that FADD−/− embryos contain elevated levels of RIP1 and exhibit massive necrosis. To investigate potential in vivo functional interaction between RIP1 and FADD, null alleles of RIP1 were crossed into FADD−/− mice. Strikingly, RIP1 deficiency allowed normal embryogenesis of FADD−/− mice. Conversely, the developmental defect of RIP1−/− lymphocytes was partially corrected by FADD deletion. Furthermore, RIP1 deficiency fully restored normal proliferation in FADD−/− T cells but not in FADD−/− B cells. FADD−/−RIP1−/− double knockout (DKO) T cells are resistant to death induced by Fas or TNFα and display reduced NF-κB activity. Therefore, our data demonstrate an unexpected cell type-specific interplay between FADD and RIP1, which is critical for the regulation of apoptosis and necrosis during embryogenesis and lymphocyte function.

Summary Programmed necrosis or necroptosis is an inflammatory form of cell death that critically requires the receptor interacting protein kinase 3 (RIPK3). Here we showed that RIPK3 controls a separate, necrosis-independent pathway of inflammation through regulating dendritic cells (DCs) cytokine expression. Ripk3−/− bone marrow derived dendritic cells (BMDCs) were highly defective in lipopolysaccharide (LPS)-induced expression of inflammatory cytokines. These effects were caused by impaired NF-κB subunit RelB and p50 activation and caspase 1-mediated processing of interleukin-1β (IL-1β). This DC-specific function of RIPK3 was critical for injury-induced inflammation and tissue repair in response to dextran sodium sulfate (DSS). Ripk3−/− mice exhibited an impaired axis of injury-induced IL-1β, IL-23 and IL-22 cytokine cascade, which was partially corrected by adoptive transfer of wild type DCs, but not Ripk3−/− DCs. These results reveal an unexpected function of RIPK3 in NF-κB activation, DC biology, innate inflammatory cytokine expression, and injury-induced tissue repair.

BackgroundNecroptosis/programmed necrosis is initiated by a macro-molecular protein complex termed the necrosome. Receptor interacting protein kinase 1 (RIPK1/RIP1) and RIP3 are key components of the necrosome. TNFα is a prototypic inducer of necrosome activation, and it is widely believed that deubiquitination of RIP1 at the TNFR-1 signaling complex precedes transition of RIP1 into the cytosol where it forms the RIP1-RIP3 necrosome. Cylindromatosis (CYLD) is believed to promote programmed necrosis by facilitating RIP1 deubiquitination at this membrane receptor complex.Methodology/Principal FindingsWe demonstrate that RIP1 is indeed the primary target of CYLD in TNFα-induced programmed necrosis. We observed that CYLD does not regulate RIP1 ubiquitination at the TNF receptor. TNF and zVAD-induced programmed necrosis was highly attenuated in CYLD-/- cells. However, in the presence of cycloheximide or SMAC mimetics, programmed necrosis was only moderately reduced in CYLD-/- cells. Under the latter conditions, RIP1-RIP3 necrosome formation is only delayed, but not abolished in CYLD-/- cells. We further demonstrate that RIP1 within the NP-40 insoluble necrosome is ubiquitinated and that CYLD regulates RIP1 ubiquitination in this compartment. Hence, RIP1 ubiquitination in this late-forming complex is greatly increased in CYLD-/- cells. Increased RIP1 ubiquitination impairs RIP1 and RIP3 phosphorylation, a signature of kinase activation.Conclusions/SignificanceOur results show that CYLD regulates RIP1 ubiquitination in the TNFα-induced necrosome, but not in the TNFR-1 signaling complex. In cells sensitized to programmed necrosis with SMAC mimetics, CYLD is not essential for necrosome assembly. Since SMAC mimetics induces the loss of the E3 ligases cIAP1 and cIAP2, reduced RIP1 ubiquitination could lead to reduced requirement for CYLD to remove ubiquitin chains from RIP1 in the TNFR-1 complex. As increased RIP1 ubiquitination in the necrosome correlates with impaired RIP1 and RIP3 phosphorylation and function, these results suggest that CYLD controls RIP1 kinase activity during necrosome assembly.

Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored solubility in aged mouse and human lenses. These findings suggest an approach to treating cataracts by stabilizing α-crystallins.

Programmed necrosis or necroptosis is controlled by the action of two serine/threonine kinases, receptor interacting protein kinase 1 (RIP1) and RIP3. The phosphorylation of RIP1 and RIP3 is critical for assembly of the necrosome, an amyloid-like complex that initiates transmission of the pro-necrotic signal. Here, we used site-directed mutagenesis to systematically examine the effects of putative phosphor acceptor sites on RIP1 and RIP3 on TNF-induced programmed necrosis. We found that mutation of individual serine residues in the kinase domain of RIP1 had little effects on RIP1 kinase activity and TNF-induced programmed necrosis. Surprisingly, alanine substitution of Ser89 enhanced RIP1 kinase activity and TNF-induced programmed necrosis without affecting RIP1-RIP3 necrosome formation. This indicates that Ser89 is an inhibitory phospho-acceptor site that can dampen the pro-necrotic function of RIP1. In addition, we show that a phosphor-mimetic mutant of RIP3, S204D, led to programmed necrosis that was refractory to RIP1 siRNA and insensitive to necrostatin-1 inhibition. Our results show that programmed necrosis is regulated by positive and inhibitory phosphorylation events.

SUMMARY DnaK is a molecular chaperone responsible for multiple aspects of proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors, we screened plant-derived extracts against a re-constituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK’s intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaJ. Together, these results highlight a “gray box” screening approach, which approximates a limited amount of the complexity expected in physiological, multi-protein systems.

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