Alternative titles; symbols
HGNC Approved Gene Symbol: RIPK1
Cytogenetic location: 6p25.2 Genomic coordinates (GRCh38) : 6:3,063,967-3,115,187 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 6p25.2 | Autoinflammation with episodic fever and lymphadenopathy | 618852 | Autosomal dominant | 3 |
| Immunodeficiency 57 with autoinflammation | 618108 | Autosomal recessive | 3 |
The RIPK1 gene encodes a cytosolic protein kinase that controls multiple signaling pathways leading to inflammation and apoptotic or necroptotic cell death. RIPK1 is present in protein complexes that mediate signal transduction from cell surface receptors (summary by Cuchet-Lourenco et al., 2018).
RIPK1 is a key signaling molecule in the programmed necrosis pathway, which plays important roles in development, tissue damage response, and antiviral immunity (Sun et al., 2012).
Two cell surface cytokine receptors, FAS (134637) and tumor necrosis factor (TNF) receptor (see TNFR1, 191190), trigger apoptosis by natural ligands or specific agonist antibodies. Both receptors contain a conserved intracellular death domain. Using a yeast 2-hybrid screen with the cytoplasmic domain of FAS as bait, Stanger et al. (1995) isolated partial cDNAs encoding a receptor-interacting protein which they designated RIP. They used the partial cDNAs to isolate mouse cDNAs corresponding to the entire Rip coding region.
Hsu et al. (1996) isolated a full-length human RIP cDNA. They reported that the predicted 671-amino acid RIP protein contains an N-terminal protein kinase domain, a C-terminal death domain, and a unique internal region that they called the intermediate domain.
Hartz (2012) mapped the RIPK1 gene to chromosome 6p25.2 based on an alignment of the RIPK1 sequence (GenBank AB208926) with the genomic sequence (GRCh37).
Stanger et al. (1995) found that overexpression of Rip in mammalian cells induced morphologic changes characteristic of apoptosis. They suggested that RIP may be an important element in the signal transduction machinery that mediates programmed cell death.
The death domain of TNFR1 triggers distinct signaling pathways leading to apoptosis and NF-kappa-B (see 164011) activation through its interaction with the death domain protein TRADD (603500). Members of the TRAF protein family have been implicated in the activation of NF-kappa-B by the TNF superfamily. By yeast 2-hybrid and coimmunoprecipitation studies using mammalian cell extracts, Hsu et al. (1996) showed that RIP interacts with TRADD, TRAF1 (601711), TRAF2 (601895), and TRAF3 (601896). RIP and TRADD interacted via their respective death domains; RIP interacted with TRAF2 via either the kinase or the intermediate domain. TRADD acted as an adaptor protein to recruit RIP to the TNFR1 complex in a TNF (191160)-dependent process. Overexpression of RIP induced both NF-kappa-B activation and apoptosis, but overexpression of the RIP death domain blocked TNF-mediated NF-kappa-B activation. Hsu et al. (1996) demonstrated that RIP is a serine/threonine protein kinase, but found that overexpression of RIP mutants lacking kinase activity could activate NF-kappa-B. They suggested that RIP kinase activity is not required for TNFR1 to signal NF-kappa-B activation. They proposed a model in which various proteins, such as TRAFs and RIP, are recruited to the TNFR1 complex by virtue of their association with TRADD. This complex would activate at least 2 distinct signaling cascades. TRADD and RIP would be involved in both apoptosis and NF-kappa-B signaling, utilizing distinct domains for both pathways.
Meylan et al. (2004) noted that TRIF (607601) is necessary for TLR3 (603029)-dependent activation of NFKB. They showed that the C-terminal RIP homotypic interaction motif (RHIM) of TRIF recruits RIP1 and RIP3 (RIPK3; 605817) via their intermediary domains. Overexpression of RIP3 resulted in dose-dependent inhibition of TRIF-induced NFKB activation. Coimmunoprecipitation and RT-PCR analysis indicated that TRIF serves as an adaptor protein linking RIP1 and TLR3 and that RIP1 mediates TLR3-induced NFKB activation. Meylan et al. (2004) concluded that RIP1 is important not only in later phases of the immune response, when TNF is active, but also at the beginning, when an antiviral immune response is engaged via TLR3 interaction with double-stranded RNA.
Alvarez et al. (2010) showed that SphK1 (603730) and the production of sphingosine-1-phosphate (S1P; see 601974) is necessary for lysine-63-linked polyubiquitination of RIP1, phosphorylation of I-kappa-B kinase (see 600664) and I-kappa-B-alpha (164008), and I-kappa-B-alpha degradation, leading to NF-kappa-B activation. These responses were mediated by intracellular S1P independently of its cell surface G protein-coupled receptors. S1P specifically binds to TRAF2 at the N-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, markedly increased recombinant TRAF2-catalyzed lysine-63-linked, but not lysine-48-linked, polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a (602961). Alvarez et al. (2010) concluded that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing cofactor for TRAF2 E3 ubiquitin ligase activity, indicating a new paradigm for the regulation of lysine-63-linked polyubiquitination. These results also highlighted the key role of SphK1 and its product S1P in TNF-alpha (191160) signaling and the canonical NF-kappa-B activation pathway important in inflammatory, antiapoptotic, and immune processes.
Gerlach et al. (2011) identified SHARPIN (611885) as a third component of the linear ubiquitin chain assembly complex (LUBAC), recruited to the CD40 (109535) and TNF receptor (see 191190) signaling complexes together with its other constituents, HOIL1 (RBCK1; 610924) and HOIP (RNF31; 612487). Mass spectrometry of TNF signaling complexes revealed RIP1 and NEMO (300248) to be linearly ubiquitinated.
By studying host responses to E. coli cytotoxic necrotizing factor-1 (CNF1) in Drosophila and human cells, Boyer et al. (2011) showed that the host indirectly sensed the pathogen via its modification and activation of RAC2 (602049). After CNF1 modified RAC2, RAC2 interacted with the innate immune adaptors Imd and RIPK1-RIPK2 (603455) in flies and human cells, respectively. Induction of the immune response in flies required CNF1 enzymatic activity, which, in mammals, catalyzes deamidation of a glutamine to glutamic acid in RAC2, abolishing GTPase activity and locking the enzyme into an active form. Modified RAC2 interacted with RIPK1 and RIPK2 to induce immune activation via NFKB (see 164011) and IL8 (146930) expression in human cells. Boyer et al. (2011) concluded that virulence factors such as CNF1 induce an immune response through this mechanism, whereas avirulent microbes fail to provoke host responses.
Using a chemical inhibitor that interrupted TNF-induced necrosis in human cell lines, Sun et al. (2012) identified MLKL (615153) as a downstream effector of RIP1 and RIP3. MLKL was phosphorylated by RIP3, and this phosphorylation was required for expansion of RIP3-positive necrotic foci and phosphorylation of downstream necrosis effectors.
Wang et al. (2012) found that RIP1, RIP3, and MLKL formed a necrosis complex in human cell lines. Upon induction of necrosis by TNF-alpha, both isoforms of PGAM5 (614939), PGAM5L and PGAM5S, interacted with the RIP1-RIP3-MLKL necrosis complex and were phosphorylated. Phosphorylated PGAM5S then recruited the mitochondrial fission factor DRP1 (DNM1L; 603850) and activated DRP1 by dephosphorylation, resulting in mitochondrial fragmentation and execution of necrosis. Blockade of phosphorylation or dephosphorylation signaling at several points in this signaling cascade, or knockdown of PGAM5 expression, blocked TNF-alpha-induced necrosis. Knockdown experiments showed that both PGAM5 isoforms and DRP1, but not RIP1, RIP3, or MLKL, were also involved in necrosis induced by reactive oxygen species or ionophore-mediated calcium shock.
Li et al. (2013) discovered that death domains in several proteins, including TRADD, FADD (602457), RIPK1, and TNFR1, were directly inactivated by NleB, an enteropathogenic E. coli type III secretion system effector known to inhibit host NF-kappa-B signaling. NleB contained an unprecedented N-acetylglucosamine (GlcNAc) transferase activity that specifically modified a conserved arginine in these death domains (arg235 in the TRADD death domain). NleB GlcNAcylation of death domains blocked homotypic/heterotypic death domain interactions and assembly of the oligomeric TNFR1 complex, thereby disrupting TNF signaling in enteropathogenic E. coli infected cells, including NF-kappa-B signaling, apoptosis, and necroptosis. Type III-delivered NleB also blocked FAS ligand (134638) and TRAIL (603598)-induced cell death by preventing formation of a FADD-mediated death-inducing signaling complex (DISC). The arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of enteropathogenic E. coli infection.
Pearson et al. (2013) independently reported that the type III secretion system (T3SS) effector NleB1 from enteropathogenic E. coli binds to host cell death-domain-containing proteins and thereby inhibits death receptor signaling. Protein interaction studies identified FADD, TRADD, and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical DISC and proteolytic activation of caspase-8 (CASP8; 601763), an essential step in death receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified arg117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defense was demonstrated using mice deficient in the FAS signaling pathway, which showed delayed clearance of the enteropathogenic E. coli-like mouse pathogen Citrobacter rodentium and reversion to virulence of an NleB mutant. Pearson et al. (2013) concluded that the activity of NleB suggested that enteropathogenic E. coli and other attaching and effacing pathogens antagonize death receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.
Dannappel et al. (2014) demonstrated that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific Ripk1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells, and premature death in mice. This pathology developed independently of the microbiota and of Myd88 (602170) signaling but was partly rescued by Tnfr1 (TNFRSF1A; 191190) deficiency. Epithelial Fadd ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific Ripk1 knockout. However, mice lacking both Ripk1 and Fadd in IECs displayed Ripk3 (605817)-dependent IEC necroptosis, Paneth cell loss, and focal erosive inflammatory lesions in the colon. Moreover, a Ripk1 kinase-inactive knockin delayed but did not prevent inflammation caused by Fadd deficiency in IECs or keratinocytes, showing that Ripk3-dependent necroptosis of Fadd-deficient epithelial cells only partly requires Ripk1 kinase activity. Epidermis-specific Ripk1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by Ripk3, but not Fadd, deficiency. Dannappel et al. (2014) concluded that these findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, the authors postulated that RIPK1 is a master regulator of epithelial cell survival, homeostasis, and inflammation in the intestine and the skin.
Takahashi et al. (2014) generated Ripk1 conditional knockout mice and showed that mice lacking Ripk1 in IECs spontaneously develop severe intestinal inflammation associated with IEC apoptosis, leading to early death. This early lethality was rescued by antibiotic treatment, Myd88 deficiency, or Tnfr1 deficiency, demonstrating the importance of commensal bacteria and Tnf in the IEC Ripk1 knockout phenotype. Casp8 deficiency, but not Ripk3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of Ripk1 in suppressing Casp8-dependent apoptosis but not Ripk3-dependent necroptosis in the intestine. Ripk1 kinase-dead knockin mice did not exhibit any sign of inflammation, suggesting that Ripk1-mediated protection resides in its kinase-independent platform function. Depletion of Ripk1 in intestinal organoid cultures sensitized them to Tnf-induced apoptosis, confirming the in vivo observations. Unexpectedly, Tnf-mediated Nfkb activation remained intact in these organoids. Takahashi et al. (2014) concluded that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NFKB (see 164011) activation.
Dying cells initiate adaptive immunity by providing antigens and apoptotic stimuli for dendritic cells, which in turn activate CD8-positive T cells through antigen cross-priming. Yatim et al. (2015) established models of apoptosis and necroptosis in which dying cells were generated through dimerization of RIPK3 and CASP8, respectively. They found that release of inflammatory mediators, such as damage-associated molecular patterns, was not sufficient for CD8-positive T-cell cross-priming. Instead, robust cross-priming required RIPK1 signaling and NFKB-induced transcription within the dying cells. Lack of NFKB signaling in necroptosis or inflammatory apoptosis reduced priming efficiency and tumor immunity. Yatim et al. (2015) proposed that coordinated inflammatory and cell death signaling pathways within dying cells are required for adaptive immunity.
Ito et al. (2016) found that optineurin (OPTN; 602432) actively suppressed RIPK1-dependent signaling by regulating its turnover. Loss of OPTN led to progressive dysmyelination and axonal degeneration through engagement of necroptotic machinery in the CNS, including RIPK1, RIPK3 (605817), and mixed lineage kinase domain-like protein (MLKL; 615153). Furthermore, RIPK1- and RIPK3-mediated axonal pathology was commonly observed in SOD1(G93A) (147450.0008) transgenic mice and pathologic samples from patients with amyotrophic lateral sclerosis (ALS; see 105400). Thus, RIPK1 and RIPK3 play a critical role in mediating progressive axonal degeneration.
Seifert et al. (2016) reported that the principal components of the necrosome, receptor-interacting proteins RIP1 and RIP3, are highly expressed in pancreatic ductal adenocarcinoma (PDA) and are further upregulated by the chemotherapy drug gemcitabine. Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype. By contrast, in vivo deletion of RIP3 or inhibition of RIP1 protected against oncogenic progression in mice and was associated with the development of a highly immunogenic myeloid and T cell infiltrate. The immune-suppressive tumor microenvironment associated with intact RIP1/RIP3 signaling depended in part on necroptosis-induced expression of the chemokine attractant CXCL1 (155730), and CXCL1 blockade protected against PDA. Moreover, cytoplasmic SF3B3 (605592), a subunit of the histone deacetylase complex, was expressed in PDA in a RIP1/RIP3-dependent manner, and MINCLE (609962), its cognate receptor, was upregulated in tumor-infiltrating myeloid cells. Ligation of MINCLE by SF3B3 promoted oncogenesis, whereas deletion of MINCLE protected against oncogenesis and phenocopied the immunogenic reprogramming of the tumor microenvironment that was induced by RIP3 deletion. Cellular depletion suggested that whereas inhibitory macrophages promote tumorigenesis in PDA, they lose their immune-suppressive effects when RIP3 or MINCLE is deleted. Accordingly, T cells, which are not protective against PDA progression in mice with intact RIP3 or MINCLE signaling, were reprogrammed into indispensable mediators of antitumor immunity in the absence of RIP3 or MINCLE. Seifert et al. (2016) concluded that their work described parallel networks of necroptosis-induced CXCL1 and MINCLE signaling that promote macrophage-induced adaptive immune suppression and thereby enable PDA progression.
Lin et al. (2016) showed that RIPK1 prevents skin inflammation by inhibiting activation of RIPK3-MLKL-dependent necroptosis mediated by Z-DNA binding protein-1 (ZBP1; 606750). ZBP1 deficiency inhibited keratinocyte necroptosis and skin inflammation in mice with epidermis-specific RIPK1 knockout. Moreover, mutation of the conserved RIP homotypic interaction motif (RHIM) of endogenous mouse RIPK1 caused perinatal lethality that was prevented by RIPK3, MLKL, or ZBP1 deficiency. Furthermore, mice expressing only RHIM-mutated RIPK1 (RIPK1(mRHIM)) in keratinocytes developed skin inflammation that was abrogated by MLKL or ZBP1 deficiency. Mechanistically, ZBP1 interacted strongly with phosphorylated RIPK3 (605817) in cells expressing RIPK1(mRHIM), suggesting that the RIPK1 RHIM prevents ZBP1 from binding and activating RIPK3. Lin et al. (2016) concluded that their results showed that RIPK1 prevents perinatal death as well as skin inflammation in adult mice by inhibiting ZBP1-induced necroptosis, and identified ZBP1 as a critical mediator of inflammation beyond its role in antiviral defence.
Newton et al. (2016) showed that RHIM in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 from activating RIPK3 upstream of MLKL. Ripk1(RHIM/RHIM) mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on thr231 and ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1(RHIM/RHIM) mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but was not known to play a role in development, also prevented perinatal lethality in Ripk1(RHIM/RHIM) mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1(RHIM/RHIM) Mlkl -/- macrophages, but not in wildtype, Mlkl -/-, or Ripk1(RHIM/RHIM)Ripk3(RHIM/RHIM) macrophages. Newton et al. (2016) concluded that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.
By mass spectrometric, coimmunoprecipitation, and mutation analyses, Feltham et al. (2018) showed that MIB2 (611141) bound to the linker region of oligomerized RIPK1. RIPK1 recruited MIB2 to TNFR signaling complex I, but MIB2 had no role in TNF-induced activation of NFKB, induction of NFKB target genes, or production of cytokines. Instead, MIB2 regulated apoptosis and protected cells from TNF-induced and RIPK1-dependent cell death. MIB2 bound RIPK1 to prevent RIPK1-induced cell death through a mechanism that was dependent on MIB2 E3 ligase activity. MIB2 ubiquitylated RIPK1 upon TNF stimulation, independently of CIAPs (see 601712), with different types of ubiquitin chains at multiple sites to repress RIPK1 kinase activity and cell death. MIB2 antagonized the lethal effects of TNF by attaching ubiquitin chains to the linker and C-terminal portion of RIPK1, thereby skewing the signaling potential of TNF toward prosurvival and proinflammation rather than cell death.
Newton et al. (2019) showed that knockin mice that express catalytically inactive caspase-8 (601763) carrying the C362A mutation die as embryos owing to MLKL (615153)-dependent necroptosis, similar to caspase-8-deficient mice. Thus, caspase-8 must cleave itself, other proteins, or both to inhibit necroptosis. Mice that express the mutant caspase-8(D212A/D218A/D225A/D387A), which cannot cleave itself, were viable, as were mice that express cFLIP (603599) or CYLD (605018) proteins that had been mutated to prevent cleavage by caspase-8. By contrast, mice that express RIPK1(D325A), in which the caspase-8 cleavage site asp325 had been mutated, died midgestation. Embryonic lethality was prevented by inactivation of RIPK1, loss of TNFR1 (191190), or loss of both MLKL and the caspase-8 adaptor FADD (602457), but not by loss of MLKL alone. Thus, RIPK1(D325A) appeared to trigger cell death mediated by TNF, the kinase activity of RIPK1, and FADD-caspase-8. Accordingly, dying endothelial cells that contained cleaved caspase-3 (600636) were abnormally abundant in yolk sacs of Ripk1(D325A/D325A) embryos. Heterozygous Ripk1(D325A/+) cells and mice were viable, but were also more susceptible to TNF-induced cell death than were wildtype cells or mice. Newton et al. (2019) concluded that their data showed that asp325 of RIPK1 is essential for limiting aberrant cell death in response to TNF, consistent with the idea that cleavage of RIPK1 by caspase-8 is a mechanism for dismantling death-inducing complexes.
Muendlein et al. (2020) showed that deficiency of the long form (L) of cFLIP (cFLIP(L)) promotes mitochondrial complex II (see 600857) formation driving pyroptosis and the secretion of IL1-beta (147720) in response to lipopolysaccharide (LPS) alone. cFLIP(L) deficiency was sufficient to drive complex II formation in response to LPS. RIP1 and CASP8 recruitment to FADD occurred as early as 2 hours after LPS addition. Muendlein et al. (2020) found that in macrophages and perhaps in other cells, if levels of cFLIP(L) are sufficiently high, CASP8 activation and pyroptosis are inhibited. When cFLIP(L) levels are low, CASP8 homodimers form readily. Fully active CASP8 cleaves and activates distant targets, and LPS-activated macrophages rapidly undergo pyroptosis and secrete IL1-beta. CASP3, CASP7 (601761), and CASP9 (602234) are dispensable for CASP8-driven pyroptosis in the absence of cFLIP(L). Instead, CASP8 likely directly activates gasdermin D (GSDMD; 617042) to drive pyroptosis and the NLRP3 (606416) inflammasome to drive IL1-beta maturation and release.
Jiao et al. (2020) showed that Z-alpha-dependent sensing of endogenous ligands induces ZBP1 (606750)-mediated perinatal lethality in mice expressing RIPK1 with a mutated RIP homotypic interaction motif (RHIM), skin inflammation in mice with epidermis-specific RIPK1 deficiency, and colitis in mice with intestinal epithelial-specific FADD (602457) deficiency. Consistently, functional Z-alpha domains were required for ZBP1-induced necroptosis in fibroblasts that were treated with caspase inhibitors or expressed RIPK1 with mutated RHIM. Inhibition of nuclear export triggered the Z-alpha-dependent activation of RIPK3 (605817) in the nucleus resulting in cell death, which suggested that ZBP1 may recognize nuclear Z-form nucleic acids. Jiao et al. (2020) found that ZBP1 constitutively bound cellular double-stranded RNA in a Z-alpha-dependent manner. Complementary reads derived from endogenous retroelements were detected in epidermal RNA, which suggested that double-stranded RNA derived from these retroelements may act as a Z-alpha-domain ligand that triggers the activation of ZBP1. Jiao et al. (2020) concluded that their results provided evidence that the sensing of endogenous Z-form nucleic acids by ZBP1 triggers RIPK3-dependent necroptosis and inflammation, which could underlie the development of chronic inflammatory conditions, particularly in individuals with mutations in RIPK1 and CASP8 (601763).
Immunodeficiency 57 with Autoinflammation
In 4 patients from 3 unrelated consanguineous families with immunodeficiency-57 with autoinflammation (IMD57; 618108), Cuchet-Lourenco et al. (2018) identified homozygous loss-of-function mutations in the RIPK1 gene (603453.0001-603453.0003). The mutations, which were found by exome sequencing, segregated with the disorder in the families. Patient cells showed absence of the RIPK1 protein. Functional studies of patient cells showed impaired mitogen-activated protein kinase activation, impaired phosphorylation of downstream signaling molecules, impaired proinflammatory signaling downstream of TNFR1 (191190) and TLR3 (603029), and defective secretion of certain cytokines. The findings were consistent with dysregulation of T-cell responses. Patient cells were also prone to necroptosis, or inflammatory cell death, particularly when stimulated with LPS. These abnormalities could be reversed by expression of wildtype RIPK1. Similar results were observed in vitro in a monocyte-like cell line with CRISPR/Cas9-mediated knockdown of RAPK1. The findings indicated that RIPK1 plays a critical role in the human immune system.
Autoinflammation with Episodic Fever and Lymphadenopathy
In 7 patients from 3 unrelated families with autoinflammation with episodic fever and lymphadenopathy (AIEFL; 618852), Lalaoui et al. (2020) identified heterozygous mutations affecting the same highly conserved residue in the RIPK1 gene: D324N (603453.0004), D324H (603453.0005), and D324Y (603453.0006). The mutations, which were found by exome sequencing and confirmed by Sanger sequencing, occurred de novo in 2 patients and was inherited in 5 affected individuals spanning 3 generations of family 2. None were present in public databases, including gnomAD. The affected residue is a essential for RIPK1 cleavage by caspase-8 and caspase-6 (CASP6; 601532), and the mutant proteins were demonstrated to be resistant to CASP-mediated cleavage in vitro. Serum and peripheral blood cells derived from patient 7 with the D324Y mutation showed increased levels of TNF, IL6 (147640), and IL1B (147720) when stimulated with LPS compared to controls; TNF levels also increased in response to poly(I:C) treatment in patient cells. However, fibroblasts derived from other patients did not show increased NFKB activation in response to TNF compared to controls. These findings suggested that RIPK1 cleavage may limit inflammation in an NFKB-independent manner. The patients did not respond to treatment with TNF inhibitors, but did respond to IL6 blockade. Detailed studies in mice with a D325A mutation, which also blocks caspase-mediated cleavage, showed a complex disruption of inflammatory signaling pathways, including sensitization to TNF-induced cell death and activation of caspase-8-mediated apoptosis, which may contribute to additional cytokine induction. The findings also indicated that inappropriate activation of these signaling pathways can lead to necroptosis.
In 4 members of a family (family 2) and in an unrelated Chinese boy (family 1) with AIEFL, Tao et al. (2020) identified heterozygous mutations affecting the same residue in the RIPK1 gene: D324H (603453.0005) and D324V (603453.0007). The mutations, which were found by exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in family 2 and occurred de novo in the Chinese boy (patient 1). In vitro studies in patient fibroblasts and transfected HEK293 cells showed that the mutations blocked caspase-8-mediated cleavage of RIPK1. Patient serum showed increased levels of pro-inflammatory cytokines and chemokines, such as IL6, TNF, and gamma-IFN, and patient immune cells showed an exaggerated inflammatory response to stimulation with LPS compared to controls. There was also evidence of activation of the NFKB and MAPK inflammatory signaling pathways. Patient immune cells showed increased sensitivity to both TNF-induced apoptosis and necroptosis compared to control cells, and this could be suppressed by inhibiting the kinase activity of RIPK1. Further detailed studies in patient cells and mouse embryonic fibroblasts indicated that the mutations result in a gain-of-function effect that promotes activation of the RIPK1 kinase, which leads to an increased inflammatory response, increased production of IL6, and hypersensitivity to apoptosis and necroptosis. In contrast, patient fibroblasts demonstrated some compensatory mechanisms to resist cell death.
Kelliher et al. (1998) introduced a RIP-null mutation in mice through homologous recombination. RIP-deficient mice appeared normal at birth but failed to thrive, displaying extensive apoptosis in both the lymphoid and adipose tissue and dying at 1 to 3 days of age. RIP-null cells were highly sensitive to TNF-alpha-induced cell death, and this sensitivity was accompanied by a failure to activate NF-kappa-B.
Zhang et al. (2011) showed that FADD (602457)-null embryos contain raised levels of RIP1 and exhibit massive necrosis. To investigate a potential in vivo functional interaction between RIP1 and FADD, null alleles of RIP1 were crossed into Fadd-null mice. Notably, RIP1 deficiency allowed normal embryogenesis of Fadd-null mice. Conversely, the developmental defect of Rip1-null lymphocytes was partially corrected by FADD deletion. Furthermore, RIP1 deficiency fully restored normal proliferation in Fadd-null T cells but not in Fadd-null B cells. Fadd-null/Rip1-null double-knockout T cells are resistant to death induced by Fas (134637) or TNF-alpha and show reduced NF-kappa-B activity. Therefore, Zhang et al. (2011) concluded that their data demonstrated 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.
Dillon et al. (2014) found that Ripk1 -/- mice died shortly after birth, but that lethality could be prevented with normal maturation in Ripk1 -/- mice that also lacked Ripk3 and either Casp8, Fadd, or Tnfr1. Mice lacking only 1 of the latter 3 genes in addition to Ripk1, or mice lacking both Ripk1 and Nik (MAP4K4; 604666), were not protected. Lethality was delayed upon disruption of Trif or Ifnar1 (107450) signaling in Ripk1 -/- Tnfr1 -/- mice. Dillon et al. (2014) concluded that RIPK1 functions to prevent perinatal lethality caused by the activity of TNFR1, FADD, and CASP8, and that it prevents perinatal lethality due to TRIF, IFN, and RIPK3 activity.
Independently, Rickard et al. (2014) found that mice lacking Ripk1, Ripk3, and Casp8 survived past weaning. Inflammation was reduced and lethality was delayed up to the time of weaning in mice lacking Ripk1 and Ripk3, Ripk1 and Mlkl (600136), or Ripk1 and Myd88. Ripk1-deficient cells failed to engraft in lethally irradiated hosts in the absence of Tnf blockade. Rickard et al. (2014) concluded that RIPK1 has an essential role in immune homeostasis and emergency hematopoiesis.
Berger et al. (2014) generated mice with a kinase-dead mutation (lys45 to ala; K45A) in Ripk1. Ripk1(K45A) mice were viable and healthy, and their macrophages were protected against necroptotic stimuli in vitro and in vivo. Crossing Ripk1(K45A) mice with Sharpin (611885)-deficient cpdm mice protected the latter from severe skin and multiorgan inflammation. Berger et al. (2014) proposed that RIPK1 is an attractive target for TNF-driven inflammatory diseases.
In 2 brothers, born of consanguineous Pakistani parents (family A), with immunodeficiency-57 (IMD57; 618108), Cuchet-Lourenco et al. (2018) identified a homozygous 4-bp deletion (TTTA) in exon 6 of the RIPK1 gene (ENST00000380409), resulting in a frameshift and premature termination. The mutation occurred in the N-terminal kinase domain. The mutation, which was found by exome sequencing, segregated with the disorder in the family and was not found in the gnomAD database. Patient cells showed complete loss of the RIPK1 protein.
In a girl, born of consanguineous Saudi Arabian parents (family B) with immunodeficiency-57 (IMD57; 618108), Cuchet-Lourenco et al. (2018) identified a homozygous 21-bp deletion in the RIPK1 gene (ENST00000380409) that removed 1 nucleotide in exon 4 and 20 in the following intron. This deletion activated an alternative splice site in intron 4, such that the RIPK1 transcript lacked the last nucleotide of exon 4 and had an insertion of 48 nucleotides from the following intron. The mutation occurred in the N-terminal kinase domain and resulted in a premature stop codon. The mutation, which was found by exome sequencing, segregated with the disorder in the family and was not found in the gnomAD database. Patient cells showed complete loss of the RIPK1 protein.
In a girl, born of consanguineous Saudi Arabian parents (family C) with immunodeficiency-57 (IMD57; 618108), Cuchet-Lourenco et al. (2018) identified a homozygous 2,064-bp deletion (chr6.3,082,952_3,085,016del, ENST00000380409) that completely removed exon 4 of the RIPK1 gene. The mutation occurred in the N-terminal kinase domain and resulted in a premature stop codon. The mutation, which was found by exome sequencing, segregated with the disorder in the family and was not found in the gnomAD database. Patient cells showed complete loss of the RIPK1 protein.
In a 10-year-old girl (P1) with autoinflammation with episodic fever and lymphadenopathy (AIEFL; 618852), Lalaoui et al. (2020) identified a de novo heterozygous c.970G-A transition in the RIPK1 gene, resulting in an asp324-to-asn (D324N) substitution at a highly conserved residue in the CASP6 (601532)/CASP8 (601763) cleavage motif. The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, was not found in public databases, including gnomAD. In vitro studies showed that the mutant RIPK1 protein was resistant to caspase-mediated cleavage.
In a mother and her 3 sons (family 2) with autoinflammation with episodic fever and lymphadenopathy (AIEFL; 618852), Tao et al. (2020) identified a heterozygous c.970G-C transversion (c.970G-C, NM_003804) in the RIPK1 gene, resulting in a D324H substitution. The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in the family; it occurred de novo in the mother. The variant was not found in public databases, including gnomAD. In vitro studies showed that the mutant RIPK1 protein was resistant to CASP8 (601763)-mediated cleavage.
In 5 affected members of a 3-generation family (family 2) with AIEFL, Lalaoui et al. (2020) identified a heterozygous asp324-to-his (D324H) substitution at a highly conserved residue in the CASP6 (601532)/CASP8 cleavage motif. The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, was not found in public databases, including gnomAD. In vitro studies showed that the mutant RIPK1 protein was resistant to caspase-mediated cleavage.
In a 13-year-old boy (P7) with autoinflammation with episodic fever and lymphadenopathy (AIEFL; 618852), Lalaoui et al. (2020) identified a de novo heterozygous asp324-to-tyr (D324Y) substitution in the RIPK1 gene occurring at a highly conserved residue in the CASP6 (601532)/CASP8 (601763) cleavage motif. The mutation, which was found by whole-exome sequencing and confirmed by Sanger sequencing, was not found in public databases, including gnomAD. In vitro studies showed that the mutant RIPK1 protein was resistant to caspase-mediated cleavage, resulting in a gain of function and inappropriate activation of inflammatory pathways.
In a 2-year-old Chinese boy (patient 1) with autoinflammation with episodic fever and lymphadenopathy (AIEFL; 618852), Tao et al. (2020) identified a de novo heterozygous c.971A-T transversion (c.971A-T, NM_003804) in the RIPK1 gene, resulting in an asp324-to-val (D324V) substitution at a highly conserved residue in the CASP8 (601763)-cleavage domain. The mutation, which was found by whole-exome sequencing, was not present in public databases, including gnomAD. In vitro studies showed that the mutant RIPK1 protein was resistant to caspase-mediated cleavage, resulting in a gain of function and inappropriate activation of inflammatory pathways.
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