Nek7 is an essential component of the NLRP3 inflammasome
IL-1β and IL-18 are inflammatory cytokines that are synthesized and released after pathogen invasion. Cytokines have several functions associated with infection, inflammation, and autoimmune processes by activating the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. IL-1β and IL-18 production is regulated by inflammasomes, which are multiprotein complexes that assemble upon detection of pathogenic or other danger signals in the cytoplasm. The composition of the inflammasome is dependent of the type of pathogen that initiates inflammasome assembly. For example, NLRP3 inflammasome formation is initiated by pathogens, DNA, single-stranded (ss) RNA, double-stranded (ds) RNA, bacterial toxins, environmental irritants, and endogenous danger signals (1, 2, 3). Formation of the NRLP3 inflammasome is initiated by aggregation of the NLRP3 protein and subsequent recruitment of the adaptor protein ASC and the cysteine protease pro-caspase-1. One of the goals of the Beutler laboratory is to identify components and factors associated with inflammasome function. The Cuties mouse strain was identified in the course of this study. The Cuties mice exhibit reduced IL-18 and IL-1β secretion, impaired NLRP3-dependent immune cell recruitment to sites of infection, and diminished association of NLRP3 with other proteins of the NLRP3 inflammasome after stimulation with nigericin and lipolysaccharide. DNA sequence analysis of the Cuties mice identified the causative mutation in the Nek7 gene. Nek7 encodes NEK7 (NIMA (never in mitosis gene a)-related expressed kinase 7), a member of the NEK family of serine/threonine kinases. NEK7 has known functions in several aspects of mitosis, the process of cell division (4, 5); the function of NEK7 in inflammasome function was unknown. Characterization of the Cuties mice determined that NEK7 binds NLRP3 to form a complex upon inflammasome stimulation, and that NEK7 functions as a switch between mitosis and NLRP3 inflammasome activation (6).
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Newly synthesized proteins enter the endoplasmic reticulum (ER) and move to the Golgi, where they can undergo post-translational modification prior to being distributed to their final location in the cell. In the ER, proteins are folded into their proper conformation with the aid of ER-resident proteins known as chaperones. Sorting of proteins in the ER is dependent on a Lys-Asp-Glu-Leu (KDEL) sequence at the C-terminus of the protein. The proper folding of proteins in the ER can be disrupted by various stresses such as ischemia, oxidative stress, and genetic mutations. These stresses can lead to the accumulation of misfolded proteins in the ER, which triggers the unfolded protein response (UPR). The UPR reduces the amount of misfolded proteins in the ER by stopping protein translation, degrading the misfolded proteins, and stimulating the production of additional chaperones. During protein translocation from the ER to the Golgi, chaperones often accompany their substrates. Retrieval of chaperones back to the ER is essential to prevent protein accumulation in the ER. KDELR1, a KDEL receptor, mediates the return of chaperones and other proteins from the Golgi to the ER by binding the KDEL sequence in the target protein. Once bound by KDELR1, the proteins are packaged into coat protein complex vesicles for retrograde transport to the ER. KDELRs also function in trafficking of proteins through the Golgi and regulate ER quality control (1). Mutations in Kdelr1 have been linked to the development of dilated cardiomyopathy in transgenic mice (2). The mutant mice exhibited accumulation of misfolded proteins in the ER due to impaired quality control in the ER. We recently identified a new strain of mice called daniel_gray that exhibited compromised antiviral immunity, reduced expression of the T cell receptor, and low numbers of lymphocytes in the blood (3). All of the phenotypes were linked to a mutation in the Kdelr1 gene. Mutation in Kdelr1 is likely to affect the retrieval of ER-resident proteins back to the ER, trafficking from the Golgi, and the regulation of ER stress. The reduced numbers of T cells were proposed to be due a sensitization of these cells to ER stress and subsequent apoptosis.
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Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) is an adaptor protein that directly binds the cytoplasmic regions of several immune response receptors, including, lymphotoxin-β receptor, CD40, TACI (transmembrane activator and CAML interactor), BCMA (B-cell maturation antigen), LMP1 (latent membrane protein 1), BAFFR (B-cell-activating factor receptor), RANK (receptor activator of NF-κB), HVEM (herpesvirus entry mediator), EDAR (ectodysplasin A receptor), XEDAR (X-linked ectodermal dysplasia receptor), CD137, and OX40 (1-4). TRAF3 also indirectly interacts with other receptors including TNFR1 and IL-1R as well as TLR3, TLR4, TLR7, and TLR9 by associating with adaptor proteins [(5); reviewed in (6)]. After receptor activation, TRAF3 promotes signaling by communicating the receptor signal to downstream signaling proteins. Downstream of the lymphotoxin-β receptor, BAFFR, and RANK receptors, TRAF3 is part of the non-canonical NF-κB (NF-κB2) signaling pathway (7, 8). The NF-κB2 signaling pathway regulates secondary lymphoid organogenesis, and thymic epithelial cell development as well as B cell development, maintenance, and antibody production by initiating the transcription of NF-κB2 target genes. Mutations in human TRAF3 are often observed in B cell malignancies including multiple myeloma, Waldenström's macroglobulinemia, non-Hodgkin lymphoma, splenic marginal zone lymphoma, B-cell chronic lymphocytic leukemia, and mantle cell lymphoma [(9-11); reviewed in (12)]. Changes in TRAF3 expression in these malignancies are linked to elevated NF-κB2 activity, which leads to enhanced B cell survival and an increased propensity for malignant transformation [reviewed in (13)]. We identified two mutations in Traf3 that caused immune deficiencies: hulk and banasplit. Both mouse strains exhibited deficient T-dependent and T-independent antibody responses to antigens as well as a reduced frequency of peripheral blood B cells. The phenotype was attributed to a loss in the ability of TRAF3 to mediate T helper cell function in antigen-specific IgG responses to T cell-dependent antigens (14, 15).
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The JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway is activated upon binding of cytokines to their receptors on the immune cell surface. Cytokine binding to cytokine receptors stimulates the activation of the JAK tyrosine kinases, which are associated with the cytoplasmic tails of the receptors. The JAK proteins phosphorylate residues on the receptor, creating a ligand for the STAT proteins. After binding to the receptor and phosphorylation by the JAK proteins, the STAT proteins are activated, promoting their movement from the cytoplasm of the cell to the nucleus, where they promote transcription (1). In the course of our immunological screening pipeline, three independent mouse strains, thistle, citron, and mount_tai, were identified that exhibited various aberrant immunological phenotypes, including reduced numbers of T cells and natural killer cells as well as increased numbers of neutrophils and macrophages. The mice also exhibited increased susceptibility to mouse cytomegalovirus (MCMV) virus. The thistle mouse exhibited a diminished T-dependent antibody response to ovalbumin administered with aluminum hydroxide. All three strains were linked to mutations in Jak3. JAK3 is expressed in natural killer (NK) cells, T cells, B cells, and intestinal epithelial cells, and is downstream of several cytokine receptors including those for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The functions of these cytokines is known: IL-2 functions in peripheral T cell homeostasis and antigen-driven T-cell expansion; IL-4 functions in B-cell maturation and isotype switching; IL-7 is necessary for T and B cell development; IL-9 serves as a growth factor for T cells and mast cells as well as a regulator of hematopoiesis; IL-15 functions in NK cell differentiation; and IL-21 effects on several immune cell types, including the regulation of humoral immune responses, B cell apoptosis, and T cell differentiation [reviewed in (2)]. Mutations in JAK3 are linked to autosomal recessive T- and NK-cell negative/B-cell positive type of severe combined immunodeficiency, which is characterized by a loss of T and NK cells [T−B+NK- SCID; (3-6)]. Patients with SCID have persistent, recurring infections due to loss of T cell-associated immunity. Gain-of-function mutations in JAK3 have also been linked to adult T cell leukemia/lymphoma, early T cell precursor acute lymphoblastic leukemia (TdT+:ALL), T-cell prolymphocytic leukemia (T-PLL), acute megakaryoblastic leukemia (AMKL), cutaneous T cell lymphoma, and extranodal nasal-type natural killer cell lymphoma (7-11).
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