Engagement of Nucleotide-binding Oligomerization Domain-containing Protein 1 (NOD1) by Receptor-interacting Protein 2 (RIP2) Is Insufficient for Signal Transduction

Following activation, the cytoplasmic pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) interacts with its adaptor protein receptor-interacting protein 2 (RIP2) to propagate immune signaling and initiate a proinflammatory immune response. This interaction is mediated by the caspase recruitment domain (CARD) of both proteins. Polymorphisms in immune proteins can affect receptor function and predispose individuals to specific autoinflammatory disorders. In this report, we show that mutations in helix 2 of the CARD of NOD1 disrupted receptor function but did not interfere with RIP2 interaction. In particular, N43S, a rare polymorphism, resulted in receptor dysfunction despite retaining normal cellular localization, protein folding, and an ability to interact with RIP2. Mutation of Asn-43 resulted in an increased tendency to form dimers, which we propose is the source of this dysfunction. We also demonstrate that mutation of Lys-443 and Tyr-474 in RIP2 disrupted the interaction with NOD1. Mapping the key residues involved in the interaction between NOD1 and RIP2 to the known structures of CARD complexes revealed the likely involvement of both type I and type III interfaces in the NOD1·RIP2 complex. Overall we demonstrate that the NOD1-RIP2 signaling axis is more complex than previously assumed, that simple engagement of RIP2 is insufficient to mediate signaling, and that the interaction between NOD1 and RIP2 constitutes multiple CARD-CARD interfaces.     

Anti-Inflammatory Effect of Pineapple Rhizome Bromelain through Downregulation of the NF-κB- and MAPKs-Signaling Pathways in Lipopolysaccharide (LPS)-Stimulated RAW264.7 Cells

Bromelain is a mixture of proteolytic enzymes derived from pineapple (Ananas comosus) fruit and stem possessing several beneficial properties, particularly anti-inflammatory activity. However, the molecular mechanisms underlying the anti-inflammatory effects of bromelain are unclear. This study investigated the anti-inflammatory effects and inhibitory molecular mechanisms of crude and purified rhizome bromelains on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. RAW264.7 cells were pre-treated with various concentrations of crude bromelain (CB) or purified bromelain (PB), and then treated with LPS. The production levels of pro-inflammatory cytokines and mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α were determined by Griess and ELISA assays. The expressions of inducible nitric oxide synthetase (iNOS), cyclooxygenase (COX)-2, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs)-signaling pathway-related proteins were examined by western blot analysis. The pre-treatment of bromelain dose-dependently reduced LPS-induced pro-inflammatory cytokines and mediators, which correlated with downregulation of iNOS and COX-2 expressions. The inhibitory potency of PB was stronger than that of CB. PB also suppressed phosphorylated NF-κB (p65), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, extracellular signal-regulated kinases, c-Jun amino-terminal kinases, and p38 proteins in LPS-treated cells. PB then exhibited potent anti-inflammatory effects on LPS-induced inflammatory responses in RAW264.7 cells by inhibiting the NF-κB and MAPKs-signaling pathways.     

Roles of NOD1 (NLRC1) and NOD2 (NLRC2) in innate immunity and inflammatory diseases

NOD1 {nucleotide-binding oligomerization domain 1; NLRC [NOD-LRR (leucine-rich repeat) family with CARD (caspase recruitment domain) 1]} and NOD2 (NLRC2) are among the most prominent members of the NLR (NOD-LRR) family –proteins that contain nucleotide-binding NACHT domains and receptor-like LRR domains. With over 20 members identified in humans, NLRs represent important components of the mammalian innate immune system, serving as intracellular receptors for pathogens and for endogenous molecules elaborated by tissue injury. NOD1 and NOD2 proteins operate as microbial sensors through the recognition of specific PG (peptidoglycan) constituents of bacteria. Upon activation, these NLR family members initiate signal transduction mechanisms that include stimulation of NF-κB (nuclear factor-κB), stress kinases, IRFs (interferon regulatory factors) and autophagy. Hereditary polymorphisms in the genes encoding NOD1 and NOD2 have been associated with an increasing number of chronic inflammatory diseases. In fact, potential roles for NOD1 and NOD2 in inflammatory disorders have been revealed by investigations using a series of animal models. In the present review, we describe recent experimental findings associating NOD1 and NOD2 with various autoimmune and chronic inflammatory disorders, and we discuss prospects for development of novel therapeutics targeting these NLR family proteins.     

Tumor Necrosis Factor (TNF) Receptor-associated Factor (TRAF)-interacting Protein (TRIP) Negatively Regulates the TRAF2 Ubiquitin-dependent Pathway by Suppressing the TRAF2-Sphingosine 1-Phosphate (S1P) Interaction

The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys⁶³-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.     

Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy

Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.     

Suppression of lysosomal-associated protein transmembrane 5 ameliorates cardiac function and inflammatory response by inhibiting the nuclear factor-kappa B (NF-κB) pathway after myocardial infarction in mice

Myocardial infarction (MI) as the remarkable presentation of coronary artery disease is still a reason for morbidity and mortality in worldwide. Lysosomal-associated protein transmembrane 5 (LAPTM5) is a lysosomal-related protein found in hematopoietic tissues and has been confirmed as a positive regulator of pro-inflammatory pathways in macrophages. However, the role of LAPTM5 in MI remains unknown. In this study, we found that both mRNA and protein expression levels of LAPTM5 were significantly elevated in MI mice. Suppression of LAPTM5 in myocardial tissues decreased cardiac fibrosis and improved cardiac function after MI. At the molecular level, downregulated LAPTM5 dramatically suppressed the macrophage activation and inflammatory response via inhibiting the activation of the nuclear factor-kappa B (NF-κB) pathway. Collectively, suppression of LAPTM5 in myocardial tissues inhibits the pro-inflammatory response and the cardiac dysfunction caused by MI. This study indicated that LAPTM5 as a pro-inflammatory factor plays a crucial role in MI disease.     

Abietic acid attenuates sepsis-induced lung injury by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway to inhibit M1 macrophage polarization

Lung injury is one of the leading causes of death in sepsis. Abietic acid (AA) has demonstrated anti-inflammatory and bacteriostatic properties. Herein, we established a mouse model of sepsis by cecal ligation and puncture, and intraperitoneally injected AA to treat. Lung injury was assessed by H&E staining and the inflammation in bronchoalveolar lavage fluid (BALF) were assessed by counting the number of inflammatory cells and detecting the content of inflammatory factors. Meanwhile, we also designed to study the effect of AA on lipopolysaccharide (LPS)-induced inflammatory response and macrophage marker gene expression in RAW264.7 cells in vitro. In this study, we found that AA inhibited LPS-induced secretion of inflammatory mediators (IL-1β, TNF-α, IL-6 and MIP-2), and decreased the expression of M1 macrophage e markers (CD16 and iNOS) and p-p65 protein, while increased the expression of M2 macrophage markers (CD206 and Arg-1) in RAW264.7 cells in vitro. In vivo, the therapy of AA not only rescued septic animals, but also attenuated lung injury in sepsis mice. Moreover, AA decreased the number of total cells, neutrophils and macrophages, the conceration of total protein, and the levels of inflammatory mediators in BALF of sepsis mice. Further, we found that AA inhibited M1 macrophage polarization and blocked nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in BALF of sepsis mice. In conclusion, Abietic acid attenuates sepsis-induced lung injury, and its mechanism may be related to reducing inflammation by inhibiting NF-κB signaling to inhibit M1 macrophage polarization.     

Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Cytoplasm-dislocalized Leukemic Nucleophosmin Protein (NPMc)

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis.     

Binding Studies of TNF Receptor Superfamily (TNFRSF) Receptors on Intact Cells

Ligands of the tumor necrosis factor superfamily (TNFSF) interact with members of the TNF receptor superfamily (TNFRSF). TNFSF ligand-TNFRSF receptor interactions have been intensively evaluated by many groups. The affinities of TNFSF ligand-TNFRSF receptor interactions are highly dependent on the oligomerization state of the receptor, and cellular factors (e.g. actin cytoskeleton and lipid rafts) influence the assembly of ligand-receptor complexes, too. Binding studies on TNFSF ligand-TNFRSF receptor interactions were typically performed using cell-free assays with recombinant fusion proteins that contain varying numbers of TNFRSF ectodomains. It is therefore not surprising that affinities determined for an individual TNFSF ligand-TNFRSF interaction differ sometimes by several orders of magnitude and often do not reflect the ligand activity observed in cellular assays. To overcome the intrinsic limitations of cell-free binding studies and usage of recombinant receptor domains, we performed comprehensive binding studies with Gaussia princeps luciferase TNFSF ligand fusion proteins for cell-bound TNFRSF members on intact cells at 37 °C. The affinities of the TNFSF ligand G. princeps luciferase-fusion proteins ranged between 0.01 and 19 nm and offer the currently most comprehensive and best suited panel of affinities for in silico studies of ligand-receptor systems of the TNF family.     

Tumor Necrosis Factor (TNF) Signaling,but Not TWEAK (TNF-like Weak Inducer of Apoptosis)-triggered cIAP1 (Cellular Inhibitor of Apoptosis Protein 1) Degradation,Requires cIAP1 RING Dimerization and E2 Binding

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-κB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-κB, however, delayed activation of NF-κB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-κB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.