Synthetic Biology Reveals the Uniqueness of the RIP Kinase Domain.

The RIP kinases (RIPKs) play an essential role in inflammatory signaling and inflammatory cell death. However, the function of their kinase activity has been enigmatic, and only recently has kinase domain activity been shown to be crucial for their signal transduction capacity. Despite this uncertainty, the RIPKs have been the subject of intense pharmaceutical development with a number of compounds currently in preclinical testing. In this work, we seek to determine the functional redundancy between the kinase domains of the four major RIPK family members. We find that although RIPK1, RIPK2, and RIPK4 are similar in that they can all activate NF-κB and induce NF-κB essential modulator ubiquitination, only RIPK2 is a dual-specificity kinase. Domain swapping experiments showed that the RIPK4 kinase domain could be converted to a dual-specificity kinase and is essentially indistinct from RIPK2 in biochemical and molecular activity. Surprisingly, however, replacement of RIPK2's kinase domain with RIPK4's did not complement a nucleotide-binding oligomerization domain 2 signaling or gene expression induction defect in RIPK2(-/-) macrophages. These findings suggest that RIPK2's kinase domain is functionally unique compared with other RIPK family members and that pharmacologic targeting of RIPK2 can be separated from the other RIPKs.

Innate immune-directed NF-κB signaling requires site-specific NEMO ubiquitination.

While the I kappa kinase (IKK) scaffolding protein NF-κB essential modulator (NEMO) binds to polyubiquitin chains to transmit inflammatory signals, NEMO itself is also ubiquitinated in response to a variety of inflammatory agonists. Although there have been hints that polyubiquitination of NEMO is essential for avoiding inflammatory disorders, the in vivo physiologic role of NEMO ubiquitination is unknown. In this work, we knock in a NEMO allele in which two major inflammatory agonist-induced ubiquitination sites cannot be ubiquitinated. We show that mice with a nonubiquitinatable NEMO allele display embryonic lethality. Heterozygous females develop inflammatory skin lesions, decreased B cell numbers, and hypercellular spleens. Embryonic lethality can be complemented by mating onto a TNFR1(-/-) background, at the cost of severe steatohepatitis and early mortality, and we also show that NEMO ubiquitination is required for optimal innate immune signaling responses. These findings suggest that NEMO ubiquitination is crucial for NF-κB activity in response to innate immune agonists.

Potentiation of caspase-1 activation by the P2X7 receptor is dependent on TLR signals and requires NF-kappaB-driven protein synthesis.

The proinflammatory cytokines IL-1beta and IL-18 are inactive until cleaved by the enzyme caspase-1. Stimulation of the P2X7 receptor (P2X7R), an ATP-gated ion channel, triggers rapid activation of caspase-1. In this study we demonstrate that pretreatment of primary and Bac1 murine macrophages with TLR agonists is required for caspase-1 activation by P2X7R but it is not required for activation of the receptor itself. Caspase-1 activation by nigericin, a K+/H+ ionophore, similarly requires LPS priming. This priming by LPS is dependent on protein synthesis, given that cyclohexamide blocks the ability of LPS to prime macrophages for activation of caspase-1 by the P2X7R. This protein synthesis is likely mediated by NF-kappaB, as pretreatment of cells with the proteasome inhibitor MG132, or the IkappaB kinase inhibitor Bay 11-7085 before LPS stimulation blocks the ability of LPS to potentiate the activation of caspase-1 by the P2X7R. Thus, caspase-1 regulation in macrophages requires inflammatory stimuli that signal through the TLRs to up-regulate gene products required for activation of the caspase-1 processing machinery in response to K+-releasing stimuli such as ATP.

Inhibitory effects of chloride on the activation of caspase-1, IL-1beta secretion, and cytolysis by the P2X7 receptor.

The P2X7 receptor (P2X7R) is an ATP-gated cation channel that activates caspase-1 leading to the maturation and secretion of IL-1beta. Because previous studies indicated that extracellular Cl- exerts a negative allosteric effect on ATP-gating of P2X7R channels, we tested whether Cl- attenuates the P2X7R-->caspase-1-->IL-1beta signaling cascade in murine and human macrophages. In Bac1 murine macrophages, substitution of extracellular Cl- with gluconate produced a 10-fold increase in the rate and extent of ATP-induced IL-1beta processing and secretion, while reducing the EC50 for ATP by 5-fold. Replacement of Cl- with gluconate also increased the potency of ATP as an inducer of mature IL-1beta secretion in primary mouse bone marrow-derived macrophages and in THP-1 human monocytes/macrophages. Our observations were consistent with actions of Cl- at three levels: 1) a negative allosteric effect of Cl-, which limits the ability of ATP to gate the P2X7R-mediated cation fluxes that trigger caspase-1 activation; 2) an intracellular accumulation of Cl- via nonselective pores induced by P2X7R with consequential repression of caspase-1-mediated processing of IL-1beta; and 3) a facilitative effect of Cl- substitution on the cytolytic release of unprocessed pro-IL-1beta that occurs with sustained activation of P2X7R. This cytolysis was repressed by the cytoprotectant glycine, permitting dissociation of P2X7R-regulated secretion of mature IL-1beta from the lytic release of pro-IL-1beta. These results suggest that under physiological conditions P2X7R are maintained in a conformationally restrained state that limits channel gating and coupling of the receptor to signaling pathways that regulate caspase-1.

Enhanced activation of Akt and extracellular-regulated kinase pathways by simultaneous occupancy of Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT7A receptors in PC12 cells.

The most commonly prescribed antidepressants, the serotonin (5-HT) selective reuptake inhibitors, increase 5-HT without targeting specific receptors. Yet, little is known about the interaction of multiple receptor subtypes expressed by individual neurons. Specifically, the effect of increases in cAMP induced by Gs-coupled 5-HT receptor subtypes on the signaling pathways modulated by other receptor subtypes has not been studied. We have, therefore, examined the activation of the extracellular-regulated kinase (ERK) and Akt pathways by Gs-coupled 5-HT7A receptors and Gq-coupled 5-HT2A receptors, which are co-expressed in discrete brain regions. Agonists for both receptors were found to activate ERK and Akt in transfected PC12 cells. 5-HT2A receptor-mediated activation of the two pathways was found to be Ca2+-dependent. In contrast, 5-HT7A receptor-mediated activation of Akt required increases in both [cAMP] and intracellular [Ca2+], while activation of ERK was inhibited by Ca2+. The activation of ERK and Akt stimulated by simultaneous treatment of cells with 5-HT2A and 5-HT7A receptor agonists was found to be at least additive. Cell-permeable cAMP analogs mimicked 5-HT7A receptor agonists in enhancing 5-HT2A receptor-mediated activation of ERK and Akt. A role was identified for the cAMP-guanine exchange factor, Epac, in this augmentation of ERK, but not Akt, activation. Our finding of enhanced activation of neuroprotective Akt and ERK pathways by simultaneous occupancy of 5-HT2A and 5-HT7A receptors may also be relevant to the interaction of other neuronally expressed Gq- and Gs-coupled receptors.

Maitotoxin induces biphasic interleukin-1beta secretion and membrane blebbing in murine macrophages.

Maitotoxin (MTX) is a potent shellfish toxin widely used as an in vitro tool for increasing intracellular Ca2+ and studying Ca2+ -dependent processes. MTX also induces membrane blebbing and nonselective pores similar to those elicited by the P2X7 receptor (P2X7R), an ATP-gated cation channel expressed in inflammatory leukocytes. We therefore tested whether MTX treatment of lipopolysaccharide-primed murine macrophages would mimic the ability of activated P2X7R to induce secretion of the proinflammatory cytokine interleukin-1beta (IL-1beta). MTX at < or = 0.6 nM predominantly induced processing and nonlytic release of mature IL-1beta (mIL-1beta), whereas >0.6 nM of MTX induced cytolytic release of unprocessed proIL-1beta. MTX-dependent release of mIL-1beta (but not cytolysis) was inhibited by the elimination of the trans-plasma membrane K+ gradient. MTX-induced cytokine release and cytolysis were both abrogated in the absence of extracellular Ca2+. On the other hand, extracellular glycine (5 mM) blocked MTX-induced cytolytic release of proIL-1beta without affecting regulated secretion of mIL-1beta. Because MTX has profound effects on plasma membrane permeability, we used time-lapse videography to examine the morphologic response of individual macrophages to MTX. MTX treatment led to biphasic propidium dye uptake and dilated blebbing coincident with cytolysis. Glycine completely blocked the second, lytic phase of dye uptake and prevented MTX-induced bleb dilation. These results indicate that the inflammatory macrophage can assemble the necessary signaling components to initiate both regulated and lytic release of IL-1beta in response to MTX. This suggests that the hyperactivation of proinflammatory cytokine secretion may be a significant component of the in vivo response to MTX during shellfish seafood poisoning.

Oxidized ATP (oATP) attenuates proinflammatory signaling via P2 receptor-independent mechanisms.

Periodate-oxidized ATP (oATP), which covalently modifies nucleotide-binding proteins, can significantly attenuate proinflammatory signaling. Although the P2X7 nucleotide receptor (P2X7R) is irreversibly antagonized by oATP, it is unclear whether anti-inflammatory actions of oATP are predominantly mediated via its actions on P2X7R. Here, we describe inhibitory effects of oATP on proinflammatory responses in three human cell types that lack expression of P2X7R: human umbilical vein endothelial cells (HUVEC), HEK293 cells, and 1321N1 astrocytes. oATP decreased by 40-70% the secretion of interleukin (IL)-8 stimulated by tumor necrosis factor-alpha (TNF-alpha) in all three cell types, by IL-1beta in HUVEC and 1321N1 cells, and by endotoxin in HUVEC. Attenuation of TNF-alpha-stimulated IL-8 secretion by oATP was similar in wild-type HEK cells or HEK cells stably expressing recombinant P2X7R. oATP also attenuated cytokine-stimulated expression of nuclear factor-kappaB-luciferase reporter genes expressed in HEK or 1321N1 cells, but did not affect the rapid downregulation of IkappaB. oATP had no effect on uridine triphosphate-induced activation of native P2Y2 receptors in HEK cells, but reduced the potency and efficacy of ADP as an agonist of native P2Y1 receptors. However, inhibition of P2Y1 receptors with the specific antagonist MRS2216 did not mimic the effects of oATP on TNF-alpha-stimulated IL-8 secretion. Although 1321N1 astrocytes lack expression of any known P2 receptor subtypes, oATP markedly inhibited ecto-ATPase activity in these cells, resulting in a significant accumulation of extracellular ATP. In summary, oATP can attenuate proinflammatory signaling by mechanisms independent of the expression or activation of known P2 receptor subtypes.

Functionalized congeners of tyrosine-based P2X(7) receptor antagonists: probing multiple sites for linking and dimerization.

Chemically funtionalized analogues of antagonists of the P2X(7) receptor, an ATP-gated cation channel, were synthesized as tools for biophysical studies of the receptor. These functionalized congeners were intended for use in chemical conjugation with retention of biological potency. The antagonists were L-tyrosine derivatives, related to [N-benzyloxycarbonyl-O-(4-arylsulfonyl)-L-tyrosyl]benzoylpiperazine (such as MRS2409, 2). The analogues were demonstrated to be antagonists in an assay of human P2X(7) receptor function, consisting of inhibition of ATP-induced K(+) efflux in HEK293 cells expressing the recombinant receptor. The analogues were of the general structure R(1)-Tyr(OR(2))-piperazinyl-R(3), in which three positions (R(1)-R(3)) were systematically varied in structure through introduction of chemically reactive groups. Each of the three positions was designed to incorporate a 3- or 4-nitrophenyl group. The nitro groups were reduced using NaBH(4)-copper(II) acetylacetonate to amines, which were either converted to the isothiocyanate groups, as potential affinity labels for the receptor, or acylated, as models for conjugation. An alternate route to N(alpha)-3-aminobenzyloxycarbonyl functionalization was devised. The various positions of functionalization were compared for effects on biological potency, and the R(2) and R(3) positions were found to be most amenable to derivatization with retention of high potency. Four dimeric permutations of the antagonists were synthesized by coupling each of the isothiocyanate derivatives to either the precursor amine or to other amine congeners. Only dimers linked at the R(2)-position were potent antagonists. In concentration-response studies, two derivatives, a 3-nitrobenzyloxycarbonyl derivative 18 and a 4-nitrotoluenesulfonate 26b, displayed IC(50) values of roughly 100 nM as antagonists of P2X(7) receptor-mediated K(+) flux.

Potent P2X7 Receptor Antagonists: Tyrosyl Derivatives Synthesized Using a Sequential Parallel Synthetic Approach.

Novel analogs of 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN-62,1) were synthesized and found to be potent antagonists in a functional assay, inhibition of ATP-induced K+ efflux in HEK293 cells expressing recombinant human P2X7 receptors. Antagonism of murine P2X7 receptors was also observed. The analogs consisted of L-tyrosine derivatives, of the general structure R1-Tyr(OR2)-piperazinyl-R3, in which three positions were systematically varied in structure through facile acylation reactions. Each of the three positions was optimized in sequence through parallel synthesis alternating with biological evaluation, leading to the identification and optimization of potent P2X7 antagonists. The optimal groups at R1 were found to be large hydrophobic groups, linked to the α-amino position through carbamate, amide, or sulfonamide groups. The benzyloxycarbonyl (Cbz) group was preferred over most sulfonamides and other acyl groups examined, except for quinoline sulfonyl. At R2, an arylsulfonate ester was preferred, and the order of potency was p-tolyl, p-methoxyphenyl, phenyl > α-naphthyl, ß-naphthyl. A benzoyl ester was of intermediate potency. Aliphatic esters and carbonate derivatives at the tyrosyl phenol were inactive, while a tyrosyl O-benzyl ether was relatively potent. The most potent P2X7 receptor antagonists identified in this study contained Cbz at the R1 position, an aryl sulfonate at the R2 position, and various acyl groups at the R3 position. At R3, t-butyloxycarbonyl- and benzoyl groups were preferred. The opening of the piperazinyl ring to an ethylene diamine moiety abolished antagonism. In concentration-response studies, a di-isoquinolinyl, Boc derivative, 4 (MRS2306), displayed an IC50 value of 40 nM as an antagonist of P2X7 receptor-mediated ion flux and was more potent than the reference compound 1. Nα-Cbz, Boc-piperazinyl derivatives, 11 (MRS2317), 22 (MRS2326), and 41 (MRS2409) were less potent than 1, with IC50 values of 200-300 nM.