Previous studies have suggested that the P2Z/P2X 7 purinergic receptor can participate in nucleotide-induced modulation of lipopolysaccharide (LPS) stimulated inflammatory mediator production. To test this hypothesis, we evaluated whether antagonism of the P2Z/P2X 7 receptor can influence LPS signaling and expression of the inducible form of nitric-oxide synthase (iNOS) in RAW 264.7 macrophages. In the present study, we demonstrate that pretreatment of RAW 264.7 macrophages with a P2Z/P2X 7 receptor antagonist, periodate oxidized adenosine 5-triphosphate (o-ATP), substantially inhibits LPS-stimulated NO production and iNOS expression without altering cell viability. This effect on LPS-induced iNOS expression is mimicked by a pyridoxal-phosphate-based antagonist (pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid) of the P2Z/P2X 7 purinergic receptor, indicating that these results are not unique to o-ATP. Additionally, o-ATP prevents cell death induced by P2Z/P2X 7 receptor agonists. To ascertain how P2Z/P2X 7 receptor antagonists influence LPS signaling, we evaluated the capacity of o-ATP to regulate LPSmediated activation of the transcription factor, nuclear factor-B, and the mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2. These experiments reveal that pretreatment of RAW 264.7 cells with o-ATP attenuates the LPS stimulation of a nuclear factor-B-like binding activity. Moreover, the activation of ERK1 and ERK2 by LPS, but not by the phorbol ester, phorbol 12-myristate 13-acetate, is also blocked in RAW 264.7 cells by o-ATP pretreatment. In summary, these data suggest that the P2Z/P2X 7 receptor modulates LPS-induced macrophage activation as assessed by iNOS expression and NO production. This report implicates the P2Z/P2X 7 receptor in the control of protein kinase cascades and transcriptional processes, and these observations are likely to be important for the development of selective purinergic receptor antagonists for the treatment of septic shock.

Toll-like receptors (TLRs) are pattern recognition receptors that sense a variety of pathogens, initiate innate immune responses, and direct adaptive immunity. All TLRs except TLR3 recruit the adaptor MyD88 to ultimately elicit inflammatory gene expression, whereas TLR3 and internalized TLR4 use TIR-domain-containing adaptor TRIF for the induction of type I interferon and inflammatory cytokines. Here, we identify the WD repeat and FYVE-domaincontaining protein WDFY1 as a crucial adaptor protein in the TLR3/4 signaling pathway. Overexpression of WDFY1 potentiates TLR3-and TLR4-mediated activation of NF-jB, interferon regulatory factor 3 (IRF3), and production of type I interferons and inflammatory cytokines. WDFY1 depletion has the opposite effect. WDFY1 interacts with TLR3 and TLR4 and mediates the recruitment of TRIF to these receptors. Our findings suggest a crucial role for WDFY1 in bridging the TLR-TRIF interaction, which is necessary for TLR signaling.

Interest in mutually exclusive pairs of bioorthogonal labeling reagents continues to drive the design of new compounds that are capable of fast and predictable reactions. The ability to easily modify S-, N-, and O-containing cyclooctynes (SNO-OCTs) enables electronic tuning of various SNO-OCTs to influence their cycloaddition rates with Type I–III dipoles. As opposed to optimizations based on just one specific dipole class, the electrophilicity of the alkynes in SNO-OCTs can be manipulated to achieve divergent reactivities and furnish mutually orthogonal dual ligation systems. Significant reaction rate enhancements of a difluorinated SNO-OCT derivative, as compared to the parent scaffold, were noted, with the second-order rate constant in cycloadditions with diazoacetamides exceeding 5.13 M–1 s–1. Computational and experimental studies were employed to inform the design of triple ligation systems that encompass three orthogonal reactivities. Finally, polar SNO-OCTs are rapidly internalized by mammalian cells and remain functional in the cytosol for live-cell labeling, highlighting their potential for diverse in vitro and in vivo applications.

Ag receptor engagement triggers lymphocyte activation and proliferation by activating several transcription factors including NF-κB. Caspase recruitment domain (CARD) containing membrane-associated guanylate kinase (MAGUK) protein 1 (CARMA1) is an essential adaptor protein that links Ag receptors to NF-κB activation. Here, we identify stress-induced-phosphoprotein 1 homology and U-box containing protein 1 (STUB1) as a CARMA1-associated protein. STUB1 constitutively interacted with CARMA1, and the interaction was intensified by TCR stimulation. Downregulation of STUB1 expression by RNAi markedly diminished TCR-induced canonical NF-κB activation and IL-2 production. Furthermore, overexpression of STUB1 enhanced the ubiquitination of CARMA1, whereas knockdown of STUB1 abolished the endogenous ubiquitination of CARMA1 induced by TCR stimulation. Subsequently, the ubiquitination of CARMA1 catalyzed by STUB1 was identified as Lys-27 linked, which is important for CARMA1-mediated NF-κB activation. These data provide the first evidence that ubiquitination of CARMA1 by STUB1 promotes TCR-induced NF-κB signaling.Keywords: CARMA1 r NF-κB pathway r Signal transduction r STUB1 r TCR Additional supporting information may be found in the online version of this article at the publisher's web-site Introduction TCR-induced activation of the transcription factor NF-κB is critical for the activation, proliferation, and differentiation of T cells [1][2][3]. Signal transduction from TCR to NF-κB activation requires the scaffold protein caspase recruitment domain (CARD) containing membrane-associated guanylate kinase (MAGUK) protein 1 (CARMA1), as evidenced by experiments on CARMA1 KO or pointmutated mice [4,5]. Upon the stimulation of TCR and CD28, CARMA1 is phosphorylated, undergoes conformational changes, and subsequently recruits B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) to assemble a signalsome, namely the CBM complex Correspondence: Dr. Yu Liu e-mail: yuliu@whu.edu.cn [6][7][8][9][10]. The CBM complex recruits TNF receptor-associated factor 6 (TRAF6) that catalyzes the ubiquitination of itself and MALT1. The ubiquitin chains formed on TRAF6 and MALT1 provide the docking sites for TGF-β activated kinase 1 (TAK1) and IκB kinase (IKK) signalsome. IKKs are subsequently activated and lead to the phosphorylation and degradation of IκBα [11,12]. NF-κB is then released and translocated to the nucleus to turn on transcription of target genes.Post-translational modification of CARMA1 is critical for its functions and the activation of NF-κB. Phosphorylation of CARMA1 by PKCθ, IKK-β, and Ca 2+ /calmodulin-dependent protein kinase II is essential for TCR-induced NF-κB activation, whereas casine kinase 1α-catalyzed phosphorylation of CARMA1 impairs its ability to activate NF-κB [9,10,[13][14][15] In an effort to understand the subtle mechanisms of T-cell activation, we previously endeavored to identify novel proteins participating in TCR signaling. By biochemical affinity purif...