Publications by authors named "Tsaffrir Zor"

26 Publications

  • Page 1 of 1

Sulfatides are endogenous ligands for the TLR4-MD-2 complex.

Proc Natl Acad Sci U S A 2021 Jul;118(30)

Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;

Many endogenous molecules, mostly proteins, purportedly activate the Toll-like receptor 4 (TLR4)-myeloid differentiation factor-2 (MD-2) complex, the innate immune receptor for lipopolysaccharide (LPS) derived from gram-negative bacteria. However, there is no structural evidence supporting direct TLR4-MD-2 activation by endogenous ligands. Sulfatides (3--sulfogalactosylceramides) are natural, abundant sulfated glycolipids that have variously been shown to initiate or suppress inflammatory responses. We show here that short fatty acid (FA) chain sulfatides directly activate mouse TLR4-MD-2 independent of CD14, trigger MyD88- and TRIF-dependent signaling, and stimulate tumor necrosis factor α (TNFα) and type I interferon (IFN) production in mouse macrophages. In contrast to the agonist activity toward the mouse receptor, the tested sulfatides antagonize TLR4-MD-2 activation by LPS in human macrophage-like cells. The agonistic and antagonistic activities of sulfatides require the presence of the sulfate group and are inversely related to the FA chain length. The crystal structure of mouse TLR4-MD-2 in complex with C16-sulfatide revealed that three C16-sulfatide molecules bound to the MD-2 hydrophobic pocket and induced an active dimer conformation of the receptor complex similar to that induced by LPS or lipid A. The three C16-sulfatide molecules partially mimicked the detailed interactions of lipid A to achieve receptor activation. Our results suggest that sulfatides may mediate sterile inflammation or suppress LPS-stimulated inflammation, and that additional endogenous negatively charged lipids with up to six lipid chains of limited length might also bind to TLR4-MD-2 and activate or inhibit this complex.
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http://dx.doi.org/10.1073/pnas.2105316118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325290PMC
July 2021

Immunoediting role for major vault protein in apoptotic signaling induced by bacterial -acyl homoserine lactones.

Proc Natl Acad Sci U S A 2021 03;118(12)

Department of Chemistry and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva 8410501, Israel;

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Here, we report the use of photoactive probes to identify MVP as a target of the -(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including A treatment of normal and cancer cells with C12 or other -acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).
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http://dx.doi.org/10.1073/pnas.2012529118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000436PMC
March 2021

A dual and conflicting role for imiquimod in inflammation: A TLR7 agonist and a cAMP phosphodiesterase inhibitor.

Biochem Pharmacol 2020 12 20;182:114206. Epub 2020 Aug 20.

Department of Biochemistry & Molecular Biology, School of Neurobiology, Biochemistry & Biophysics, Life Sciences Faculty, Tel Aviv University, Tel Aviv 69978, Israel. Electronic address:

The Toll-like receptor 7 (TLR7) agonist imiquimod is an antitumor and antiviral drug used for the treatment of skin indications such as basal cell carcinoma, squamous cell carcinoma, and genital warts caused by the human papilloma virus. We show that imiquimod has TLR7-independent activity in which it directly inhibits phosphodiesterase (PDE), leading to cAMP increase, PKA-mediated CREB phosphorylation and subsequent CRE-dependent reporter transcription. The activation of the cAMP pathway by imiquimod is synergistically amplified by the β-adrenergic receptor agonist, isoproterenol. PDE inhibition is implied from cAMP measurements and CRE-reporter assays in intact RAW264.7 macrophages and HEK293T cells, and also directly demonstrated in-vitro using macrophages lysate. Moreover, molecular docking simulated the binding of imiquimod in the active site of PDE4B, enabled by the high molecular similarity between imiquimod and the adenine moiety of cAMP. As expected from the known anti-inflammatory role of cAMP inducers in stimulated macrophages, PDE inhibition by imiquimod results in reduced expression of the key pro-inflammatory cytokine TNFα, and enhanced expression of the key anti-inflammatory cytokine IL-10, compared to a different TLR7 agonist, loxoribine, as well as to the TLR4 agonist LPS. To conclude, our results indicate that the widely used inflammatory drug, imiquimod, is not only a TLR7 agonist, but also harbors a novel anti-inflammatory function as a PDE inhibitor. This off-target affects the desired therapeutic inflammatory activity of imiquimod and may be accountable for adverse side effects.
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http://dx.doi.org/10.1016/j.bcp.2020.114206DOI Listing
December 2020

Exclusive Temporal Stimulation of IL-10 Expression in LPS-Stimulated Mouse Macrophages by cAMP Inducers and Type I Interferons.

Front Immunol 2019 6;10:1788. Epub 2019 Aug 6.

Department of Biochemistry & Molecular Biology, School of Neurobiology, Biochemistry & Biophysics, Tel Aviv University, Tel Aviv, Israel.

Expression of the key anti-inflammatory cytokine IL-10 in lipopolysaccharide (LPS)-stimulated macrophages is mediated by a delayed autocrine/paracrine loop of type I interferons (IFN) to ensure timely attenuation of inflammation. We have previously shown that cAMP synergizes with early IL-10 expression by LPS, but is unable to amplify the late type I IFN-dependent activity. We now examined the mechanism of this synergistic transcription in mouse macrophages at the promoter level, and explored the crosstalk between type I IFN signaling and cAMP, using the β-adrenergic receptor agonist, isoproterenol, as a cAMP inducer. We show that silencing of the type I IFN receptor enables isoproterenol to synergize with LPS also at the late phase, implying that autocrine type I IFN activity hinders synergistic augmentation of LPS-stimulated IL-10 expression by cAMP at the late phase. Furthermore, IL-10 expression in LPS-stimulated macrophages is exclusively stimulated by either IFNα or isoproterenol. We identified a set of two proximate and inter-dependent cAMP response element (CRE) sites that cooperatively regulate early IL-10 transcription in response to isoproterenol-stimulated CREB and that further synergize with a constitutive Sp1 site. At the late phase, up-regulation of Sp1 activity by LPS-stimulated type I IFN is correlated with loss of function of the CRE sites, suggesting a mechanism for the loss of synergism when LPS-stimulated macrophages switch to type I IFN-dependent IL-10 expression. This report delineates the molecular mechanism of cAMP-accelerated IL-10 transcription in LPS-stimulated murine macrophages that can limit inflammation at its onset.
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http://dx.doi.org/10.3389/fimmu.2019.01788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691811PMC
October 2020

The cAMP Pathway Amplifies Early MyD88-Dependent and Type I Interferon-Independent LPS-Induced Interleukin-10 Expression in Mouse Macrophages.

Mediators Inflamm 2019 17;2019:3451461. Epub 2019 Apr 17.

Department of Biochemistry & Molecular Biology, School of Neurobiology, Biochemistry & Biophysics, Life Sciences Faculty, Tel Aviv University, Tel Aviv 69978, Israel.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine, secreted by macrophages and other immune cells to attenuate inflammation. Autocrine type I interferons (IFNs) largely mediate the delayed expression of IL-10 by LPS-stimulated macrophages. We have previously shown that IL-10 is synergistically expressed in macrophages following a costimulus of a TLR agonist and cAMP. We now show that the cAMP pathway directly upregulates IL-10 transcription and plays an important permissive and synergistic role in early, but not late, LPS-stimulated IL-10 mRNA and protein expression in mouse macrophages and in a mouse septic shock model. Our results suggest that the loss of synergism is not due to desensitization of the cAMP inducing signal, and it is not mediated by a positive crosstalk between the cAMP and type I IFN pathways. First, cAMP elevation in LPS-treated cells decreased the secretion of type I IFN. Second, autocrine/paracrine type I IFNs induce IL-10 promoter reporter activity only additively, but not synergistically, with the cAMP pathway. IL-10 promoter reporter activity was synergistically induced by cAMP elevation in macrophages stimulated by an agonist of either TLR4, TLR2/6, or TLR7, receptors which signal via MyD88, but not by an agonist of TLR3 which signals independently of MyD88. Moreover, MyD88 knockout largely reduced the synergistic IL-10 expression, indicating that MyD88 is required for the synergism displayed by LPS with cAMP. This report delineates the temporal regulation of early cAMP-accelerated vs. late type I IFN-dependent IL-10 transcription in LPS-stimulated murine macrophages that can limit inflammation at its onset.
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http://dx.doi.org/10.1155/2019/3451461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501241PMC
December 2019

3-Aminobenzamide Prevents Concanavalin A-Induced Acute Hepatitis by an Anti-inflammatory and Anti-oxidative Mechanism.

Dig Dis Sci 2018 12 8;63(12):3382-3397. Epub 2018 Sep 8.

Department of Biochemistry and Molecular Biology, Life Sciences Faculty, Tel-Aviv University, 69978, Tel-Aviv, Israel.

Background And Aims: Concanavalin A is known to activate T cells and to cause liver injury and hepatitis, mediated in part by secretion of TNFα from macrophages. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been shown to prevent tissue damage in various animal models of inflammation. The objectives of this study were to evaluate the efficacy and mechanism of the PARP-1 inhibitor 3-aminobenzamide (3-AB) in preventing concanavalin A-induced liver damage.

Methods: We tested the in vivo effects of 3-AB on concanavalin A-treated mice, its effects on lipopolysaccharide (LPS)-stimulated macrophages in culture, and its ability to act as a scavenger in in vitro assays.

Results: 3-AB markedly reduced inflammation, oxidative stress, and liver tissue damage in concanavalin A-treated mice. In LPS-stimulated RAW264.7 macrophages, 3-AB inhibited NFκB transcriptional activity and subsequent expression of TNFα and iNOS and blocked NO production. In vitro, 3-AB acted as a hydrogen peroxide scavenger. The ROS scavenger N-acetylcysteine (NAC) and the ROS formation inhibitor diphenyleneiodonium (DPI) also inhibited TNFα expression in stimulated macrophages, but unlike 3-AB, NAC and DPI were unable to abolish NFκB activity. PARP-1 knockout failed to affect NFκB and TNFα suppression by 3-AB in stimulated macrophages.

Conclusions: Our results suggest that 3-AB has a therapeutic effect on concanavalin A-induced liver injury by inhibiting expression of the key pro-inflammatory cytokine TNFα, via PARP-1-independent NFκB suppression and via an NFκB-independent anti-oxidative mechanism.
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http://dx.doi.org/10.1007/s10620-018-5267-1DOI Listing
December 2018

Synthesis and evaluation of a tag-free photoactive phospho-ceramide analogue-1 (PCERA-1) probe to study immunomodulation in macrophages.

Chem Commun (Camb) 2017 Mar;53(27):3842-3845

Dept. of Chemistry and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva, Israel.

Phospho-ceramide analogue-1 (PCERA-1), a synthetic analogue of ceramide-1-phosphate (C1P), has been previously shown to act as a potent modulator of macrophage activity and inflammation. We have developed an efficient synthesis of PCERA-1 from readily available starting materials, and designed and prepared derivatives of this analogue, including a photoaffinity probe to tag and identify putative proteins that bind PCERA-1.
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http://dx.doi.org/10.1039/c6cc09849eDOI Listing
March 2017

Exogenous ceramide-1-phosphate (C1P) and phospho-ceramide analogue-1 (PCERA-1) regulate key macrophage activities via distinct receptors.

Immunol Lett 2016 Jan 3;169:73-81. Epub 2015 Dec 3.

Department of Biochemistry & Molecular Biology, Life Sciences Institute, Tel-Aviv University, Tel-Aviv 69978, Israel. Electronic address:

Inflammation is an ensemble of tightly regulated steps, in which macrophages play an essential role. Previous reports showed that the natural sphingolipid ceramide 1-phosphate (C1P) stimulates macrophages migration, while the synthetic C1P mimic, phospho-ceramide analogue-1 (PCERA-1), suppresses production of the key pro-inflammatory cytokine TNFα and amplifies production of the key anti-inflammatory cytokine IL-10 in LPS-stimulated macrophages, via one or more unidentified G-protein coupled receptors. We show that C1P stimulated RAW264.7 macrophages migration via the NFκB pathway and MCP-1 induction, while PCERA-1 neither mimicked nor antagonized these activities. Conversely, PCERA-1 synergistically elevated LPS-dependent IL-10 expression in RAW264.7 macrophages via the cAMP-PKA-CREB signaling pathway, while C1P neither mimicked nor antagonized these activities. Interestingly, both compounds have the capacity to additively inhibit TNFα secretion; PCERA-1, but not C1P, suppressed LPS-induced TNFα expression in macrophages in a CREB-dependent manner, while C1P, but not PCERA-1, directly inhibited recombinant TNFα converting enzyme (TACE). Finally, PCERA-1 failed to interfere with binding of C1P to either the cell surface receptor or to TACE. These results thus indicate that the natural sphingolipid C1P and its synthetic analog PCERA-1 bind and activate distinct receptors expressed in RAW264.7 macrophages. Identification of these receptors will be instrumental for elucidation of novel activities of extra-cellular sphingolipids, and may pave the way for the design of new sphingolipid mimics for the treatment of inflammatory diseases, and pathologies which depend on cell migration, as in metastatic tumors.
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http://dx.doi.org/10.1016/j.imlet.2015.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4837700PMC
January 2016

Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities.

Nucleic Acids Res 2016 Apr 3;44(6):e51. Epub 2015 Dec 3.

Mina and Evrard Goodman life science faculty, Bar Ilan University, Ramat-Gan, 5290002, Israel

Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.
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http://dx.doi.org/10.1093/nar/gkv1327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824076PMC
April 2016

The bacterial quorum-sensing signal molecule N-3-oxo-dodecanoyl-L-homoserine lactone reciprocally modulates pro- and anti-inflammatory cytokines in activated macrophages.

J Immunol 2013 Jul 29;191(1):337-44. Epub 2013 May 29.

Department of Biochemistry and Molecular Biology, Life Sciences Institute, Tel Aviv University, Tel Aviv 69978, Israel.

The bacterial molecule N-3-oxo-dodecanoyl-l-homoserine lactone (C12) has critical roles in both interbacterial communication and interkingdom signaling. The ability of C12 to downregulate production of the key proinflammatory cytokine TNF-α in stimulated macrophages was suggested to contribute to the establishment of chronic infections by opportunistic Gram-negative bacteria, such as Pseudomonas aeruginosa. We show that, in contrast to TNF-α suppression, C12 amplifies production of the major anti-inflammatory cytokine IL-10 in LPS-stimulated murine RAW264.7 macrophages, as well as peritoneal macrophages. Furthermore, C12 increased IL-10 mRNA levels and IL-10 promoter reporter activity in LPS-stimulated RAW264.7 macrophages, indicating that C12 modulates IL-10 expression at the transcriptional level. Finally, C12 substantially potentiated LPS-stimulated NF-κB DNA-binding levels and prolonged p38 MAPK phosphorylation in RAW264.7 macrophages, suggesting that increased transcriptional activity of NF-κB and/or p38-activated transcription factors serves to upregulate IL-10 production in macrophages exposed to both LPS and C12. These findings reveal another part of the complex array of host transitions through which opportunistic bacteria downregulate immune responses to flourish and establish a chronic infection.
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http://dx.doi.org/10.4049/jimmunol.1300368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691282PMC
July 2013

Species selective diazirine positioning in tag-free photoactive quorum sensing probes.

Chem Commun (Camb) 2013 Jul 24;49(52):5826-8. Epub 2013 May 24.

Department of Chemistry and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er-Sheva 84105, Israel.

The synthesis and comparison of activities of 'tag-free' probes with diazirines at various positions are described. Remarkable differences in their effects on P. aeruginosa and on human bronchial epithelial cells were observed, supporting the efforts to isolate and identify receptors for N-acyl homoserine lactones.
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http://dx.doi.org/10.1039/c3cc43092hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723129PMC
July 2013

Identification of elements that dictate the specificity of mitochondrial Hsp60 for its co-chaperonin.

PLoS One 2012 4;7(12):e50318. Epub 2012 Dec 4.

Department of Biochemistry and Molecular Biology, Tel Aviv University, Tel Aviv, Israel.

Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-chaperonin.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050318PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514286PMC
June 2013

Hyaluronan-modified and regular multilamellar liposomes provide sub-cellular targeting to macrophages, without eliciting a pro-inflammatory response.

J Control Release 2012 Jun 12;160(2):388-93. Epub 2011 Oct 12.

Department of Biochemistry and Molecular Biology, the George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

Macrophages, pivotal cells in onset and progression of inflammation, can benefit from sub-cellular drug targeting to the molecular loci of drug action, whether cell membrane or cell interior. Postulating manipulation of liposome size and surface properties can provide sub-cellular targeting, we studied: thermodynamics of liposome-macrophage binding; liposome cellular localizations; liposome safety including pro-inflammatory cytokine production. We aimed at extending the body of knowledge on interactions of regular unilamellar (RL-ULV) and multilamellar (RL-MLV) liposomes with macrophages. We investigated, for the first time, the interactions of hyaluronan (HA) surface-modified liposomes (HA-ULV and HA-MLV) with macrophages, with respect to multiple equilibria binding combined with cellular localization. Macrophages bound all four liposome types, substantially-favoring the two MLV species over the two ULV species, and internalizing only RL-MLV. Three macrophage-internalization inhibitors (2-deoxyglucose, LY294002 and Wortmannin) reduced RL-MLV internalization but not binding affinity nor binding capacity. Both MLV types were not detrimental to cell proliferation, nor did they elicit TNF-α production in resting and in LPS-activated macrophages. Moreover, a 24-hour exposure of LPS-activated macrophages to HA-MLV reduced TNF-α production by 40%, indicating potential for anti-inflammatory activity. In conclusion RL-MLV and HA-MLV are the liposomes of choice for delivering anti-inflammatory drugs to the macrophage surface or its interior, according to the loci of drug action.
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http://dx.doi.org/10.1016/j.jconrel.2011.10.008DOI Listing
June 2012

The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 modulates cytokine expression in macrophages via p50 nuclear factor κB inhibition, in a PI3K-independent mechanism.

Biochem Pharmacol 2012 Jan 12;83(1):106-14. Epub 2011 Oct 12.

Department of Biochemistry and Molecular Biology, Life Sciences Institute, Tel-Aviv University, Israel.

The Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 (LY2), has been previously reported to inhibit nuclear factor κB (NFκB) activity, in a PI3K-independent mechanism. The goals of the current research were to determine the specificity of LY2 regarding NFκB subunits, and to identify relevant modulation of cytokine expression in LPS-stimulated macrophages. We found that LY2 specifically diminished the level of p50, but not p65, NFκB in the nucleus of LPS-stimulated mouse RAW264.7 macrophages and human THP-1 monocytes. This activity of LY2 was mimicked by its PI3K-inert analog LY303511 (LY3), but not by another PI3K inhibitor - wortmannin. We further show that LY2 inhibited LPS-induced IL-10 expression by RAW264.7 macrophages, in a PI3K-independent mechanism. Moreover, using a deletion mutant of an IL-10 promoter reporter gene we demonstrate that the activity of the NFκB enhancer site at the IL-10 promoter is regulated by LY2 in a PI3K-independent manner. Finally, both LY2 and LY3 elevated TNFα production in the LPS tolerant state which is regulated by p50 NFκB homodimers, but not before tolerance development. The effects of LY2 and LY3 on p50 translocation and on cytokine production in LPS-stimulated macrophages are thus consistent with specific PI3K-independent inhibition of p50 NFκB homodimer activity by LY2.
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http://dx.doi.org/10.1016/j.bcp.2011.09.025DOI Listing
January 2012

A ceramide analog inhibits cPLA(2) activity and consequent PGE(2) formation in LPS-stimulated macrophages.

Immunol Lett 2011 Mar 30;135(1-2):136-43. Epub 2010 Oct 30.

Department of Biochemistry and Molecular Biology, Life Sciences Institute, Tel-Aviv University, Tel-Aviv 69978, Israel.

Prostaglandin E(2) (PGE(2)) is an important mediator of the inflammatory response. Phospho-ceramide analogue-1 (PCERA-1), a synthetic phospholipid-like molecule, was previously reported to modulate pro- and anti-inflammatory cytokine production. We show here that PCERA-1 inhibited LPS-stimulated PGE(2) production in RAW264.7 macrophages, without affecting COX-2 expression. Furthermore, PCERA-1 efficiently suppressed arachidonic acid (AA) release in response to LPS. The dephosphorylated derivative of PCERA-1, ceramide analogue-1 (CERA-1), mimicked the inhibitory effect of PCERA-1 on AA release and PGE(2) production in macrophages. Inhibition of PGE(2) production by CERA-1 was completely rescued by addition of exogenous AA. Importantly, PCERA-1 and ceramide-1-phosphate (C1P) stimulated the enzymatic activity of cPLA(2)α in an in vitro assay, whereas CERA-1 and ceramide inhibited both basal and C1P-stimulated cPLA(2)α activity. Collectively, these results indicate that CERA-1 suppresses AA release and subsequent PGE(2) production in LPS-stimulated macrophages by direct interaction with cPLA(2), and suggest that ceramide may similarly counteract C1P effect on cPLA(2) activity in cells. The suppression of PGE(2) production is suggested to contribute to the anti-inflammatory action of PCERA-1.
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http://dx.doi.org/10.1016/j.imlet.2010.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3026855PMC
March 2011

Linearization of the bradford protein assay.

J Vis Exp 2010 Apr 12(38). Epub 2010 Apr 12.

Department of Biochemistry, Tel-Aviv University.

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.
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http://dx.doi.org/10.3791/1918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164080PMC
April 2010

Role of CREB in modulation of TNFalpha and IL-10 expression in LPS-stimulated RAW264.7 macrophages.

Mol Immunol 2010 Apr 19;47(7-8):1396-403. Epub 2010 Mar 19.

Department of Biochemistry, Life Sciences Institute, Tel-Aviv University, Tel-Aviv 69978, Israel.

The role of CREB in LPS signaling is controversial. The objective of this study was to evaluate the effect of LPS on phosphorylation and transcriptional activation of CREB, in comparison to isoproterenol, a beta-adrenergic receptor agonist. We show here that LPS elevates intra-cellular cAMP level in RAW264.7 macrophages, with slower kinetics and lower magnitude than isoproterenol. The two agents stimulated CREB phosphorylation on Ser-133 to a similar extent, but with a different mechanism; rapid and mostly PKA-mediated for isoproterenol; slow and MSK1-mediated for LPS. Interestingly, LPS-stimulated phosphorylation of CREB did not result in transcriptional activation of a CRE-regulated luciferase reporter, in contrast to stimulation by isoproterenol. Furthermore, inhibitors of p38 and MSK1, but not PKA, completely blocked the production of IL-10 and TNFalpha in LPS-stimulated macrophages. Distinctively, the PKA inhibitor H89 blocked the suppressive effect of isoproterenol on TNFalpha production, as well as its stimulatory effect on IL-10 induction, in LPS-stimulated macrophages. Likewise, while over-expression of dominant negative CREB had no effect on LPS-stimulated TNFalpha production, it blocked the suppressive effect of isoproterenol on TNFalpha production in the LPS-stimulated macrophages. Our results thus indicate that PKA-mediated phosphorylation of CREB promotes TNFalpha suppression and IL-10 induction, whereas the same phosphorylation event initiated by LPS and mediated by MSK1 is non-functional for transcriptional modulation.
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http://dx.doi.org/10.1016/j.molimm.2010.02.015DOI Listing
April 2010

The ceramide-1-phosphate analogue PCERA-1 modulates tumour necrosis factor-alpha and interleukin-10 production in macrophages via the cAMP-PKA-CREB pathway in a GTP-dependent manner.

Immunology 2010 Mar 16;129(3):375-85. Epub 2009 Nov 16.

Department of Biochemistry, Life Sciences Institute, Tel-Aviv University, Tel-Aviv, Israel.

The synthetic phospho-ceramide analogue-1 (PCERA-1) down-regulates production of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) and up-regulates production of the anti-inflammatory cytokine interleukin-10 (IL-10) in lipopolysaccharide (LPS) -stimulated macrophages. We have previously reported that PCERA-1 increases cyclic adenosine monophosphate (cAMP) levels. The objective of this study was to delineate the signalling pathway leading from PCERA-1 via cAMP to modulation of TNF-alpha and IL-10 production. We show here that PCERA-1 elevates intra-cellular cAMP level in a guanosine triphosphate-dependent manner in RAW264.7 macrophages. The cell-permeable dibutyryl cAMP was able to mimic the effects of PCERA-1 on cytokine production, whereas 8-chloro-phenylthio-methyladenosine-cAMP, which specifically activates the exchange protein directly activated by cAMP (EPAC) but not protein kinase A (PKA), failed to mimic PCERA-1 activities. Consistently, the PKA inhibitor H89 efficiently blocked PCERA-1-driven cytokine modulation as well as PCERA-1-stimulated phosphorylation of cAMP response element binding protein (CREB) on Ser-133. Finally, PCERA-1 activated cAMP-responsive transcription of a luciferase reporter, in synergism with the phosphodiesterase (PDE)-4 inhibitor rolipram. Our results suggest that PCERA-1 activates a G(s) protein-coupled receptor, leading to elevation of cAMP, which acts via the PKA-CREB pathway to promote TNF-alpha suppression and IL-10 induction in LPS-stimulated macrophages. Identification of the PCERA-1 receptor is expected to set up a new target for development of novel anti-inflammatory drugs.
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http://dx.doi.org/10.1111/j.1365-2567.2009.03188.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2826682PMC
March 2010

Distinct receptor-mediated activities in macrophages for natural ceramide-1-phosphate (C1P) and for phospho-ceramide analogue-1 (PCERA-1).

Mol Cell Endocrinol 2010 Jan 23;314(2):248-55. Epub 2009 May 23.

Department of Biochemistry, Life Sciences Institute, Tel-Aviv University, Tel-Aviv, Israel.

Ceramide-1-phosphate (C1P) is known as a second messenger regulating a multitude of processes including cell growth, apoptosis and inflammation. Exciting recent findings now suggest that C1P can stimulate macrophages migration in an extra-cellular manner via a G protein-coupled receptor (GPCR). Interestingly, a synthetic C1P analog, named phospho-ceramide analogue-1 (PCERA-1), was recently described as a potent in-vivo anti-inflammatory agent, and was suggested to act on macrophages in an extra-cellular manner via a GPCR. Here we summarize and compare the receptor-mediated as well as receptor-independent activities of natural C1P and its synthetic analog. We also provide experimental data in support of distinct C1P and PCERA-1 receptors.
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http://dx.doi.org/10.1016/j.mce.2009.05.007DOI Listing
January 2010

Synergistic IL-10 induction by LPS and the ceramide-1-phosphate analog PCERA-1 is mediated by the cAMP and p38 MAP kinase pathways.

Mol Immunol 2009 Jun 10;46(10):1979-87. Epub 2009 Apr 10.

Department of Biochemistry, Life Sciences Institute, Tel-Aviv University, Tel-Aviv 69978, Israel.

Expression of the anti-inflammatory cytokine IL-10 can be induced either by TLR agonists such as lipopolysaccharide (LPS), or by various endogenous stimuli, in particular those acting via a cAMP-dependent signaling pathway. We have previously reported that the synthetic phospho-ceramide analogue-1 (PCERA-1) increases cAMP level and subsequently down-regulates production of TNFalpha and up-regulates production of IL-10 in LPS-stimulated macrophages. The objective of this study was to determine the mechanism of activity of PCERA-1 and the role of cAMP in LPS-induced IL-10 production. We show here that PCERA-1 induces IL-10 production in synergism with various TLR agonists in mouse RAW264.7 macrophages. Cooperativity is evident both at the mRNA and protein levels. IL-10 production by LPS and PCERA-1 is mediated by the cAMP pathway and by the p38 MAP kinase. Phosphorylation of p38 is cooperatively accomplished by LPS and PCERA-1 or other cAMP inducers. Furthermore, the activity of PCERA-1 can be partially mimicked by a cell-permeable analog of cAMP, and blocked by the protein kinase A (PKA) inhibitor H89. Finally, in the absence of PCERA-1, the residual IL-10 induction by LPS depends on the basal cAMP level as it can be largely elevated by the phosphodiesterase (PDE)-4 inhibitor rolipram. Our results thus indicate that IL-10 induction by LPS critically depends on basal cAMP level, and that a co-stimulus by a TLR agonist and a cAMP-elevating agent results in synergistic PKA-dependent and p38-dependent IL-10 production.
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http://dx.doi.org/10.1016/j.molimm.2009.03.009DOI Listing
June 2009

Modulation of TNFalpha, IL-10 and IL-12p40 levels by a ceramide-1-phosphate analog, PCERA-1, in vivo and ex vivo in primary macrophages.

Immunol Lett 2009 Mar 29;123(1):1-8. Epub 2009 Jan 29.

Department of Biochemistry, Life Sciences Institute, Tel-Aviv University, Tel-Aviv 69978, Israel.

Phospho-ceramide analog-1 (PCERA-1) has been described as a potent in vivo suppressor of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha), and thus as a putative drug for the treatment of inflammatory diseases. However, the in vivo cell target of PCERA-1 has not been identified, and its in vivo effect on secretion of other relevant cytokines has not been reported. We have previously shown that PCERA-1 suppresses lipopolysaccharide (LPS)-induced TNFalpha production in RAW264.7 macrophages in vitro. We therefore hypothesized that PCERA-1 targets TNFalpha production by primary macrophages. In this study we thus investigated the effect of PCERA-1 on LPS-induced release of TNFalpha, interleukin (IL)-10 and IL-12p40, in vivo, and ex vivo. We found that PCERA-1 suppressed production of the pro-inflammatory cytokines, TNFalpha and IL-12p40, and increased production of the anti-inflammatory cytokine, IL-10, in LPS-challenged mice, and in primary peritoneal macrophages as well as bone marrow-derived macrophages (BMDM) stimulated with LPS and interferon (IFN)-gamma. These activities of PCERA-1 were independent of each other. In contrast, PCREA-1 only slightly affected TNFalpha production in the whole blood assay, where LPS-induced cytokines are mainly produced by monocytes. Moreover, isolated blood monocytes were inert to PCERA-1, but acquired responsiveness to PCERA-1 upon macrophage colony stimulating factor (M-CSF)-induced differentiation into macrophages. Pharmacokinetic analysis in mice showed that while the volume of distribution of PCERA-1 is low, the drug was rapidly exchanged between the peritoneum and the systemic circulation. Together, these results suggest that sensitivity to PCERA-1 increases upon differentiation of blood monocytes into tissue macrophages, and imply a mechanistic role for peritoneal macrophages in the in vivo anti-inflammatory activity of PCERA-1. Finally, we show that the mechanism of activity of PCERA-1 and prostaglandin E2 (PGE2) is distinct, and that PCERA-1 signaling is not mediated by EP2, a PGE2 receptor which is also activated by oxidized phospholipids. The independent and reciprocal modulation of production of TNFalpha and IL-12p40, vs. IL-10, suggests that PCERA-1 may be a candidate drug for the treatment of inflammation-linked diseases.
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http://dx.doi.org/10.1016/j.imlet.2008.12.011DOI Listing
March 2009

A ceramide-1-phosphate analogue, PCERA-1, simultaneously suppresses tumour necrosis factor-alpha and induces interleukin-10 production in activated macrophages.

Immunology 2009 May;127(1):103-15

Department of Molecular and Structural Biochemistry, Life Sciences Institute, Tel-Aviv University, Tel-Aviv, Israel.

Tight regulation of the production of the key pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) is essential for the prevention of chronic inflammatory diseases. In vivo administration of a synthetic phospholipid, named hereafter phospho-ceramide analogue-1 (PCERA-1), was previously found to suppress lipopolysaccharide (LPS)-induced TNF-alpha blood levels. We therefore investigated the in vitro anti-inflammatory effects of PCERA-1. Here, we show that extracellular PCERA-1 potently suppresses production of the pro-inflammatory cytokine TNF-alpha in RAW264.7 macrophages, and in addition, independently and reciprocally regulates the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Specificity is demonstrated by the inability of the phospholipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) to perform these activities. Similar TNF-alpha suppression and IL-10 induction by PCERA-1 were observed in macrophages when activated by Toll-like receptor 4 (TLR4), TLR2 and TLR7 agonists. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that, while PCERA-1 does not block activation of nuclear factor (NF)-kappaB and mitogen-activated protein kinases by LPS, it elevates the intracellular cAMP level. In conclusion, the anti-inflammatory activity of PCERA-1 seems to be mediated by a cell membrane receptor, upstream of cAMP production, and eventually TNF-alpha suppression and IL-10 induction. Thus, identification of the PCERA-1 receptor may provide new pharmacological means to block inflammation.
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http://dx.doi.org/10.1111/j.1365-2567.2008.02928.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2678186PMC
May 2009

Identification of small-molecule antagonists that inhibit an activator: coactivator interaction.

Proc Natl Acad Sci U S A 2004 Dec 7;101(51):17622-7. Epub 2004 Dec 7.

Department of Peptide Biology and Regulatory Biology Laboratories, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037-1002, USA.

Phosphorylation of the cAMP response element binding protein (CREB) at Ser-133 in response to hormonal stimuli triggers cellular gene expression via the recruitment of the histone acetylase coactivator paralogs CREB binding protein (CBP) and p300 to the promoter. The NMR structure of the CREB:CBP complex, using relevant interaction domains called KID and KIX, respectively, reveals a shallow hydrophobic groove on the surface of KIX that accommodates an amphipathic helix in phospho (Ser-133) KID. Using an NMR-based screening approach on a preselected small-molecule library, we identified several compounds that bind to different surfaces on KIX. One of these, KG-501 (2-naphthol-AS-E-phosphate), targeted a surface distal to the CREB binding groove that includes Arg-600, a residue that is required for the CREB:CBP interaction. When added to live cells, KG-501 disrupted the CREB: CBP complex and attenuated target gene induction in response to cAMP agonist. These results demonstrate the ability of small molecules to interfere with second-messenger signaling cascades by inhibiting specific protein-protein interactions in the nucleus.
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http://dx.doi.org/10.1073/pnas.0406374101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC539725PMC
December 2004

Solution structure of the KIX domain of CBP bound to the transactivation domain of c-Myb.

J Mol Biol 2004 Mar;337(3):521-34

Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

The hematopoietic transcription factor c-Myb activates transcription of target genes through direct interactions with the KIX domain of the co-activator CBP. The solution structure of the KIX domain in complex with the activation domain of c-Myb reveals a helical structure very similar to that adopted by KIX in complex with the phosphorylated kinase inducible domain (pKID) of CREB. While pKID contains two helices, alphaA and alphaB, which interact with KIX, the structure of bound c-Myb reveals a single bent amphipathic helix that binds in the same hydrophobic groove as the alphaB helix of pKID. The affinity of c-Myb for KIX is lower than that of pKID, and relies more heavily on optimal interactions of the single helix of c-Myb with residues in the hydrophobic groove. In particular, a deep hydrophobic pocket in KIX accounts for more than half the interactions with c-Myb observed by NMR. A bend in the alpha-helix of c-Myb enables a critical leucine side-chain to penetrate into this pocket more deeply than the equivalent leucine residue of pKID. The components that mediate the higher affinity of pKID for KIX, i.e. the phosphate group and the alphaA helix, are absent from c-Myb. Results from isothermal titration calorimetry, together with the structural data, point to a key difference between the two complexes in optimal pH for binding, as a result of differential pH-dependent interactions with histidine residues of KIX. These results explain the structural and thermodynamic basis for the observed constitutive versus inducible activation properties of c-Myb and CREB.
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http://dx.doi.org/10.1016/j.jmb.2004.01.038DOI Listing
March 2004

Cooperativity in transcription factor binding to the coactivator CREB-binding protein (CBP). The mixed lineage leukemia protein (MLL) activation domain binds to an allosteric site on the KIX domain.

J Biol Chem 2002 Nov 29;277(45):43168-74. Epub 2002 Aug 29.

Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

The interactions between cAMP-response element-binding protein (CREB)-binding protein (CBP) and gene-specific transcription factors play an important role in activation of transcription from numerous genes. Cooperative interactions between CBP and multiple transcriptional activators may provide a mechanism for synergistic increases in transcriptional activation. Here we report the characterization of ternary complexes formed by the KIX domain of CBP and the transactivation domain of the trithorax group protein mixed lineage leukemia protein (MLL), together with either the phosphorylated kinase-inducible domain (pKID) of CREB or the activation domain from c-Myb. Isothermal titration calorimetry experiments show that KIX in complex with the MLL activation domain binds the c-Myb activation domain and pKID domain with 2-fold higher affinity than does the KIX domain alone. Thus, the activation domains of Myb and MLL or of pKID and MLL bind cooperatively to KIX. The thermodynamics of these interactions imply different mechanisms of binding cooperativity for the two ternary complexes; the KIX.MLL.pKID complex is stabilized by entropy increases, whereas the enhancement of Myb binding in the presence of the MLL activation domain is due to more favorable enthalpy. NMR experiments show that the MLL-binding site on KIX is distinct from the surface that binds the pKID and c-Myb activation domains. Our data indicate that KIX can directly mediate cooperative interactions between pairs of transcriptional regulatory proteins. In the case of MLL and c-Myb, both proteins are involved in proliferation of hematopoietic cells and leukemogenesis, and synergistic interactions mediated by CBP may play a functional role.
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http://dx.doi.org/10.1074/jbc.M207660200DOI Listing
November 2002

Roles of phosphorylation and helix propensity in the binding of the KIX domain of CREB-binding protein by constitutive (c-Myb) and inducible (CREB) activators.

J Biol Chem 2002 Nov 23;277(44):42241-8. Epub 2002 Aug 23.

Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

cAMP-response element-binding protein (CREB)-binding protein (CBP) is a general transcriptional co-activator that mediates interactions between transcription factors and the basal transcription machinery. To obtain insights into the mechanism by which the KIX domain of CBP can recognize the transactivation domains of many different transcription factors, we have used NMR and biochemical analyses to study the interactions of KIX with the transactivation domain from the constitutive activator c-Myb and with the kinase-inducible transactivation domain (KID) from CREB. NMR chemical shift mapping shows that both activation domains bind to the same surface of KIX. In the unbound state, both the phosphorylated KID and c-Myb activation domains are only partly structured, and binding to KIX is coupled with folding to form an amphipathic helix. Helix-destabilizing mutations significantly impair binding, whereas mutations that increase the intrinsic secondary structure content of the free phosphorylated KID peptide have only a small influence on binding affinity. Low affinity but specific binding of unphosphorylated KID to KIX was measured by ITC and was also observed in Western blot assays and by a fluorescence resonance energy transfer experiment in living cells. The large increase in the affinity for phosphorylated KID is due to favorable intermolecular interactions involving the phosphate moiety. After induction by phosphorylation, CREB is able to compete effectively with other transcriptional activators for binding to CBP.
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http://dx.doi.org/10.1074/jbc.M207361200DOI Listing
November 2002
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