Lavens, Delphine

Peelman, Frank

Piessevaux, Julie

Tavernier, Jan

English

Ghent University Academic Bibliography

fore the identiﬁcation, biological characterization of endogenous Cynomolgus IFN-asubtypes and their accurate detection are of utmost importance for predicting theclinical outcome of effective human therapeutics. We have isolated full length IFN-a coding sequences from Cynomolgus genomic DNA using PCR methods. Sequenceanalysis revealed 11 unique IFN-a subtypes which were transiently expressed in Verocells. The conditioned supernatants were tested in parallel with puriﬁed, E. coli pro-duced Cynomolgus IFN-a2 for accurate quantitation. All samples tested on Cynomol-gus cells exhibited strong antiviral activity. Additionally, the biological activities ofthe IFN proteins secreted in the tissue culture supernatants were characterized in sev-eral human cell-based assays including A549/EMCV cytopathic protective effect, OV-CAR-3 antiproliferative and caspase-3/7 activation assays. All subtypes displayedactivity on these separate human cell lines suggesting reasonable species cross-reac-tivity. Analysis in a human IFN-a ELISA further supported these observations. Sepa-rately, we analyzed the activity in a human cell-based reporter assay (iLiteTM HumanIFN-a Kit) including the use of an anti-IFN-a receptor neutralizing antibody. This as-say correlated with the other human cell-based assays, providing an additional, morerapid measure of activity. Moreover, the activity could be potently neutralized by theanti-human IFNAR monoclonal antibody further suggesting similarities betweenCynomolgus and human IFN-a receptor proteins. Therefore these reagents shouldprove useful in the detection and biological evaluation of Cynomolgus IFN-a proteinsused for the better understanding of human viral diseases and their treatments.doi:10.1016/j.cyto.2008.07.289226Multiple roles for the SOCS boxJulie Piessevaux, Delphine Lavens, Frank Peelman, Jan Tavernier, Department of MedicalProtein Research, VIB, Department of Biochemistry, Faculty of Medicine and Health Sciences,Ghent University, A. Baertsoenkaai 3, 9000 Ghent, BelgiumSOCS proteins play a critical role in the negative feedback inhibition of cytokinesignal transduction pathways. SOCS protein contain a C-terminal SOCS box domain.A similar SOCS box domain is found at the C-terminus of over 70 human proteins.The SOCS box domain is usually coupled to protein interaction module(s) that interactwith a protein target. The SOCS box can interact with elongin and cullin molecules,leading to formation of an E3 ligase complex and proteasomal degradation of the tar-get. In this way, SOCS proteins can direct activated cytokine receptor complexes toproteasomal degradation. We recently demonstrated that the SOCS box of SOCS pro-teins is implicated in the interaction of all SOCS proteins with SOCS2, 6 and 7. Thisinteraction can lead to degradation of the targeted SOCS protein. SOCS2, 6 and 7are thus able to control degradation of other SOCS proteins. We further demonstratedthat the interaction of Cis with its substrate is critically dependent upon the integrityof its SOCS box and its interaction with elongins. The SOCS box thus emerges as a ver-satile module controlling cytokine signaling via multiple mechanisms.doi:10.1016/j.cyto.2008.07.290227Design of high afﬁnity interleukin-6 and gp130 variants: Implications for cyto-kine-receptor assemblyNina Adam1, Stephanie Tenhumberg 1, Georg H. Waetzig 2, Athena Chalaris 1,Björn Rabe 1, Jan Suthaus 1, Dirk Seegert 2, Jürgen Scheller 1, Stefan Rose-John 1,Joachim Grötzinger 1, 1 Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel,Germany, 2CONARIS Research Institute AG, Kiel, GermanyBinding of human Interleukin-6 (IL-6) to its speciﬁc receptor IL-6R is a prere-quisite for the activation of the signal transducing receptor glycoprotein 130(gp130).IL-6 binds to the IL-6R via site I and makes contact to gp130 via site IIand site III. The mode of assembly and the stochiometry of the signalling complexis not completely understood. We have used two different models to shed morelight on the molecular make-up of the signalling IL-6R complex. In the ﬁrst modelwe have analyzed the viral IL-6 (vIL-6) protein from Human herpes virus 8whichunlike human IL-6 can bind to gp130 directly. We have identiﬁed the amino acidswithin vIL-6, which are responsible for direct binding to gp130 and we show thatthese amino acids when transferred into the human IL-6 protein also render humanIL-6 independent of the IL-6R. In the second model we have identiﬁed amino acidresidues within the gp130 molecule, which are of particular importance for bindingto the IL-6/IL-6R complex. We have identiﬁed three amino acids, which, whenmutated, give rise to gp130 molecules with higher binding afﬁnities. Surprisingly,amino acids within vIL-6, important for direct gp130 binding and amino acids gov-erning gp130 binding to the IL-6/IL-6R lie within site III, which has previously beenthought to be responsible for the dimerization of two preassembled IL-6/IL-6R/gp130 trimers. We believe that the importance of site III for the assembly of theIL-6 receptor complex is underestimated and we will present an alternative modelof receptor complex assembly which proposes a tetrameric instead of a hexamericIL6/IL6R/gp130 signaling complex.doi:10.1016/j.cyto.2008.07.291228Erythropoietin and glucocorticoids exert antagonistic effects on STAT-5 phos-phorylation in primary human erythroblastsEmilia Stellacci 1, Antonella Di Noia 2, Katija Jelicic 2, Maria Zingariello 6,Angela Di Baldassarre 6, Giovanni Migliaccio 2, Ronald Hoffman 4,5, Angela Battistini 1,Anna Rita Migliaccio 3,4,5, 1Departments of Infectious, Parasitic and ImmunomediatedDiseases, Istituto Superiore Sanita`, Rome, Italy, 2Cell Biology and Neurosciences, IstitutoSuperiore Sanita`, Rome, Italy, 3Hematology, Oncology and Molecular Medicine, IstitutoSuperiore Sanita`, Rome, Italy, 4Department of Medicine, Mount Sinai School of Medicine,New York, NY, USA, 5Myeloproliferative Research Consortium (MPD-RC),6Department of Biomorphology, University of Chieti-Pescara, Chieti, ItalyExtensive clinical studies have established a direct correlation between num-bers of red cells present in the blood and the concentration of erythropoietin.Erythropoietin interacts with a speciﬁc receptor, EPO-R, on the surface of erythroidcells. In the mouse, in addition to EPO and EPO-R, the glucocorticoid receptor (GR)controls erythropoiesis following stress. It is generally accepted that glucocorticoidsenter cells and interact with GR within the cytoplasm. This binding induce receptordimerization, STAT-5-phosphorylation and formation of GR/STAT-5 complexes, thatmigrate to the nucleus, bind to speciﬁc consensus sequences and activate theexpression of target genes. In erythroid cells, GR stimulation regulates the expres-sion of a subset of genes associated with induction of proliferation. Recent evi-dence indicates the existence of rapid glucocorticoid signalling mediated bymembrane associated receptors. The possibility that GR might be associated withthe plasma membrane of erythroid cells has not been investigated to date. In thisstudy we describe a new biochemical pathway that might contribute to the revers-ible inhibition exerted by glucocorticoids on erythroid maturation and that maycontribute to the genesis of polycythemia vera (PV). We show that dexamethasoneantagonizes the effects of erythropoietin on maturation, but not on survival, ofhuman proerythroblasts. While erythropoietin and dexamethasone alone induceSTAT-5-phosphorylation, the combination was ineffective in promoting STAT-5-phosphorylation in normal erythroblasts. By contrast, STAT-5-phosphorylationwas observed in erythroblasts from PV patients stimulated with erythropoietinand dexamethasone in combination. Growth factor deprivation induced signiﬁcantlevels of STAT-5-phosphorylation in erythroblasts from normal subjects and fromPV patients. A signiﬁcant correlation exists between levels of STAT-5-phosphoryla-tion induced by growth factor deprivation and numbers of proerythroblasts gener-ated in vitro from different donors. These data suggest that glucocorticoidsinterfere with EPO-induced maturation by antagonizing the STAT-5-phosphoryla-tion activity of EPO-R and that this antagonism is altered in proerythroblasts fromPV patients.doi:10.1016/j.cyto.2008.07.292229Does S100A12 activate mast cells and monocytes/macrophages via rage?Jesse Goyette, Weixing Yan, Su Yin Lim, Nick Di Girolamo, Carolyn L. Geczy, Centre forInfection and Inﬂammation Research, School of Medical Sciences, University of New SouthWales, Sydney, NSW 2052, AustraliaS100A12 is considered a DAMP thought to exert proinﬂammatory functionsthrough ligation of RAGE1. Earlier experiments were based on effects of bovineS100A12 on BV-2 cells (murine microglial cells)1. Monocytes and THP-1 cellsexpress RAGE. Using a homologous system, we assessed 9 batches of LPS-freerecombinant S100A12, but found no induction of IL-1b, IL-6, IL-8 or TNF inPBMCs, or of IL-8 by undifferentiated/differentiated THP-1 cells. Although weshowed that S100A12 undergoes structural changes with zinc, to form quatra-mers and hexamers, zinc did not alter the inability of S100A12 to activate mono-cytes. S100A12 is chemotactic for monocytes and mast cells (MC), induces MCdegranulation and MC-dependent inﬂammation in vivo. Responses were partiallyblocked by sRAGE, a RAGE antagonist, although MC did not express RAGE mRNAor protein, suggesting an alternate receptor. S100A12 provoked chemotaxis ofTHP-1 cells in a biphasic manner, with optimal concentrations at 10–9 and10–12 M; both were pertussis toxin sensitive. sRAGE only reduced migration ofTHP-1 cells in response to 10-9 M S100A12. PD98059, a selective ERK1/2 inhib-itor which reduces RAGE-mediated responses, did not affect S100A12-mediatedTHP-1 cell migration at either concentration. Thus monocytes and MC may sharea common G-protein coupled receptor that mediates S100A12-induced chemo-taxis. These results suggest that the ‘RAGE/S100 proinﬂammatory axis’ theoryshould be re-evaluated. Because S100A12 binds zinc we investigated its inﬂuenceAbstracts / Cytokine 43 (2008) 291–295 293

Multiple roles for the SOCS box

https://biblio.ugent.be/publication/532841/file/3113071

Multiple roles for the SOCS box

Abstract

Similar works

Full text

Available Versions

Ghent University Academic Bibliography