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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010New SOCS1-KIR Mimetic Peptides Through the Screening ofFocused Simplified Combinatorial LibrariesDaniela Marasco 1,2 , Nunzianna Doti 2 , Pasqualina L. Scognamiglio 1 ,Stefania Madonna 3 , Menotti Ruvo 2 , Carlo Pedone 1,2 ,and Cristina Albanesi 31 Department of Biological Sciences, School of Biotechnological Sciences, University“Federico II”, Naples, 80134, Italy; 2 Institute of Biostructures and Bioimaging –IBB-CNR,Naples, 80134, Italy; 3 IDI-IRCCS, Roma, 00167, ItalyIntroductionSuppressor Of Cytokine Signalling (SOCS) proteins are negative feedback regulators ofseveral pathways involved in immune response, particularly the JAK/STAT (Januskinase/Signal Transducer and Activator of Transcription) [1]. Usually their basal levels arelow, but they can be selectively induced by cytokines, such as IFN . SOCS1 inhibits IFNsignalling for its capacity to bind and inactivate JAK2 protein and consequently to blockthe IFNγ-induced tyrosine phosphorylation of IFN receptor (IFN R) and STAT1activation. It has been demonstrated that keratinocytes avoid the detrimental consequencesof an excessive stimulation by IFN- over-expressing SOCS1 thus hindering the expressionof many pro-inflammatory genes, including those involved in skin diseases, such aspsoriasis and allergic contact dermatitis (ACD) [2]. A three-dimensional model of thecomplex between SOCS-1 and JAK2 [3] shows that the Kinase Inhibitory Region (KIR) ofSOCS-1 protrudes towards the catalytic region of JAK2 and occupies the ATP binding site.In this work we identify new peptides mimicking KIR-SOCS-1 binding activity through anELISA-based screening of a focused simplified combinatorial peptide library [4].Results and DiscussionThrough direct binding ELISAs, we confirmed the ability of KIR region (52-67) of SOCS1to bind to JAK2 catalytic site (1001-1013) both in the phosphorylated (Tyr1007), namedpJAK2, and in the non-phosphorylated form, JAK2) (Figure 1). We quantified thisinteraction in terms of KD values of about 100 µM (Table 1).Then to investigate crucial residues of KIR 52-67 region involved in JAK2recognition, we synthesized Ala-scanning peptides. Their binding capacities to both JAK2peptides were analyzed through direct ELISAs. Data analysis suggested that the keyresidues of KIR for JAK2 recognition are localized in 52-61 region. Then we synthesizedand analyzed the binding properties of this shorter sequence, named New-KIR. ELISAexperiments provided K D values in the low micromolar range. Therefore the deletion of sixamino acids in the C-terminal region of KIR led to new-KIR, able to bind to JAK2 catalyticsite with a 4-fold greater affinity.1.00.8Abs, 490nm0.60.4KIR vs JAK2KIR vs pJAK2CP vs JAK2CP vs pJAK20.20.00 100 200 300 400 500 600 700Peptide concentration, ( M)Fig. 1. ELISA direct binding between KIR peptide 52-67vs JAK2 and p-JAK2.538