The "sneaking-ligand" approach: cell-type specific inhibition of the classical NF-κB pathway.
The intracellular delivery of molecules across the plasma membrane represents a major obstacle. The conjugation of cell-permeable peptides (CPPs) to proteins promotes the uptake and internalization. However, uptake of CPPs is receptor independent and not cell-type specific. Recently, we established the "sneaking-ligand" approach which is based on multimodular recombinant fusion proteins that consist of three modules connected with serine-glycine linkers. Module one is responsible for receptor-mediated endocytosis; module two supports translocation into the cytoplasm so that the effector module three can interact with its binding partner in the cytoplasm. For NF-κB inhibition, we described an NF-κB inhibitor that targets selectively the activated endothelium via an oligopeptide motif. Upon E-selectin-mediated endocytosis, the Pseudomonas exotoxin A domain II (ETAII) translocates the NEMO-binding peptide to the cytoplasm interfering with IκB kinase complex assembly. Inflammatory autoimmune diseases are triggered, but also resolved by a variety of cell types. Therefore, the inhibition of NF-κB should be restricted to those cells that are crucially involved in the pathogenesis of inflammatory diseases. A general blockade of NF-κB may result in severe immunosuppression and possibly in organ dysfunction or damage. The "sneaking-ligand" approach could minimize the risks of therapeutic interventions and identify disease-relevant cell types. Here we describe the recombinant expression and purification of the E-selectin-specific "sneaking-ligand construct" (SLC1) and its ability to inhibit cytokine-induced NF-κB activation in vitro.
Department of Rheumatology and Clinical Immunology and Centre of Chronic Immunodeficiency, University Medical Centre and University of Freiburg, Hugstetter Street 55, Freiburg, 79106, Germany, Bettina.Sehnert@uniklinik-freiburg.de.
Bettina
B
Sehnert
Harald
H
Burkhardt
Stefan
S
Dübel
Reinhard E
RE
Voll
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D016466:CHO Cells; D057846:Cell-Penetrating Peptides; D002846:Chromatography, Affinity / Q000379:methods; D003412:Cricetulus; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D016328:NF-kappa B / Q000378:metabolism*; D009928:Organ Specificity / Q000187:drug effects; D011489:Protein Denaturation; D054730:Protein Interaction Domains and Motifs; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects
D057846:Cell-Penetrating Peptides; D019040:E-Selectin; D008024:Ligands; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins