Cell permeable ITAM constructs for the modulation of mediator release in mast cells.
Spleen tyrosine kinase (Syk) is essential for high affinity IgE receptor (FcεRI) mediated mast cell degranulation. Once FcεRI is stimulated, intracellular ITAM motifs of the receptor are diphosphorylated (dpITAM) and Syk is recruited to the receptor by binding of the Syk tandem SH2 domain to dpITAM, resulting in activation of Syk and, eventually, degranulation. To investigate intracellular effects of ITAM mimics, constructs were synthesized with ITAM mimics conjugated to different cell penetrating peptides, i.e. Tat, TP10, octa-Arg and K(Myr)KKK, or a lipophilic C(12)-chain. In most constructs the cargo and carrier were linked to each other through a disulfide bridge, which is convenient for combining different cargos with different carriers and has the advantage that the cargo and the carrier may be separated by reduction of the disulfide once it is intracellular. The ability of these ITAM constructs to label RBL-2H3 cells was assessed using flow cytometry. Fluorescence microscopy showed that the octa-Arg-SS-Flu-ITAM construct was present in various parts of the cells, although it was not homogeneously distributed. In addition, cell penetrating constructs without fluorescent labels were synthesized to examine degranulation in RBL-2H3 cells. Octa-Arg-SS-ITAM stimulated the mediator release up to 140%, indicating that ITAM mimics may have the ability to activate non-receptor bound Syk.
Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands.
Joeri
J
Kuil
Marcel J E
MJ
Fischer
Nico J
NJ
de Mol
Rob M J
RM
Liskamp
D020816:Amino Acid Motifs; D000818:Animals; D040761:Biomimetic Materials / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D002470:Cell Survival; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D008407:Mast Cells / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D000072377:Syk Kinase
D047908:Intracellular Signaling Peptides and Proteins; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606898:Syk protein, rat