ABSTRACT
Bioactive Peptides ................................................................................ 277
16.2.1 Peptide Synthesis .................................................................... 277
16.2.2 Cell Culture .............................................................................. 278
16.2.3 Measurement of Translocation Kinetics .................................. 278
16.2.4 Measurement of b-Hexoseaminidase from RBL-2H3 Cells ........................................................................ 278
16.2.5 Measurement of Phospholipase D (PLD) Activity ................ 279
16.2.6 Detection of p42/44 MAPK Phosphorylation ........................ 279
16.2.7 MTT Assay of Cellular Viability ............................................ 279
16.3 Results .................................................................................................... 280
16.3.1 Translocation Kinetics of TP10-Gia3 346-355 .......................... 280
16.3.2 TP10 Chimerae-Induced Secretion of
b-Hexoseaminidase .................................................................. 280 16.3.3 Mechanisms of Cys0PKC238-249-Induced Secretion of
b-Hexoseaminidase .................................................................. 280 16.3.4 Intracellular Delivery of Gia3
346-355 Does Not Modulate
b-Hexoseaminidase Secretion from RBL-2H3 Cells .............. 280 16.3.5 Intracellular Translocation of Gia3
346-355 Activates a Signal
Transduction Cascade that Terminates in the
Dual Phosphorylation of P42/44 MAPK ................................ 283
16.3.6 TP10 and TP10 Chimerae Do Not Reduce
Cellular Viability ...................................................................... 285
16.4 Discussion .............................................................................................. 285
16.4.1 TP10 as an Efficient Vector for the Intracellular
Delivery of Peptides ................................................................ 285
16.4.2 Biological Activities of Peptide Cargoes ................................ 287
Acknowledgments ............................................................................................ 289
References ........................................................................................................ 289
A fundamental paradigm of intracellular signal transduction is that of protein-protein
interactions. The molecular interfaces and regulatory domains that mediate these
protein-protein interactions are therefore potential targets for mimetic peptides
that can selectively modulate signal transduction. Moreover, attachment of peptide
fragments, corresponding to these molecular interfaces, to cell-penetrating peptide
(CPP) vectors not only provides a vehicle for the introduction of peptidomimetic
probes into the cytosolic environment, but offers the distinct advantage of
intracellular delivery of peptide cargoes into intact and living cellular systems.1,2
Furthermore, the bioassays used herein represent membrane translocation, protein
binding, and activation/inhibition of biological responses and are therefore perhaps a
more rigorous assessment of the utility of our TP10 chimerae to act as signal
transduction modulators (STMs).
