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24 Cell-Penetrating Peptides: Processes and Applications
2.5 Experimental Procedures ...............................................................................40
2.5.1 AntpHD and Penetratin......................................................................40
2.5.1.1 AntpHD and Penetratin Labeling.......................................40
2.5.1.1.1 FITC Labeling Postsynthesis ...........................40
2.5.1.1.2 Biotinylation Postsynthesis ..............................41
2.5.1.2 Internalization of AntpHD or Penetratin Peptides.............41
2.5.1.3 Detection of Internalized AntpHD or Penetratin ...............42
2.5.1.3.1 Detection of Peptides in Live Cells .................42
2.5.1.3.2 Detection of Biotin-Labeled Peptides..............42
2.5.1.3.3 Detection of Radioactive Peptides
Detection...........................................................42
2.5.2 AntpHD and Penetratin-Coupled Cargoes ........................................42
2.5.2.1 Cargo–Vector Linkage ........................................................42
2.5.2.1.1 Oligonucleotides and PNAs .............................42
2.5.2.1.2 Peptide ..............................................................43
2.5.2.1.3 Direct Linkage..................................................44
2.5.2.1.4 Fusion Proteins in Bacteria ..............................44
2.5.2.1.5 Decoupling Reaction ........................................44
2.5.2.2 Internalization and Detection of Vector–Cargo
Fusion Molecules................................................................44
2.5.2.2.1 Oligonucleotide Internalization........................45
2.5.2.2.2 Peptide Internalization......................................45
2.5.3 Comments...........................................................................................46
2.5.3.1 Oligopeptides ......................................................................46
2.5.3.2 Oligonucleotides .................................................................47
2.5.3.3 Peptide Nucleic Acids ........................................................47
2.5.3.4 Drugs...................................................................................47
References................................................................................................................47
2.1 INTRODUCTION
Homeoproteins are transcription factors involved in several biologic processes, primarily
during development. They were first discovered in Drosophila and later in
all metazoans and plants. They bind DNA through a 60-amino-acid-long sequence,
the homeodomain. This homeodomain is highly conserved across homeoproteins
and species and composed of three α-helices, the third of which is more particularly
dedicated to recognition of the DNA target site. 1 In addition to their role in pattern
formation during development, homeoproteins can act later to refine or maintain
neuronal connections in invertebrates and vertebrates. Indeed, mutations in homeogenes
lead to modifications in axon pathfinding, 2-4 and synapse formation. 5-7 This
suggests that homeoproteins probably help to define the important plasticity characterizing
the vertebrate nervous system.
2.2 HOMEOPROTEIN-DERIVED PEPTIDIC VECTORS
2.2.1 THE ANTENNAPEDIA HOMEODOMAIN
To analyze the role of homeoproteins in neuronal morphogenesis, we developed a
protocol aimed at antagonizing transcriptional activity of endogenous homeoproteins.