Antennapedia homeobox peptide regulates neural morphogenesis.

Abstract

We synthesized the 60-amino acid polypeptide corresponding to the sequence of the Drosophia antennapedia gene homeobox. This peptide (pAntp) recognized the consensus motif for binding to the promoter region ofHox-1.3. pAntp mechanically introduced into mammalian nerve cells provoked a dramatic morphological differentiation of the neuronal cultures. Moreover, pAntp directly added to already differentiated neuronal cultures penetrated the cells and further augmented their morphological differentiation. Examination of live and fixed neurons in classical and confocal fluorescence microscopy demonstrated that pAntp was captured at all regions of the nerve cells and accumulated in the nuclei. In addition, the effect of pAntp on neurite extension was blocked in the presence of the protein synthesis inhibitor cycloheximide. Thus, our results demonstrate that neurons possess an efficient uptake system for the antennapedia homeobox peptide and suggest that binding of pAntp to consensus motifs present in nerve cell nuclei influences neuronal morphogenetic programs.Studies on developmental mutants in Drosophila have demonstrated that several DNA binding proteins encoded by homeotic genes are endowed with morphogenetic functions (1, 2). In Drosophila at least, the expression of specific homeotic genes is responsible for the formation of cell assemblies exhibiting precise and defined morphologies. The DNA binding properties ofthese proteins is due to a sequence of about 60 amino acids called the homeobox.The homeobox sequences have been highly conserved during evolution and genes containing homeobox sequences are present in all vertebrates, including mammals (3, 4). In vertebrates, as in Drosophila, homeobox gene expression is not limited to the period during which the general features of body organization are established. In particular, a number of these genes are expressed in the nervous system rather late during development and, in some cases, through adulthood (5-7).In vitro studies have shown that the regulatory effect of homeotic proteins on gene expression depends on the specific binding of the homeobox domain to consensus DNA sequences found in the promotors or enhancers of several reporter genes and homeobox-containing genes (8-10). It has also been clearly demonstrated that the 60-amino acid homeobox polypeptide alone, isolated from the flanking regions, which are necessary for the activation or repression of transcription, has per se a high affinity for such consensus motifs (11).Thus, one possible way to study directly, within the live cell, the role of a homeotic protein family, as defined by the primary structure of the homeobox (e.g., antennapedia-or engrailed-like) and by the recognition of identical binding sites, on neural development would be to synthesize and inject the 60-amino acid homeotic polypeptides into the cells. In fact, such peptides could act as competitive inhibitors of endogeneous homeotic proteins with similar binding specificities.In the experiments presented herein we have used this...

Efficient Delivery of Antennapedia Homeodomain Fused to CTL Epitope with Liposomes into Dendritic Cells Results in the Activation of CD8+ T Cells

“…Several vectors have been reported to have this ability, including toxins (43,44), the protein transduction domain from the HIV, TAT protein (45), and AntpHD (26). Their common attribute is their capacity to deliver peptides directly in the cytosol in a receptor-dependent manner for toxins (44) or following spontaneous crossing of cell membranes in the case of TAT (46) and AntpHD (47). They all differ from exogenous proteins in terms of their internalization mechanisms, which commonly use phagocytosis/endocytosis.…”

Cell-penetrating and cell-targeting peptides in drug delivery

“…In particular, two of these molecules have been extensively studied in order to define the structural or sequence elements needed for the translocation: the Tat protein from the HIV-1 virus [1,2] and the Drosophila melanogaster Antennapedia homeodomain [3]. A study on the structure-activity relationship was carried out on these two small proteins of 101 and 60 amino acids, respectively, and the minimal domain needed for translocation was defined [4][5][6].…”

Sequence Defined Disulfide-Linked Shuttle for Strongly Enhanced Intracellular Protein Delivery

“…The first observed protein that was naturally able to translocate through the intact phospholipid bilayer was the HIV-1 TAT protein. 3,4 In the meantime, it has been discovered that natural homeoproteins such as the Antennapedia homeobox can cross cell membranes and internalize into cells as part of their physiological function, 5 and numerous homeoprotein-derived natural and artificial transduction peptides have been identified. 6−9 Thus, the intracellular delivery of active proteins, named protein transduction, now is considered as an interesting option and alternative to gene therapy, substituting for dysfunctional cellular proteins.…”