Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties.

Abstract

In previous reports we have demonstrated that the 60-aa peptide corresponding to the homeodomain of the Drosophila protein Antennapedia (pAntp) translocates through the membrane of neurons in culture, accumulates in neuronal nuclei, and promotes neurite growth. To analyze the importance of specific pAntp DNA-binding properties in this phenomenon we have constructed three mutant versions of pAntp that differ in their ability to translocate through the membrane and to bind specifically the cognate sequence for homeodomains present in the promoter of HoxAS. We demonstrate that removing two hydrophobic residues of the third helix inhibits pAntp internalization and suppresses its neurotrophic activity. We also show that pAntp neurotrophic activity is lost when mutations are introduced in positions preserving its penetration and nuclear accumulation but abolishing its capacity to bind specifically the cognate DNA-binding motif for homeoproteins. Our results strongly suggest that pAntp neurotrophicity requires both its internalization and its specific binding to homeobox cognate sequences. We propose that homeoproteins might regulate important events in the morphological differentiation of the postmitotic neuron.

Protein transduction: an alternative to genetic intervention?

“…The homeodomain of antennapedia (AntpHD) is capable of translocating across neuronal membranes and is conveyed to the nucleus. [4][5][6] The third helix of the homeodomain, comprising just 16 amino acids, also possesses this unusual property, 2,7 enabling small molecules to be taken up with AntpHD or with the AntpHD peptide into live cells 8 and has formed the basis for the family of peptides. 3 Internalisation is independent of the ␣-helix configuration of the third domain of AntpHD, and occurs both at 37°C and 4°C and thus is not receptor-mediated.…”

Promoter‐specific regulation of gene expression by an exogenously added homeodomain that promotes neurite growth

“…Only pAntp enhanced neurite outgrowth; pAntp50A ~s well as other variants devoid of DNA-binding activity and/or not internalized by the cells were devoid of neurotrophic effect [4]. These results suggested that the mode of action of pAntp involves a direct competition for sequences present in the promoters of genes normally regulated by homeoproteins, consequently affecting their transcription.…”

“…pAntp and pAntp50A (GlnS°---)Ala 5°) were prepared in E. coli as described previously [4]. Their internalization was tested as described in Joliot et al [1].…”

“…Gel retardation experiments were as described in [4]. The HindlII BamHI fragment of pTHCR was gel-purified and end-labelled with the Klenow DNA polymerase in the presence of [32p]dCTP.…”

“…To analyse the specificity of pAntp neurotrophic effect we generated several mutant peptides. One of these mutants, pAntp50A, carries a Glutamine to Alanine mutation in position 9 of the third helix [4]. pAntp50A can still translocate through membranes, accumulates in the nucleus but does not bind with high affinity to the homeodomain target sequences recognized by pAntp [4].…”