Induction of raft‐like domains by a myristoylated NAP‐22 peptide and its Tyr mutant

Abstract

The N‐terminally myristoylated, 19‐amino acid peptide, corresponding to the amino terminus of the neuronal protein NAP‐22 (NAP‐22 peptide) is a naturally occurring peptide that had been shown by fluorescence to cause the sequestering of a Bodipy‐labeled PtdIns(4,5)P2 in a cholesterol‐dependent manner. The present work, using differential scanning calorimetry (DSC), extends the observation that formation of a PtdIns(4,5)P2‐rich domain is cholesterol dependent and shows that it also leads to the formation of a cholesterol‐depleted domain. The PtdIns(4,5)P2 used in the present work is extracted from natural sources and does not contain any label and has the native acyl chain composition. Peptide‐induced formation of a cholesterol‐depleted domain is abolished when the sole aromatic amino acid, Tyr11 is replaced with a Leu. Despite this, the modified peptide can still sequester PtdIns(4,5)P2 into domains, probably because of the presence of a cluster of cationic residues in the peptide. Cholesterol and PtdIns(4,5)P2 also modulate the insertion of the peptide into the bilayer as revealed by 1H NOESY MAS/NMR. The intensity of cross peaks between the aromatic protons of the Tyr residue and the protons of the lipid indicate that in the presence of cholesterol there is a change in the nature of the interaction of the peptide with the membrane. These results have important implications for the mechanism by which NAP‐22 affects actin reorganization in neurons.

Tracking peptide–membrane interactions: Insights from in situ coupled confocal-atomic force microscopy imaging of NAP-22 peptide insertion and assembly

“…The ability of NAP-22 to influence the distribution of PtdIns(4,5)P 2 and cholesterol in membranes provides a mechanism by which this protein can influence the actin cytoskeleton (Epand et al, 2003a(Epand et al, ,b, 2001(Epand et al, , 2004. Since NAP-22 sequesters both cholesterol and PtdIns(4,5)P 2 into domains, it has been suggested that the protein recruits PtdIns(4,5)P 2 into raft-like domains and increases cytoskeleton/plasma membrane interactions through rafts that induce spatial rearrangements of the cytoskeleton (Epand et al, 2005a). In a bilayer that phase-segregates into l o (enriched in Chol and SM) and DOPC-enriched (l d ) domains, it was found that the fulllength NAP-22 inserts almost exclusively into the l o domain (Khan et al, 2003).…”

“…Inset: corresponding section analyses along A-A 0 . PtdIns(4,5)P 2 (Epand et al, 2005a(Epand et al, ,c, 2004. As the NAP-22 peptide concentration increases in the l d domain, it is likely that the peptide would start to recruit more cholesterol from the SM/Chol domain.…”

“…Corresponding to the amino terminus of the NAP-22 protein, the smaller NAP-22 peptide has a weaker ability to sequester cholesterol from a mixture of SOPC and cholesterol (Epand et al, 2005a), and thus may be less able to selectively partition into l o versus l d domains. This peptide has also been shown to sequester PtdIns(4,5)P 2 into domains in a cholesteroldependent manner in an SOPC/cholesterol mixture (Epand et al, 2004).…”

“…Recently, it was demonstrated that NAP-22 peptide inserts into membranes, sequesters PtdIns(4,5)P 2 in a cholesteroldependent manner, and induces formation of a cholesterol-depleted domain (Epand et al, 2004(Epand et al, , 2005a. The formation of membrane domains containing both cholesterol and PtdIns(4,5)P 2 is also promoted by a peptide segment of caveolin (Wanaski et al, 2003).…”

Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation

“…Myristate inserts hydrophobically into the lipid bilayer whereas basic amino acids form electrostatic interactions with the headgroups of acidic membrane phospholipids including PI (4,5)P 2 . There is evidence that in resting cells PI (4,5)P 2 is sequestered in cholesterol-rich domains by interaction with the hydrophobic/ basic motif of myristoylated proteins such as MARCKS, NAP-22, CAP-23, GAP-43 (Laux et al, 2000;Epand et al, 2005;McLaughlin & Murray, 2005). The modulation of availability of free PI (4,5)P 2 by these proteins regulates a number of cell signaling cascades in cells.…”