RESUMEN
Rho GTPases are a multifunctional family of proteins that are localized at cellular membranes via an isoprenyl group covalently linked to a C-terminal cysteine. Close to this primary site of membrane anchoring there is often found an additional polybasic region (PBR), which plays a secondary role in membrane binding and targeting of the complex. Here, peptides derived from the PBRs of the Rho family proteins Rac1 (K(183)KRKRK), TCL (K(198)KKKKR) and Cdc42 (P(182)KKSRR) were prepared with hexalysine (K(6)) and hexaarginine (R(6)) to study their interactions with multilamellar vesicles of phosphatidylglycerol (DOPG) and headgroup-deuterated dimyristoylphosphatidylcholine (DMPC-d(4)) using (2)H and (31)P NMR. The membranes retained their lamellar architecture after peptide binding, but the (2)H NMR line shapes for DMPC-d(4) indicated that the bound peptides altered the orientation of the choline headgroups, consistent with a change in membrane surface charge. Rac1 and TCL peptides appeared to affect the headgroup orientation similarly to K(6), although the perturbations were weaker and unlike those induced by the Cdc42 peptide and R(6). Magic-angle spinning (31)P NMR spectra of the membranes showed significant and selective broadening of the peak for DMPC after addition of the peptides, with R(6) and the Cdc42 peptide having the greatest effect. The selective broadening may be a consequence of the lipids separating into short-lived domains enriched in peptide-bound DOPG and peptide-free DMPC. These results illustrate that a complex relationship exists between the sequence of PBRs and their behaviour at membrane surfaces, which may have implications for the cellular functions and localization of Rho GTPases.
Asunto(s)
Lípidos de la Membrana/química , Membranas Artificiales , Péptidos/química , Proteína de Unión al GTP cdc42/química , Proteína de Unión al GTP rac1/química , Proteínas de Unión al GTP rho/química , Humanos , Lípidos de la Membrana/metabolismo , Péptidos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Host-derived anti-infective proteins represent an important source of sequences for designing antimicrobial peptides (AMPs). However such sequences are often long and comprise diverse amino acids with uncertain contribution to biological effects. Previously, we identified a simple highly cationic peptide derivative of human apolipoprotein E (apoEdp) that inhibited a range of microorganisms. Here, we have dissected the protein chemistry underlying this activity. We report that basic residues and peptide length of around 18 residues were required for activity; however, the Leu residues can be substituted by several other residues without loss of activity and, when substituted with Phe or Trp, resulted in peptides with increased potency. These apoEdp-derived AMPs (apoE-AMPs) showed no cytotoxicity and minimal haemolytic activity, and were active against HIV and Plasmodium via an extracellular target. CXCR4 and CCR5 strains of HIV were inhibited though an early stage in viral infection upstream of fusion, and a lack of inhibition of vesicular stomatitis virus G protein pseudotyped HIV-1 suggested the anti-HIV activity was relatively selective. Inhibition of Plasmodium invasion of hepatocytes was observed without a direct action on Plasmodium integrity or attachment to cells. The Trp-substituted apoE-AMP adhered to mammalian cells irreversibly, explaining its increased potency; NMR experiments confirmed that the aromatic peptides also showed stronger perturbation of membrane lipids (relative to apoEdp). Our data highlight the contribution of specific amino acids to the activity of apoEdp (and also potentially unrelated AMPs) and suggest that apoE-AMPs may be useful as lead agents for preventing the early stages of HIV and Plasmodium cellular entry.