Incorporation of α-methylated amino acids into Apolipoprotein A-I mimetic peptides improves their helicity and cholesterol efflux potential.

Apolipoprotein A-I (ApoA-I) mimetic peptides are potential therapeutic agents for promoting the efflux of excess cellular cholesterol, which is dependent upon the presence of an amphipathic helix. Since α-methylated Ala enhances peptide helicity, we hypothesized that incorporating other types of α-methylated amino acids into ApoA-I mimetic peptides may also increase their helicity and cholesterol efflux potential. The last helix of apoA-I, peptide 'A' (VLESFKVSFLSALEEYTKKLNT), was used to design peptides containing a single type of α-methylated amino acid substitution (Ala/Aα, Glu/Dα, Lys/Kα, Leu/Lα), as well as a peptide containing both α-methylated Lys and Leu (6α). Depending on the specific residue, the α-helical content as measured by CD-spectroscopy and calculated hydrophobic moments were sometimes higher for peptides containing other types of α-methylated amino acids than those with α-methylated Ala. In ABCA1-transfected cells, cholesterol efflux to the peptides showed the following order of potency: 6α>Kα≈Lα≈Aα≫Dα≈A. In general, α-methylated peptides were resistant to proteolysis, but this varied depending on the type of protease and specific amino acid substitution. In summary, increased helicity and amphilicity due to α-methylated amino acid substitutions in ApoA-I mimetic peptides resulted in improved cholesterol efflux capacity and resistance to proteolysis, indicating that this modification may be useful in the future design of therapeutic ApoA-I mimetic peptides.

Alterations in the HLA-B*57:01 Immunopeptidome by Flucloxacillin and Immunogenicity of Drug-Haptenated Peptides.

Neoantigen formation due to the interaction of drug molecules with human leukocyte antigen (HLA)-peptide complexes can lead to severe hypersensitivity reactions. Flucloxacillin (FLX), a ß-lactam antibiotic for narrow-spectrum gram-positive bacterial infections, has been associated with severe immune-mediated drug-induced liver injury caused by an influx of T-lymphocytes targeting liver cells potentially recognizing drug-haptenated peptides in the context of HLA-B*57:01. To identify immunopeptidome changes that could lead to drug-driven immunogenicity, we used mass spectrometry to characterize the proteome and immunopeptidome of B-lymphoblastoid cells solely expressing HLA-B*57:01 as MHC-I molecules. Selected drug-conjugated peptides identified in these cells were synthesized and tested for their immunogenicity in HLA-B*57:01-transgenic mice. T cell responses were evaluated in vitro by immune assays. The immunopeptidome of FLX-treated cells was more diverse than that of untreated cells, enriched with peptides containing carboxy-terminal tryptophan and FLX-haptenated lysine residues on peptides. Selected FLX-modified peptides with drug on P4 and P6 induced drug-specific CD8+ T cells in vivo. FLX was also found directly linked to the HLA K146 that could interfere with KIR-3DL or peptide interactions. These studies identify a novel effect of antibiotics to alter anchor residue frequencies in HLA-presented peptides which may impact drug-induced inflammation. Covalent FLX-modified lysines on peptides mapped drug-specific immunogenicity primarily at P4 and P6 suggesting these peptide sites as drivers of off-target adverse reactions mediated by FLX. FLX modifications on HLA-B*57:01-exposed lysines may also impact interactions with KIR or TCR and subsequent NK and T cell function.

CCR5 N-terminal region plays a critical role in HIV-1 inhibition by Toxoplasma gondii-derived cyclophilin-18.

Molecular mimicry of chemokine ligands has been described for several pathogens. Toxoplasma gondii produces a protein, cyclophilin-18 (C-18), which binds to the human immunodeficiency virus (HIV) co-receptor CCR5 and inhibits fusion and infection of T cells and macrophages by R5 viruses but not by X4 viruses. We recently identified structural determinants of C-18 required for anti-HIV activity (Yarovinsky, F., Andersen, J. F., King, L. R., Caspar, P., Aliberti, J., Golding, H., and Sher, A. (2004) J. Biol. Chem. 279, 53635-53642). Here we have elucidated the fine specificity of CCR5 residues involved in binding and HIV inhibitory potential of C-18. To delineate the regions of CCR5 involved in C-18 binding, we analyzed C-18 inhibition of cells expressing CXCR4/CCR5 chimeric receptors and CCR5 with a truncated N terminus (Delta2-19). These experiments identified a critical role for the N terminus of CCR5 in C-18 binding and anti-HIV activity. Studies with a large panel of CCR5 N-terminal peptides, including Tyr-sulfated analogues, truncated peptides, and alanine-scanning mutants, suggested that each of the 12-17 amino acids in the N terminus of CCR5 are essential for C-18 binding and inhibitory activity. Tyr sulfation did not improve C-18 reactivity. This finding is of interest because the same CCR5 N-terminal region was shown previously to play a key role in binding of HIV-1 envelope glycoproteins. The elucidation of the functional C-18-binding mechanism may help in the rational design of novel antiviral agents against HIV.