Intragenic antimicrobial peptides (IAPs) from human proteins with potent antimicrobial and anti-inflammatory activity

Following the treads of our previous works on the unveiling of bioactive peptides encrypted in plant proteins from diverse species, the present manuscript reports the occurrence of four proof-of-concept intragenic antimicrobial peptides in human proteins, named Hs IAPs. These IAPs were prospected using the software Kamal, synthesized by solid phase chemistry, and had their interactions with model phospholipid vesicles investigated by differential scanning calorimetry and circular dichroism. Their antimicrobial activity against bacteria, yeasts and filamentous fungi was determined, along with their cytotoxicity towards erythrocytes. Our data demonstrates that Hs IAPs are capable to bind model membranes while attaining α-helical structure, and to inhibit the growth of microorganisms at concentrations as low as 1μM. Hs02, a novel sixteen residue long internal peptide (KWAVRIIRKFIKGFISNH2) derived from the unconventional myosin 1h protein, was further investigated in its capacity to inhibit lipopolysaccharide-induced release of TNF-α in murine macrophages. Hs02 presented potent anti-inflammatory activity, inhibiting the release of TNF-α in LPSprimed cells at the lowest assayed concentration, 0.1 μM. A three-dimensional solution structure of Hs02 bound to DPC micelles was determined by Nuclear Magnetic Resonance. Our work exemplifies how the human genome can be mined for molecules with biotechnological potential in human health and demonstrates that IAPs are actual alternatives to antimicrobial peptides as pharmaceutical agents or in their many other putative applications

Conformational and functional effects induced by D- and L-amino acid epimerization on a single gene encoded peptide from the skin secretion of Hypsiboas punctatus.

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy (1)H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways

Structural studies on L- and D- Phenylseptin peptide isomers by Ion Mobility Mass Spectrometry (IM-MS) and Nuclear Magnetic Resonance (NMR).

<p>(A) L-Phes and D-Phes were individually analyzed showing that each one (M+3H⁺ = 652.04 m/z) can assume at least two major conformations with distinct amounts of each type. L-Phes conformations at 10.45 and 12.89 ms and D-Phes conformations at 10.80 and 12.72 ms. D-Phes has its major conformation at 10.80 ms. Experiments were performed on a Synapt HDMS instrument (Quadrupole Ion Mobility High-Definition mass spectrometry – Waters Co. MA, USA) equipped with nano-electrospray ionization. All spectra were acquired with a direct infusion of 1 µL·min-1 of in a range m/z 300 up to 2000. Precursor charge state: 3. Tolerance: 0.1 Da. (B) and (C) The 20 lowest-energy structures for both peptides. The hydrophobic residues are represented in gold yellow, the hydrophilic residues in green. (D) The lowest-energy Phenylseptins showing Phenylalanine enatiomerization, the aromatic phenylalanine are in dark red and (E) The alignment of lowest-energy L- and D-Phes structure in the presence of 60% TFE viewed along the helix axis and from the side.</p

UFLC analysis and molecular mass determination of Phenylseptin mixture.

<p>(A) The accurate molecular masses and purity of Phes peptides were determined by MALDI-TOF/MS and the observed molecular mass was 1954.2 Da for both molecules. (B) Analytical chromatographic profile of natural (black line) and synthetic L-Phes (red dash line) and D-Phes (blue dash line). The two peptides were mixed in similar molar concentrations and load into an Ultra Fast Liquid Chromatography using a Shimpack-XR-ODS column under a linear gradient of acetonitrile at a flow rate of 0.4 mL·min-1. The two distinct fractions eluted around 11 and 12 minutes corresponded to L-Phes and D-Phes, respectively.</p

One single gene encoding Phes peptide.

<p>(A) Nucleotide sequences of clone encoding precursor of selected Phenylseptin peptides. The putative signal peptide (in box), acidic spacer (dash underline) and mature peptide (bold underline), C-terminal codon for Glycine (blue underline) and stop codon (asterisk) are indicated. The nucleotide sequences were deposited in the NCBI Nucleotide Sequence Database under HQ012497 annotated accession code. (B) Predicted amino acid sequence alignment of Phenylseptin with previously sequenced Hylidae peptides aurein, ranateurin, brevinin and gaegurin. Sequence alignments were done using CLUSTAL W software and were edited with the BIOEDIT software.</p