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

Abstract

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 (KWAVRIIRKFIKGFIS-NH2) 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 LPS-primed 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.

Fig 1

a. Helical wheel Edmundson plot of the peptides Hs01, Hs02, Hs03 and Hs04 highlighting their amphiphilic character. In green are hydrophobic amino acid residues, in blue, polar uncharged residues, in dark blue, negatively charged residues, and in red are positively charged residues, and b. Structure of the N subunit of the human Anaphase-Promoting Complex (PDB:4UI9). In detail is the α-helical segment spanning the amino acid residues from 285 to 304, which correspond to the IAP Hs03.

Fig 2. Hs IAPs transition to α-helical structure upon interaction with model phospholipid membranes and induce disturbances in the P´_β→L_α phase transition of vesicles.

a. Far-UV CD scans were performed to evaluate the secondary structure of Hs IAPs in buffer alone (40 μM solution peptide in phosphate buffer), represented as a black line, and after the addition of 2 mM DMPC, represented as a red line, or in the presence of 2 mM 2:1 DMPC:DMPG, blue line. b. Heating thermal scans of DMPC and 2:1 DMPC:DMPG LUVs enriched with 4 mol% IAPs. Black line corresponds to experimental data. Red line corresponds to a non-two state model fitting with two components of the main phase transition of model vesicles. Hs IAPs and the corresponding LUV compositions are indicated in each inset of the figure.

Fig 3. Constellation plot of the dendrogram obtained from HCA categorizes peptides in discrete clusters.

The transition temperature (Tm), enthalpy (ΔH) and van’t Hoff enthalpy (ΔH_vh) of the broad and sharp components of DMPC and 2∶1 DMPC:DMPG LUV thermograms following the addition of the Hs IAPs were obtained by adjusting peaks to a non-two state transition with two components. Data for Hs01, 02, 03 and 04 were appended to 52 other IAPs and AMPs and jointly submitted to a novel principal component analysis followed by hierarchical clustering (HCA analysis) of the first five principal components. The corresponding dendrogram is depicted in the form of a constellation plot in dimensionless units.

Fig 4

a. Cytotoxicity evaluation in the mouse BALB/cN macrophage (J774A.1) cell line after exposure for 24 h at 0.1, 1.0 and 10 μM concentrations. DMEM was used as a negative control. Cells were analyzed by flow cytometry (20,000 events/sample). b. Evaluation of the cell death mechanism mediated by Hs02 in J774A.1 cells after exposure for 24 h at 0.1, 1.0 and 10 μM concentrations using annexin-V FITC (apoptosis marker) and propidium iodide (PI, necrosis marker) staining. Cells were analyzed by flow cytometry (20,000 events/sample). The values are expressed as mean ± SEM. * p<0.05 and **** p<0.0001 versus DMEM untreated control group. # p<0.05 versus apoptosis staining for each respective group.

Fig 5. TNF-α secretion and lipid droplet biogenesis evaluation.

a. Murine peritoneal macrophages from C57BL/6 mice were pre-treated or not with LPS (500 ng/ml) for 4h and then stimulated or not with Hs IAPs (0.1, 1.0 and 10 μM), incubated for 24h and TNF-α levels measured by ELISA b. In parallel, murine peritoneal macrophages from C57BL/6 mice were pre-treated or not with LPS (500 ng/ml) for 4h and then stimulated or not with Hs IAPs (0.1 μM), incubated for 24h and lipid droplets were quantitated by flow cytometry (B). The values are expressed as mean ±SEM. * p<0.05 and **** p<0.0001 versus DMEM control group.

Fig 6

a. Ribbon representation of the lowest energy structure of Hs02 (left) and backbone alignment of the final 10 lowest-energy structures of Hs02 (right) in the presence of DPC-d38 micelles. b. Surface hydrophobic feature of Hs02 peptide. Side chain amino acid properties are showed in the following color code: red for the positively charged residues, cyan for the polar residues and green for the hydrophobic residues. The structure on the right side shows the molecule rotated 180° around the vertical axis (PDB: 6MBM).