Cell-Penetrating Milk-Derived Peptides with a Non-Inflammatory Profile

Abstract

Milk-derived peptides are known to confer anti-inflammatory effects. We hypothesised that milk-derived cell-penetrating peptides might modulate inflammation in useful ways. Using computational techniques, we identified and synthesised peptides from the milk protein Alpha-S1-casein that were predicted to be cell-penetrating using a machine learning predictor. We modified the interpretation of the prediction results to consider the effects of histidine. Peptides were then selected for testing to determine their cell penetrability and anti-inflammatory effects using HeLa cells and J774.2 mouse macrophage cell lines. The selected peptides all showed cell penetrating behaviour, as judged using confocal microscopy of fluorescently labelled peptides. None of the peptides had an effect on either the NF-κB transcription factor or TNFα and IL-1β secretion. Thus, the identified milk-derived sequences have the ability to be internalised into the cell without affecting cell homeostatic mechanisms such as NF-κB activation. These peptides are worthy of further investigation for other potential bioactivities or as a naturally derived carrier to promote the cellular internalisation of other active peptides.

Keywords: NF-κB; TNFα; cell-penetrating peptides; macrophages; milk.

Figure 1

Representative confocal images of HeLa cells treated with 5-FAM (green) labelled synthetic peptides (peptides 15, 16, 17 and 18) at 1, 30 and 60 min. Nuclei are stained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue).

Figure 2

Representative confocal images of HeLa cells treated with 5-FAM (green) labelled-synthetic peptides (peptides 19, 20 and 22) and 5-FAM control at 1, 30 and 60 min. Nuclei are stained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue).

Figure 3

Top: Effect of peptides 17, 19 and 22 and scrambled peptides (suffix “s”) on NF-κB activity measured by luciferase assay without LPS stimulation; bottom left: NF-kB secretion by luciferase assay with LPS stimulation. Untx refers to untreated cells. Data represent mean ± SEM for n = 3 independent experiments.

Figure 4

Top: Effects of peptides 17, 19, and 20 (and scrambled peptides denoted with suffix “s”) on TNFα secretion with LPS stimulation; bottom right: Effects of peptides on IL-1β secretion with LPS stimulation. Untx refers to untreated cells. Data represent mean ± SEM for n = 3 independent experiments.