Moonlighting peptides with emerging function

Abstract

Hunter-killer peptides combine two activities in a single polypeptide that work in an independent fashion like many other multi-functional, multi-domain proteins. We hypothesize that emergent functions may result from the combination of two or more activities in a single protein domain and that could be a mechanism selected in nature to form moonlighting proteins. We designed moonlighting peptides using the two mechanisms proposed to be involved in the evolution of such molecules (i.e., to mutate non-functional residues and the use of natively unfolded peptides). We observed that our moonlighting peptides exhibited two activities that together rendered a new function that induces cell death in yeast. Thus, we propose that moonlighting in proteins promotes emergent properties providing a further level of complexity in living organisms so far unappreciated.

Figure 1. Antibacterial and mitochondrial swelling activities of Iztli peptides.

The activity for Iztli peptide IP1 (20 µM) (▪ in A, black bars in B) against the bacteria E. coli (DH10B) was tested in two ways: following the optical cell density of the culture at 600 nm (A) and counting the colony forming units (CFU) (B). Three controls are included: LB with no peptide (▪ in A, white bars in B), α-pheromone (25 µM; ▪ in A, gray bars in B) and the six residues at the N-terminus of IP1 (fIP1) (119 µM; ⋄ in A). The results of 4 experiments are presented for plots in A and B. The bars represent standard deviations. Mitochondrial swelling was followed at 540 nm (C) in the presence of Iztli peptide IP1 (▪ 117 µM); three controls were used: distilled water (▪), α-pheromone (▪ 160 µM) and fIP1 (⋄ 100 µM), Polyethylene glycol 3.4 kDa was added at 180 seconds. Only the IP1 data is shown here; the 3 other IP peptides display similar activity than IP1.

Figure 2. Pheromone activity of Iztli peptides.

The pheromone-like activity of Iztli peptide IP1 was determined in two ways: detecting the activation of Fus1-GFP and observing the Shmoo phenotype. The image presents MatA cells in media without pheromone (A) and those presenting both Shmoo phenotype (small protrution on the cells indicated with the arrow) and the Fus1-GFP fluorescence observed after 1 hr of induction in the presence of either the α-pheromone (B) or the Iztli peptide IP1. (C) This is a compose image from DIC and fluorescence microscopy. Images were generated using a confocal microscope Olympus FluoView FV1000 with a magnification of 60×. The scale line is 5 µm.

Figure 3. Antifungal activity of Iztli peptides.

The activity of the Iztli peptide IP1 (10 µM) (▪ in A, black bars in B) against the MatA cells from Saccharomyces cerevisiae (BY4741) is presented in two formats: Optical cell density (A) and CFU (B). Cells were grown in YPD. We used three controls: No peptide (▪ in A, white bars in B), α-pheromone (10 µM) (▪ in A, gray bars in B) and the six residues at the N-terminus (fIP1) (60 µM) (⋄ in A). The results of 4 experiments are presented for plots in A and B. The error bars represent standard deviations. The strain BY4741 ΔSTE2 lacking the receptor for the α-pheromone was tested against the Iztli peptides (C): IP1 ▪ 10 µM, IP2 ▪ 10 µM for antifungal activity. For these experiments we used no peptide ▪ and α-pheromone ▪ (10 µM) as controls.

Figure 4. Secondary structure of Iztli peptides in TFE.

The observed conformation of the four Iztli Peptides (IP1 dashed-dotted line, IP2 solid line, IP3 dashed line and IP4 dotted line) in 50% Trifluroethanol is shown. CD spectra of each peptide in water were subtracted from the spectra of corresponding peptide in TFE.