An Antimicrobial Peptide Induces FIG1-Dependent Cell Death During Cell Cycle Arrest in Yeast.

Although most antibiotics act on cells that are actively dividing and non-dividing cells such as in microbe sporulation or cancer stem cells represent a new paradigm for the control of disease. In addition to their relevance to health, such antibiotics may promote our understanding of the relationship between the cell cycle and cell death. No antibiotic specifically acting on microbial cells arrested in their cell cycle has been identified until the present time. In this study we used an antimicrobial peptide derived from α-pheromone, IP-1, targeted against MATa Saccharomyces cerevisiae cells in order to assess its dependence on cell cycle arrest to kill cells. Analysis by flow cytometry and fluorescence microscopy of various null mutations of genes involved in biological processes activated by the pheromone pathway (the mitogen-activated protein kinase pathway, cell cycle arrest, cell proliferation, autophagy, calcium influx) showed that IP-1 requires arrest in G0/G1 in order to kill yeast cells. Isolating cells in different cell cycle phases by elutriation provided further evidence that entry into cell cycle arrest, and not into G1 phase, is necessary if our peptide is to kill yeast cells. We also describe a variant of IP-1 that does not activate the pheromone pathway and consequently does not kill yeast cells that express the pheromone's receptor; the use of this variant peptide in combination with different cell cycle inhibitors that induce cell cycle arrest independently of the pheromone pathway confirmed that it is cell cycle arrest that is required for the cell death induced by this peptide in yeast. We show that the cell death induced by IP-1 differs from that induced by α-pheromone and depends on FIG1 in a way independent of the cell cycle arrest induced by the pheromone. Thus, IP-1 is the first molecule described that specifically kills microbial cells during cell cycle arrest, a subject of interest beyond the process of mating in yeast cells. The experimental system described in this study should be useful in the study of the mechanisms at play in the communication between cell cycle arrest and cell death on other organisms, hence promoting the development of new antibiotics.

Cell penetrating peptides and cationic antibacterial peptides: two sides of the same coin.

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.

Moonlighting peptides with emerging function.

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.