Binding and crossing: Methods for the characterization of membrane-active peptides interactions with membranes at the molecular level
Introduction
Membrane-active peptides include the large families of antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs). These two families have long been studied separately, most likely because historically speaking AMPs came first. The first antimicrobial peptide active against Gram positive bacteria, gramicidin, has indeed been evidenced from Bacillus brevis in the late 1930's [1]. Sixty years later, the first endogenous cell-penetrating peptide derived from the Antennapedia drosophila homeoprotein was reported [2]. Homeoproteins are transcription factors active throughout development and in adulthood, endowed with paracrine activities and cell-penetrating properties [3]. Apart from their physiological role, it is suggested that they can be used as therapeutic proteins, in particular against neurodegenerative diseases [4]. The peptide motif responsible for the membrane penetration properties has been identified in early nineties [5]. The two types of peptides are present in the whole living kingdom. Antimicrobial peptides are part of the innate immune system, some active at the level of the membrane, when others interact with intracellular molecules (proteins, DNA, RNA). We will focus herein only on peptides active at the level of the membrane. Over the last 30 years, numerous cell-penetrating peptides have been identified either in natural proteins such as the HIV Tat protein [6], or rationally designed [7].
A global overview of the members of these two families, their sequences, structures and properties can be found in the data bases that respectively exist for AMPs and CPPs (http://www.camp3.bicnirrh.res.in/exLinks.php; http://crdd.osdd.net/raghava/cppsite/).
A large number of these membrane-active peptides are short (<40Ā amino acids) cationic and/or amphipathic sequences that are essentially unstructured in buffered solutions but can organize into well-defined Ī±-helices or Ī²-strands when they interact with membrane components [8]. On the one side, AMPs disrupt membrane integrity and lead to lytic effects in bacteria. On the other side, CPPs do enter into cells keeping the membrane intact and are able to carry cargo molecules inside cells for therapeutic or biotechnological purposes [9].
Interestingly, over the last 15-years, a few studies have started to bridge the gap between AMPs and CPPs. It has been evidenced that some CPPs can indeed also be AMPs and reciprocally. For a complete overview, see Refs. [[10], [11], [12]]. This observation implies that the properties of animal cell membrane penetration and those of bacterial cell membrane permeabilization can belong to the same peptide sequence. Therefore, it may be possible to combine a host cell entry property with antimicrobial activity, which is important for therapeutical aspects. For example, it has been shown that pVEC (LLIILRRRIRKQAHAHSK-amide, net charge +6) and TP10 (AGYLLGKINLKALAALAKKIL-amide, net charge +4), which were first reported as cell-penetrating peptides are also AMPs [13]. Interestingly, at micromolar concentrations lower that those killing bacteria, the two fluorescent peptides translocate into microbial cells. It is also the case for Penetratin and Tat which are active against Gram positive and negative bacteria and are also taken up at concentrations lower than the MIC [14,15]. Reciprocally, it has been shown that Magainin-2 [16] and buforin-2 [17] are AMPs endowed with translocation properties. It was also possible to transform an original cell-penetrating peptide Pep-1 (KETWWETWWTEWSQPKKKRKV, net charge +3) into a potent AMP, Pep1-K (KKTWWKTWWTKWSQPKKKRKV, net charge +9), for which the minimal inhibitory concentrations (MIC) against different Gram positive and negative bacterial strains are similar to those of melittin [18]. However, in contrast with melittin that creates large pores in bacterial membrane mimics, Pep1-K peptide induces cell membrane depolarization via small channel formation that permits ions but not calcein leakage. Similarly, it has recently been suggested that some AMPs can induce formation of short-lived membrane water bridges [19,20].
With regard to the membrane that peptides with AMP and CPP activity recognize, they differ in terms of lipids. Before reaching the lipid membrane, AMPs and CPPs are initially attracted by the negatively charged cell-surface. AMPs are attracted to negative microbial surfaces, via lipopolysaccharides (LPS) in Gram-negative and lipoteichoic acids (LTA) in Gram-positive bacteria. Similarly, CPPs are captured at the cell-surface by negatively charged glycosaminoglycans (GAGs).
The bacterial cell wall contains as major phospholipids the negatively charged phosphatidyl glycerol (PG) and cardiolipin (CL), and zwitterionic phosphatidylethanolamine (PE) in different proportions according to the bacteria type [21]. One important property of the bacterial membrane is that the lipid content is highly dynamic and can change with the cell growth conditions (temperature, planktonic or sessile population, ageing etc.) [22,23]. For example, bacteria remodel the fluidity of their membrane through incorporation of more unsaturated fatty acids as growth temperature decreases. Furthermore, resistance to AMPs can also occur by lipid modification, which often decreases the net negative charge on the bacterial cell surface, for example by addition of lysine on PG [24]. Besides, the animal cell membrane contains principally phosphatidylcholine (PC) and sphingomyelin (SM) in the outer membrane leaflet [25,26]. The inner leaflet, in contrast, involves phosphatidylethanolamine (PE) and the negatively charged phosphatidylserine (PS) and phosphatidylinositol (PI). In a living system, cell types differ in the membrane content of proportions of lipids, which lead to specific physico-chemical behaviours. Animal cell membrane asymmetry depends on the activity of flippases and floppases which are ATP-driven pumps [27]. Phospholipid scrambling occurs quickly with dysfunction of these pumps and externalizes PS, which is recognizes as a stress signal. The content in lipids of animal cell membranes is highly dynamic and can be modified by diet or environmental changes [28,29]. Rational modification of lipids in cell membranes is currently an emerging therapeutical strategy.
The initial steps leading to the activity of AMPs and CPPs are very similar: 1) capture at the cell-surface; 2) interaction with specific membrane partners; 3) translocation/crossing the cell membrane. To understand fully at the molecular level these steps and the crossing of biological membranes, we need dedicated techniques. In the following paragraphs, we will describe some unusual ones that are not yet fully exploited to study membrane-active peptides [30].
Section snippets
Droplet interface bilayers
Cell Penetrating peptides (CPPs) can cross cell membrane by endocytosis and/or by direct translocation through the bilayer of the plasma membrane. The proportion of each mechanism is dependent on the peptides and on the condition of its internalization such as target cell type, peptide concentration, presence of a cargo to deliver, etc. [31]. The existence of the direct translocation mechanism and its importance to deliver directly a cargo into the cytosol of a cell lead many authors to study
Fluorescence techniques
To detect the entry of CPPs into cells (live cells if possible), the most common technique is to label the CPPs with a fluorescent probe and to detect it through epifluorescence or confocal microscopy [50,51]. Though powerful and convenient, this technique suffers from some pitfalls, among which the potential alteration of the peptide physical-chemical properties by the introduction of a fluorescent probe, the difficulty to distinguish the membrane bound peptide from the internalized peptide
Biomembrane force probe
A key question to understand the mechanism of action of membranotropic peptides is to determine their partners at the membrane, which can be lipids, proteins or saccharides. It is also desirable to determine the kinetics and strength of these interactions as the peptides adhere to some partners at the membrane, insert in the membrane possibly by forming complexes with other partners and finally cross the membrane possibly driven by their interactions with final partners. Many techniques can be
Photolabelling studies
Chemobiological tools can also be used to identify key binding partners for CPP-cell membrane interactions and internalization. Photocrosslinking also known as photolabelling or photoaffinity labelling (PAL) is an example of such a tool, and has recently been applied to polyarginines and Penetratin. The aim of PAL is to freeze non-covalent interactions by creating a covalent bond between interacting partners, to maintain this interaction during the downstream analysis steps. This approach
Conclusion
Herein, we provided an overview of classical and contemporary methods to characterize the interactions of membrane-active peptides with model or biological membranes. The advantages and limitations of each technique are summarised in Table 1.
BFP and photolabelling approaches can help deciphering direct interacting partners of membranotropic peptides. The strength of BFP is that it allows to work with whole cells and provide quantitative information. Photolabelling is more challenging, though
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