Selective Moonlighting Cell-Penetrating Peptides

Abstract

Cell penetrating peptides (CPPs) are molecules capable of passing through biological membranes. This capacity has been used to deliver impermeable molecules into cells, such as drugs and DNA probes, among others. However, the internalization of these peptides lacks specificity: CPPs internalize indistinctly on different cell types. Two major approaches have been described to address this problem: (i) targeting, in which a receptor-recognizing sequence is added to a CPP, and (ii) activation, where a non-active form of the CPP is activated once it interacts with cell target components. These strategies result in multifunctional peptides (i.e., penetrate and target recognition) that increase the CPP's length, the cost of synthesis and the likelihood to be degraded or become antigenic. In this work we describe the use of machine-learning methods to design short selective CPP; the reduction in size is accomplished by embedding two or more activities within a single CPP domain, hence we referred to these as moonlighting CPPs. We provide experimental evidence that these designed moonlighting peptides penetrate selectively in targeted cells and discuss areas of opportunity to improve in the design of these peptides.

Keywords: cell-penetrating peptide; computational biology; machine learning; moonlight protein; multifunctional protein; protein function prediction.

Figure 1

Strategies to provide selectivity to CPPs. Two strategies were tested to provide selectivity to CPPs, namely targeting and activation. The CPP sequence in blue, non-CPP conferring function sequence in red and ligand and cleavage site in green for (A) targeting and (B) activation, respectively.

Figure 2

Internalization of selective CPPs on yeast cells. MATa (gray) and MATα (golden) cells were exposed to α-NLS-C (left panel), NLS-α-CE (middle panel) and chimera (right panel) peptides. The degree of internalization is proportional to fluorescence intensity. The data is presented as box plots, where the second and third quartiles are presented below and above the dark horizontal line representing the median. The data represented in this plot is included as Table S1 and corresponds with 10,000 cells analyzed for each condition. These experiments were repeated at least 3 times for each condition.

Figure 3

Internalization of selective CPPs on mixtures of yeast cells. MATa: RPL9A-GFP (gray) and MATα (golden) cells were combined in a single vessel and exposed to α-NLS-C (left panel), NLS-α-CE (middle panel) and chimera (right panel) peptides. The degree of internalization is proportional to fluorescence intensity. The data is presented as box plots, where the second and third quartiles are presented below and above the dark horizontal line representing the median. The data represented in this plot is included as Table S2 and corresponds with 10,000 cells analyzed for each condition. These experiments were repeated at least 3 times for each condition.

Figure 4

Internalization of α-NLS-C followed by confocal microscopy. (A,C) present the accumulated and normalized fluorescence intensities captured for each cell observed in the microscope for MATa and MATα, respectively. (B,D) present the cells MATa and MATα at the beginning of the experiment, respectively. Two sets of experiments were conducted; in this image we provide a summary of one of those experiments. The set of images captured each 3 min during 1 h are available at http://bis.ifc.unam.mx/ironbios/selmoonCPP/ (accessed on 21 July 2021) as Supplementary File S1.

Figure 5

Protein expression levels of neprilysin in HEK293T, HEK293T-NEP and HeLa cell lines. The top panel shows a representative Western blot comparing NEP expression level in HEK293T cells parental (Wt) and HEK293T transfected cells stably over-expressing NEP (HEK293T-NEP) and HeLa cells. The bottom graph shows a densitometric analysis of the corresponding expression (n = 6 for HEK293T-NEP cells and n = 10 for the other cell lines). All paired comparisons reported p values < 0.05, hence all are considered significantly different. Raw images for the Western blots are presented as Figure S1A,B.

Figure 6

Internalization of activatable CPPs in mammalian cells. Normalized fluorescence intensity found in 2700 cells for each peptide. (A) Cell dependent internalization for HEK293T (red), HEK293T-NEP (blue) and HeLa (green). (B) Peptide dependent internalization for TatNep (red), TatNepNoCPP (blue) and D-TatNepNoCPP (green). Statistically similar distributions between the experiments are indicated with yellow circles or blue triangles (p < 0.5, see Methods). The data represented in these boxplots are included in Table S4 and as Supplementary File S2 at http://bis.ifc.unam.mx/ironbios/selmoonCPP (accessed on 21 July 2021). The data presented correspond with 1000 images per condition and 3 replicates.

Figure 7

Confocal microscopy of mammalian cells exposed to selective CPPs. Orthogonal projections of cells exposed to fluorescent CPPs (5 μM) to estimate internalization in HEK293T, HEK293T-NEP and HeLa cell lines are presented. Original stack images (“xy” coordinates) in the middle and “yz” and “xz” projections at the right and bottom, respectively. Yellow (punctuated), blue (diffuse) and white (punctuated and diffuse) arrows indicate examples of the three distinct fluorescent patterns observed. White bars represent 25 μm.