Investigating the impact of thiol reactivity and disulfide formation on cellular uptake of cell-permeable peptides.

Cell-penetrating peptides (CPPs) have been explored as versatile tools to transport various molecules into cells. The uptake mechanism of CPPs is still not clearly understood and most probably depends on several factors like the nature of the CPP itself, the attached cargo, the investigated cell system, and other experimental conditions, such as temperature and concentration. One of the first steps of internalization involves the interaction of CPPs with negatively charged molecules present at the outer layer of the cell membrane. Recently, thiol-mediated uptake has been found to support the effective translocation of sulfhydryl-bearing substances that would actually not be cell-permeable. Within this work, we aimed to understand the relevance of thiol reactivity for the uptake mechanism of cysteine-containing CPPs that we have developed previously in our group. Therefore, we compared the two peptides, sC18-Cys and CaaX-1, in their single reduced and dimeric disulfide versions. Cytotoxicity, intracellular accumulation, and impact on the internalization process of the disulfides were investigated in HeLa cells. Both disulfide CPPs demonstrated significantly stronger cytotoxic effects and membrane activity compared with their reduced counterparts. Notably, thiol-mediated uptake could be excluded as a main driver for translocation, showing that peptides like CaaX-1 are most likely taken up by other mechanisms.

Cell-permeable peptide-based delivery vehicles useful for subcellular targeting and beyond.

Personal medicine aims to provide tailor-made diagnostics and treatments and has been emerged as a promising but challenging strategy during the last years. This includes the active delivery and localization of a therapeutic compound to a targeted site of action within a cell. An example being targeting the interference of a distinct protein-protein interaction (PPI) within the cell nucleus, mitochondria or other subcellular location. Therefore, not only the cell membrane has to be overcome but also the final intracellular destination has to be reached. One approach which fulfills both requirements is to use short peptide sequences that are able to translocate into cells as targeting and delivery vehicles. In fact, recent progress in this field demonstrates how these tools can modulate the pharmacological parameters of a drug without compromising its biological activity. Beside classical targets that are addressed by various small molecule drugs such as receptors, enzymes, or ion channels, PPIs have received increasing attention as potential therapeutic targets. Within this review, we will provide a recent update on cell-permeable peptides targeting subcellular destinations. We include chimeric peptide probes that combine cell-penetrating peptides (CPPs) and a targeting sequence, as well peptides having intrinsic cell-permeability and which are often used to target PPIs.

A Peptide Inhibitor of the Human Cytomegalovirus Core Nuclear Egress Complex.

The replication of human cytomegalovirus (HCMV) involves a process termed nuclear egress, which enables translocation of newly formed viral capsids from the nucleus into the cytoplasm. The HCMV core nuclear egress complex (core NEC), a heterodimer of viral proteins pUL50 and pUL53, is therefore considered a promising target for new antiviral drugs. We have recently shown that a 29-mer peptide presenting an N-terminal alpha-helical hook-like segment of pUL53, through which pUL53 interacts with pUL50, binds to pUL50 with high affinity, and inhibits the pUL50-pUL53 interaction in vitro. Here, we show that this peptide is also able to interfere with HCMV infection of cells, as well as with core NEC formation in HCMV-infected cells. As the target of the peptide, i.e., the pUL50-pUL53 interaction, is localized at the inner nuclear membrane of the cell, the peptide had to be equipped with translocation moieties that facilitate peptide uptake into the cell and the nucleus, respectively. For the resulting fusion peptide (NLS-CPP-Hook), specific cellular and nuclear uptake into HFF cells, as well as inhibition of infection with HCMV, could be demonstrated, further substantiating the HCMV core NEC as a potential antiviral target.

Mitochondria shed their outer membrane in response to infection-induced stress.

The outer mitochondrial membrane (OMM) is essential for cellular homeostasis. Yet little is known of the mechanisms that remodel it during natural stresses. We found that large "SPOTs" (structures positive for OMM) emerge during Toxoplasma gondii infection in mammalian cells. SPOTs mediated the depletion of the OMM proteins mitofusin 1 and 2, which restrict parasite growth. The formation of SPOTs depended on the parasite effector TgMAF1 and the host mitochondrial import receptor TOM70, which is required for optimal parasite proliferation. TOM70 enabled TgMAF1 to interact with the host OMM translocase SAM50. The ablation of SAM50 or the overexpression of an OMM-targeted protein promoted OMM remodeling independently of infection. Thus, Toxoplasma hijacks the formation of SPOTs, a cellular response to OMM stress, to promote its growth.

Cell-permeable CaaX-peptides affect K-Ras downstream signaling and promote cell death in cancer cells.

Cysteine prenylation is a post-translational modification that is used by nature to control crucial biological functions of proteins, such as membrane trafficking, signal transduction, and apoptosis. It mainly occurs in eukaryotic proteins at a C-terminal CaaX box and is mediated by prenyltransferases. Since the discovery of prenylated proteins, various tools have been developed to study the mechanisms of prenyltransferases, as well as to visualize and to identify prenylated proteins. Herein, we introduce cell-permeable peptides bearing a C-terminal CaaX motif based on Ras sequences. We demonstrate that intracellular accumulation of those peptides in different cells is controlled by the presence of their CaaX motif and that they specifically interact with intracellular prenyltransferases. As proof of concept, we further highlight their utilization to alter downstream signaling of Ras proteins, particularly of K-Ras-4B, in pancreatic cancer cells. Application of this strategy holds great promise to better understand and regulate post-translational cysteine prenylation.