Cationic cell-penetrating peptides induce ceramide formation via acid sphingomyelinase: Implications for uptake

Abstract

Cationic cell-penetrating peptides (CPP) are receiving increasing attention as molecular transporters of membrane-impermeable molecules. Import of cationic CPP occurs both via endocytosis and — at higher peptide concentrations — in an endocytosis-independent manner via localized regions of the plasma membrane. At present, this endocytosis-independent import of cationic CPP is not well understood, but has been shown to be sensitive to various pharmacological inhibitors, suggesting a role of an unidentified enzymatic activity. Here, we demonstrate that the direct translocation of cationic CPP depends on a CPP-induced translocation of acid sphingomyelinase (ASMase) to the outer leaflet of the plasma membrane and ceramide formation. The involvement of ASMase in uptake was confirmed by a pharmacological inhibition of ASMase by imipramine and a subsequent rescue of uptake through external addition of sphingomyelinase, and by using ASMase-deficient cells. We also found that the threshold for direct CPP translocation can be lowered through addition of sphingomyelinase and that sphingomyelinase enhances the translocation of R9 coupled to low-molecular weight cargos, but not high-molecular weight cargos. In conclusion, we show that a previously poorly understood mechanism of cationic CPP import depends on the ASMase-dependent formation of ceramide on the outer leaflet of the plasma membrane. To our knowledge, this is the first illustration that a class of delivery vectors operates through the induction of an enzymatic activity that changes the lipid composition of the plasma membrane.

Introduction

Interest in cell-penetrating peptides (CPP) as tools to deliver membrane-impermeable therapeutic molecules continues to grow. This development is illustrated by the increasing efforts of the scientific community to elucidate the chemical, physical and biological principles underlying the activity of CPP and CPP-based delivery systems [1], [2], [3], [4] and the steep rise in the number of therapeutic strategies that are being pursued with these systems [5], [6]. It is becoming progressively clear that instead of a single mechanism that is valid for all CPP, multiple modes-of-action exist with respect to the route of internalization and intracellular trafficking, that depend on the cell line [7], the CPP [8], and the cargo [9], [10]. Although endocytosis is now considered the major internalization route of cationic CPP [4], [11], [12], [13], [14], direct translocation for cationic CPP has also been observed, mainly at high [8], [15], [16], [17], but also at low concentrations [18], [19].

For arginine-rich CPP the potential for direct translocation is thought be related to the ability of the guanidinium moieties of arginines to form bidentate hydrogen bonds with membrane lipids [20] and is underscored by the ability of the arginine-rich TAT peptide to induce pores in artificial membranes, as was observed by small angle X-ray scattering [21]. Moreover, molecular-dynamics simulations indicate the potential of arginine-rich peptides to form transient pores in membranes in the presence of an electrochemical gradient and provide a mechanistic hypothesis for direct membrane translocation [22].

Nevertheless, biological details remain poorly understood and it is not clear to which degree in vitro models manage to reflect the complex molecular environment of a cell. At higher concentrations, direct cytoplasmic import of cationic CPP has been shown to depend on spatially confined nucleation zones. This rapid import is sensitive to the PKCδ inhibitor rottlerin and to chlorpromazine [8]. Available evidence indicates that the plasma membrane remains intact.

In a search for a common denominator of the observed pharmacological profile, we discovered that both molecules act as inhibitors of acid sphingomyelinase (ASMase) activity [23], [24], [25]. This finding provided us with a rationale for investigating a possible involvement of ASMase in the CPP uptake via direct translocation.

Acid sphingomyelinase has traditionally been known in relation to Niemann–Pick disease, a lysosomal storage disorder [26]. More recently, an important role for this enzyme in ceramide-mediated signal transduction pathways has emerged, linking its activity to a variety of common diseases, among which are cancer, cardiovascular diseases and diabetes [27]. A major function of this enzyme is the hydrolysis of sphingomyelin to ceramide and phosphorylcholine following a trigger-initiated translocation from lysosomes to the outer leaflet of the plasma membrane. The details of the translocation mechanism are unknown at present. Ceramide is a lipid with a well-established role in the induction of antiproliferative and apoptotic responses in a variety of cancer cells [28]. As a part of this role, ceramide greatly affects the structure and properties of cellular membranes. In the plasma membrane ceramide-enriched membrane domains modulate signaling [29], [30]. In the mitochondrial outer membrane ceramide increases permeability [31]. Here, we provide evidence for a role of ceramide formation by ASMase at the plasma membrane in the rapid cytoplasmic import of cationic CPP. This finding gives rise to the concept that cationic CPP enhance their own uptake by initiating a positive feed-back loop that involves an enzymatic alteration of the lipid composition of the plasma membrane.