Cell-penetrating Protein Research Articles

Evaluation of Chikusetsusaponin IV a Isolated fromAlternanthera philoxeroidesfor Its Potency Against Viral Replication

Chikusetsusaponin IVa and calenduloside E were isolated from the whole plant of Alternanthera philoxeroides (Mart.) Griseb (Amaranthaceae) and evaluated for their antiviral activities. Chikusetsusaponin IVa showed antiviral activities against HSV-1, HSV-2, human cytomegalovirus, measles virus, and mumps virus with selectivity indices (CC (50)/IC (50)) of 29, 30, 73, 25, and 25, respectively. On the other hand, calenduloside E showed no antiviral effects against any of the viruses tested. The mode of HSV-2 action of chikusetsusaponin IVa was determined under different experimental conditions. The anti-HSV-2 target of the compound might be mainly related to direct inactivation of virus particles and to the inhibition of release of progeny viruses from infected cells, but it is not related to inhibition of viral attachment, cell penetration, and viral protein synthesis. This compound also provided in vivo efficacy in a mouse model of genital herpes caused by HSV-2. These results demonstrate that chikusetsusaponin IVa might be a candidate of antiherpetic agents.

Cell-penetrating peptides and proteins: new inhibitors of allergic airways diseaseThis review is an invited paper from 2007 ICRH Leadership in Science: a Forum for Trainees and New Investigators.

Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) are peptides that have the ability to efficiently traverse cellular membranes, either alone or in association with molecular cargo. Several naturally occurring PTDs, including those from HIV TAT and Drosophila antennapedia, have this unique activity. Synthetic CPPs, such as polyarginine, also have the ability to enter cells and transport a variety of cargo. While the precise mechanism(s) of cellular entry for individual CPPs may vary, it is likely that uptake is mediated, at least in part, through endocytosis. Moreover, biological activity of cell-penetrating peptides and proteins has been clearly demonstrated in a number of in vitro and in vivo studies. Recently, cell-penetrating proteins targeting the Ras GTPase and the phospholipid kinase PI3K (phosphoinositide 3-kinase) have been shown to inhibit eosinophil trafficking and survival in vitro. These proteins, as well as CPPs targeting the STAT-6 transcription factor or the T-cell costimulatory molecule CTLA-4 (cytotoxic T lymphocyte-associated antigen-4), have also been tested in animal models of asthma. Data from several groups, including ours, indicate that these molecules inhibit airway eosinophilic inflammation, airway hyperresponsiveness (AHR), and mucus production in experimental allergic airways disease. Thus, CPPs targeting these and other signaling molecules may also effectively inhibit allergic airways disease in humans.

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

Effect of Cell-Based Intercellular Delivery of Transcription Factor GATA4 on Ischemic Cardiomyopathy

Recent loss-of-function studies highlight the importance of the transcription factor GATA4 in the myocardial response to injury in the adult heart. However, the potential effects of gain-in-function of GATA4 overexpression, and transcription factors in general, is hindered by the fact that transcription factors are neither secreted nor taken up by cells. Chimeric proteins incorporating motifs of cell-penetrating proteins are secreted from cells and internalized by surrounding cells. We engineered a chimeric protein consisting of GATA4 and the cell-penetrating protein VP22. Cardiac fibroblasts stably transfected with the GATA4:VP22, GFP:VP22, or green fluorescent protein (GFP) constructs were transplanted into Lewis rats 1 month after left anterior descending ligation. GATA4:VP22 expression in the border zone was associated with increased cardiac myosin expression and cardiac myocyte size (30 mum versus 19 mum, P<0.01). Compared with the GFP:VP22 control group, 6 weeks after cardiac fibroblast transplantation (10 weeks after myocardial infarction), animals that received GATA4:VP22-expressing cardiac fibroblasts demonstrated increased cardiac function and less negative remodeling. These data demonstrate a novel strategy for transcription factor delivery to injured myocardium and indicate that the delivery of GATA4 locally to the infarct border zone induces multiple local effects in the border zone cardiac myocytes resulting in beneficial ventricular remodeling and improved global left ventricular function.

RECENT APPROACHES TO INTRACELLULAR DELIVERY OF DRUGS AND DNA AND ORGANELLE TARGETING

Intracellular delivery of various drugs, including DNA, and drug carriers can sharply increase the efficiency of various treatment protocols. However, the receptor-mediated endocytosis of drugs, drug carriers, and DNA results in their lysosomal delivery and significant degradation. The problem can be solved and therapy efficacy still further increased if the approaches for direct intracytoplasmic delivery that bypass the endocytic pathway are developed. This is especially important for many anticancer drugs (proapoptotic drugs whose primary action site is the mitochondrial membrane) and gene therapy (nuclear or mitochondrial genomes should be targeted). This review considers several current approaches for intracellular drug delivery: the use of pH-sensitive liposomes, the use of cell-penetrating proteins and peptides, and the use of immunoliposomes targeting intracellular antigens. Among intracellular targets, nuclei (gene therapy), mitochondria (proapoptotic cancer therapy and targeting of the mitochondrial genome), and lysosomes (lysosomal targeting of enzymes for the therapy of the lysosomal storage diseases) are considered. Examples of successful intracellular and organelle-specific delivery of biologically active molecules, including DNA, are presented; unanswered questions, challenges, and future trends are also discussed.

Protein Transduction: Cell Penetrating Peptides and Their Therapeutic Applications

Cell penetrating proteins or peptides (CPPs) have the ability to cross the plasma membranes of mammalian cells in an apparently energy- and receptor-independent fashion. Although there is much debate over the mechanism by which this "protein transduction" occurs, the ability of CPPs to translocate rapidly into cells is being exploited to deliver a broad range of therapeutics including proteins, DNA, antibodies, oligonucleotides, imaging agents and liposomes in a variety of situations and biological systems. The current review looks at the delivery of many such molecules by various CPPs, and their potential therapeutic application in a wide range of areas. CPP ability to deliver different cargoes in a relatively efficient and non-invasive manner has implications as far reaching as drug delivery, gene transfer, DNA vaccination and beyond. Although many questions remain to be answered and limitations on the use of CPPs exist, it is clear that this emerging technology has much to offer in a clinical setting.

Specific Delivery of Corroles to Cells via Noncovalent Conjugates with Viral Proteins

Corroles are amphiphilic macrocycles that can bind and transport metal ions, and thus may be toxic to cells. We predicted that anionic corroles would poorly enter cells due to the negatively charged cell membrane, but could be ideal tumor-targeted drugs if appropriate carriers enabled delivery into tumor cells. In this work, we test the hypothesis that recombinant cell penetrating proteins of the adenovirus (Ad) capsid form noncovalent conjugates with corroles to facilitate target-specific delivery and cell death. Corroles mixed with recombinant proteins were tested for conjugate assembly, cell penetration, stability, targeted binding, and cell killing in vitro. Sulfonated corroles entered cells only with carrier proteins, and formed stable complexes with recombinant Ad capsid proteins. ErbB receptor-targeted conjugates were cytotoxic to ErbB2-positive but not ErbB2-negative breast cancer cells, whereas molar equivalents of free corrole had no effect on these cells. Sulfonated corroles are cytotoxic to ErbB2-positive breast cancer cells when delivered by a targeted cell penetrating protein. The relatively low dose required to accomplish this compared to untargeted compounds suggests that corroles may lend themselves to targeted therapy. Importantly, the amphiphilicity of corroles enables a unique approach to bioconjugate formation whereby the carrier and drug form a stable complex by noncovalent assembly.

Engineered Cell Therapy for Sustained Local Myocardial Delivery of Nonsecreted Proteins

Novel strategies for the treatment of congestive heart failure have taken the form of gene and cell therapy to induce angiogenesis, optimize calcium handling by cardiac myocytes, or regenerate damaged myocardial tissue. Arguably both gene- and cell-based therapies would be benefited by having the ability to locally deliver specific transcription factors and other usually nonsecreted proteins to cells in the surrounding myocardial tissue. The herpes simplex virus type 1 (HSV-1) tegument protein VP22 has been shown to mediate protein intercellular trafficking to mammalian cells and finally localize into the nucleus, which makes it a useful cargo-carrying functional protein in cell-based gene therapy. While VP22 has been studied as a means to modulate tumor growth, little is known about the distribution and transport kinetics of VP22 in the heart and its potential application in combination with autologous cell transplantation for the delivery of proteins to myocardial tissue. The aim of this study was to evaluate the efficacy of VP22 fusion protein intercellular trafficking combined with autologous cell transplantation in the heart. In an in vitro study untransfected rat heart cells were cocultured with stably transfected rat cardiac fibroblasts (RCF) with fusion constructs of VP22. The control experiment was untransfected rat heart cells co-plated with RCF stably transfected with enhanced green fluorescence protein (eGFP). The Lewis rat model was selected for in vivo study. In the in vitro studies there was a 14-fold increase in the number of GFP-positive cells 48 h after initiating coculture with VP22-eGFP RCF compared to eGFP RCF. In the rat model, transplantation of VP22-eGFP expressing RCF led to VP22-eGFP fusion protein delivery to an area of myocardial tissue that was 20-fold greater than that observed when eGFP RCF were transplanted. This area appeared to reach a steady state between 7 and 10 days after transplantation. The VP22-eGFP area consisted of eGFP-positive endothelium, smooth muscle cells, and cardiac myocytes with delivery to an area of approximately 1 mm2 of myocardial tissue. Our data suggest a viable strategy for the delivery of proteins that are not naturally secreted or internalized, and provide the first insight into the feasibility and effectiveness of cell-penetrating proteins combined with cell transplantation in the heart.

Prediction of Cell-Penetrating Peptides

Cell-penetrating peptides, CPPs, are used as delivery vectors for pharmacologically interesting substances, such as antisense oligonucleotides, proteins and peptides. We present a general principle for designing cell-penetrating peptides derived from naturally occurring proteins as well as from randomly generated polyamino acid sequences. Thereby, we introduce a novel pharmacological principle for identification of cell-penetrating peptides for which the applications can be numerous, including cellular transduction vectors and mimics of intracellular protein–protein interactions. The methods of identifying a CPP comprises assessing the averaged bulk property values of the defined sequence, and ensuring that they fall within the bulk property value interval obtained from the training set. Despite this simplistic approach, the search criteria proved useful for finding CPP properties in either proteins or random sequences. We have experimentally verified cell-penetrating properties of 10–20-mer peptides derived from naturally occurring proteins as well as from random poly-amino acids. We note that since CPPs can be found in part of the protein sequences that may govern protein interactions, it is possible to produce cell-penetrating protein agonists or antagonists.

Translocation of Histone Proteins Across Lipid Bilayers and Mycoplasma Membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).

Non-endocytic penetration of core histones into petunia protoplasts and cultured cells: a novel mechanism for the introduction of macromolecules into plant cells

The results of the present work demonstrate that core histones are able to penetrate the plasma membrane of plant cells. Confocal microscopy has revealed that incubation of petunia protoplasts with fluorescently labeled core histones resulted in cell penetration and nuclear import of the externally added histones. Intracellular accumulation was also confirmed by an ELISA-based quantitative method using biotin-labeled histones. Penetration into petunia protoplasts and cultured cells was found to be non-saturable, occurred at room temperature and at 4 °C and was not inhibited by Nocodazole. Furthermore, penetration of the biotinylated histone was neither blocked by the addition of an excess of free biotin molecules, nor by non-biotinylated histone molecules. All these results clearly indicate that the observed uptake is due to direct translocation through the cell plasma membrane and does not occur via endocytosis. Our results also show that the histones H2A and H4 were able to mediate penetration of covalently attached BSA molecules demonstrating the potential of the histones as carriers for the delivery of macromolecules into plant cells. To the best of our knowledge, the findings of the present paper demonstrate, for the first time, the activity of cell penetrating proteins (CPPs) in plant cells.