Noncovalent protein transduction in plant cells by macropinocytosis

* Protein delivery across cellular membranes or compartments is primarily limited by low biomembrane permeability. * Many protein transduction domains (PTDs) have previously been generated, and covalently cross-linked with cargoes for cellular internalization. * An arginine-rich intracellular delivery (AID) peptide could rapidly deliver fluorescent proteins or beta-galactosidase enzyme into plant and animal cells in a noncovalent fashion. The possible mechanism of this noncovalent protein transduction (NPT) may involve macropinocytosis. * The NPT via a nontoxic AID peptide provides a powerful tool characterized by its simplicity and quickness to have active proteins function in living cells in vivo. This should be of broad utility for functional enzyme assays and protein therapies in both plant biology research as well as biomedical applications.     

Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells

Plasma membranes of plant or animal cells are generally impermeable to peptides or proteins. Many basic peptides have previously been investigated and covalently cross-linked with cargoes for cellular internalization. In the current study, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to deliver fluorescent proteins or beta-galactosidase enzyme into animal and plant cells, as well as animal tissue. Cellular internalization and transdermal delivery of protein could be mediated by effective and nontoxic AID peptides in a neither fusion protein nor conjugation fashion. Therefore, noncovalent AID peptides may provide a useful strategy to have active proteins function in living cells and tissues in vivo.     

A Bacterial Indole-3-acetyl-L-aspartic Acid Hydrolase Inhibits Mung Bean (Vigna radiata L.) Seed Germination Through Arginine-rich Intracellular Delivery

Indole-3-acetyl-L-aspartic acid (IAA-Asp) is a natural product in many plant species and plays many important roles in auxin metabolism and plant physiology. IAA-Asp hydrolysis activity is, therefore, believed to affect plant physiology through changes in IAA metabolism in plants. We applied a newly discovered technique, arginine-rich intracellular delivery (AID), to deliver a bacterial IAA-Asp hydrolase into cells of mung bean (Vigna radiata) seeds and measured its effects on mung bean seed germination. IAA-Asp hydrolase inhibited seed germination about 12 h after the enzyme was delivered into cells of mung bean seeds both covalently and noncovalently. Mung bean seed germination was delayed by 36 h when the enzyme protein was noncovalently attached to the AID peptide and longer than 60 h when the enzyme protein was covalently attached to the AID peptide. Root elongation of mung bean plants was inhibited as much as 90% or 80%, respectively, when the IAA-Asp hydrolase was delivered with the AID peptide by covalent or noncovalent association. Further thin-layer chromatography analysis of plant extracts indicated that the levels of IAA increased about 12 h after treatment and reached their peak at 24 h. This result suggests that IAA-Asp hydrolase may increase IAA levels and inhibit seed germination of mung bean plants and that the AID peptide is a new, rapid, and efficient experimental tool to study the in vivo activity of enzymes of interest in plant cells.     

Intracellular Delivery of Proteins via Fusion Peptides in Intact Plants

In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology.     

Protein transport in human cells mediated by covalently and noncovalently conjugated arginine-rich intracellular delivery peptides

Generally, biomacromolecules, such as DNA, RNA, and proteins, cannot freely permeate into cells from outside the membrane. Protein transduction domains (PTDs) are peptides containing a large number of basic amino acids that can deliver macromolecules into living cells. Arginine-rich intracellular delivery (AID) peptides are more effective than other PTD peptides at carrying large molecules across cellular membranes. In the present study, we demonstrated that AID peptides are able to deliver cargo proteins into living cells in both covalent and noncovalent protein transductions (CNPT) synchronously. Human A549 cells were treated with a fluorescent protein (FP) that was noncovalently premixed with another AID-conjugated FP, which emitted a different color. After the delivery of carrier AID-FP and cargo FP into cells, the emission and merge of florescence were observed and recorded with a confocal microscope, while the internalization efficiency was quantitatively analyzed with a flow cytometer. The optimal molecular ratio between carrier AID-FP and cargo FP for CNPT is about 1:1/3. Fluorescence resonance energy transfer (FRET) assay further confirmed AID-conjugates can physically interact with its cargo FPs in CNPT in cells. Potential uptake mechanisms of CNPT may involve a combination of multiple internalization pathways. After delivery, intracellular distributions of AID-conjugates and FPs may possibly colocalize with lysosomes. These results will facilitate the understanding of multiple mechanisms of PTDs, and provide a powerful tool for simultaneously delivering several proteins or compounds in protein internalization.     

Cell Membrane Diversity in Noncovalent Protein Transduction

Crossing of the plasma membrane for all macromolecules without energy, receptors or any artificial methods was thought to be difficult. Our previous studies demonstrated that arginine-rich intracellular delivery (AID) peptides are able to deliver macromolecules, such as proteins, RNAs and DNAs, into either animal or plant cells. Cellular internalization could be mediated by effective and nontoxic AID peptides in either a covalent or noncovalent protein transduction (NPT) manner. AID peptides were so versatile that the procedure seemed to replace the current artificial transfection methods. However, the utilization of AID peptides has been limited to animal or plant systems so far. None has proposed that AID peptides could work in other species. Here, we select some representative organisms to screen whether NPT mediated by AID peptides works in them. They include cyanobacteria, bacteria, archaea, algae, fungi and yeasts. The results reveal that not all living beings possess this capability of protein transduction. Interestingly, all species of prokaryotes tested, which were thought to be highly diverse from the animal and plant systems, appear to be capable of NPT. The mechanism of AID-mediated NPT in cyanobacteria is in a classical endocytosis- and energy-independent pathway and may involve macropinocytosis. In contrast, green algae and multicellular fungi of the eukaryotes are impermeable to protein passage. Our results bring an interesting clue to the reexamination of the phylogeny of both algae and fungi.     

Non-natural cell surface receptors: synthetic peptides capped with N-cholesterylglycine efficiently deliver proteins into Mammalian cells

Protein toxins such as shiga toxin and cholera toxin penetrate into cells by binding small molecule-based cell surface receptors localized to cholesterol and sphingolipid-rich lipid raft subdomains of cellular plasma membranes. Molecular recognition between these toxins and their receptors triggers endocytic protein uptake through endogenous membrane trafficking pathways. We report herein the synthesis of functionally related non-natural cell surface receptors comprising peptides capped with N-cholesterylglycine as the plasma membrane anchor. The peptide moieties of these receptors were based on high-affinity epitopes of anti-hemaglutinin antibodies (anti-HA), anti-Flag antibodies, and a moderate-affinity Strep Tag II peptide ligand of the streptavidin protein from Streptomyces avidini. These non-natural receptors were directly loaded into plasma membranes of Jurkat lymphocytes to display peptides from lipid rafts on the cell surface. Molecular recognition between these receptors and added cognate anti-HA, anti-Flag, or streptavidin proteins resulted in rapid clathrin-mediated endocytosis; fluorescent target proteins were completely internalized within 4-12 h of protein addition. Analysis of protein uptake by epifluorescence microscopy and flow cytometry revealed intracellular fluorescence enhancements of 100-fold to 200-fold (10 microM non-natural receptor) with typically >99% efficiency. This method enabled intracellular delivery of a functional Escherichia coli beta-galactosidase enzyme conjugated to Protein A from Staphylococcus aureus. We termed this novel delivery strategy "synthetic receptor targeting", which is an efficient method to enhance macromolecular uptake by decorating mammalian cells with chemically defined synthetic receptors that access the molecular machinery controlling the organization of cellular plasma membranes.     

A peptide carrier for the delivery of biologically active proteins into mammalian cells.

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.     

Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.     

Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway

Facilitating the uptake of molecules into living cells is of substantial interest for basic research and drug delivery applications. Arginine-rich peptides have been shown to facilitate uptake of high molecular mass cargos into cells, but the mechanism of uptake is complex and may involve multiple receptors. In this report, we show that a derivative of the aminoglycoside antibiotic neomycin, in which all of the ammonium groups have been converted into guanidinium groups, can carry large (>300 kDa) bioactive molecules across cell membranes. Delivery occurs at nanomolar transporter concentrations and under these conditions depends entirely on cell surface heparan sulfate proteoglycans. Conjugation of guanidinoneomycin to the plant toxin saporin, a ribosome-inactivating agent, results in proteoglycan-dependent cell toxicity. In contrast, an arginine-rich peptide shows both heparan sulfate-dependent and -independent cellular uptake. The high selectivity of guanidinoneomycin for heparan sulfate suggests the possibility of exploiting differences in proteoglycan compositions to target delivery to different cell types.     

Natural and grafted cyclotides in cancer therapy: An insight

Cyclotides is a rapidly growing class of plant‐derived cyclic peptides exhibiting several bioactivities with potential applications in the agricultural and pharmaceutical sectors. Both natural and grafted cyclotides have shown promise in cancer therapy. Approximately 70 natural cyclotides belonging to three plant families (Fabaceae, Rubiaceae, and Violaceae) have shown cytotoxicity against several cancer cell lines. Cyclotides exhibit considerable stability against thermal and enzymatic proteolysis, owing to their unique structure with knotted topology and head to tail cyclization. Further, their small size, high stability, oral bioavailability, and tolerance to amino acid substitution in structural loops make them an ideal platform for designing peptide‐based drugs for cancer. Thus, cyclotides provide ideal scaffolds for bioactive epitope grafting and facilitating drug delivery in cancer treatment. Many anticancer linear peptides have been grafted in cysteine knotted cyclic framework of cyclotide for enhancing their cell permeability across cellular membranes, thereby improving their delivery and pharmacokinetics. The present review comprehensively discusses the distribution, toxicity, and anticancer bioactivity of natural cyclotides. Further, it systematically elaborates on the role and action of epitopes' into grafted cyclotides in targeting cancer. The review also encompasses related patents landscape study and future challenges in peptide‐based cancer therapy.     

Cell-penetrating peptides derived from viral capsid proteins

Cell-penetrating peptides (CPP) can translocate across the cell membrane and have been extensively studied for the delivery of proteins, nucleic acids, and therapeutics in mammalian cells. However, characterizations of CPP in plants have only recently been initiated. We showed that the intact virion and a recombinant capsid protein (CaP) from a plant-infecting nonenveloped icosahedral RNA virus, Brome mosaic virus (BMV), can penetrate the membranes of plant protoplasts but are trapped by the extracellular matrix. Furthermore, a 22-residue peptide derived from the N-terminal region of the CaP (CPNT) can enter barley protoplasts and cells of intact barley and Arabidopsis roots. An inhibitor of the macropinocytosis reduced CPNT entry, while treatment with NiCl(2) changed the cellular localization of CPNT. CPNT increased uptake of the green flourescent protein (GFP) into the cell when covalently fused to GFP or when present in trans of GFP. The BMV CPNT overlaps with the sequence known to bind BMV RNA, and it can deliver BMV RNAs into cells, resulting in viral replication, as well as deliver double-stranded RNAs that can induce gene silencing.     

Oligoarginine vectors for intracellular delivery: design and cellular-uptake mechanisms

Intracellular delivery using membrane-permeable peptide vectors is a recently developed methodology that has been employed successfully to transport various bioactive molecules into cells to modify cell functions. The efficient delivery of proteins, peptides, nucleic acids, liposomes, and so on has been accomplished using this methodology by conjugation of a peptide vector with the cargo molecules. The potentials of this approach for medical and pharmaceutical applications has also attracted our attention. Arginine-rich peptides, including a basic peptide segment derived from the human immunodeficiency virus type 1 (HIV-1) Tat protein, are categorized into one of the most frequently used peptide vectors, and the efforts of designing novel vectors have been ongoing. Internalization of these peptides has previously been regarded as not employing endocytosis. However, recent reevaluations have demonstrated the significant involvement of endocytosis in the cellular uptake of these peptides. These arginine-rich peptide vectors share many common features in internalization. However, there seem to be certain simultaneous dissimilarities observed in the modes of internalization among these peptides. In this review, the structural features of these arginine-rich peptide vectors have been focused on and the current understandings of their internalization mechanisms are summarized.     

Generation of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in cis or trans

We describe the synthesis and cellular delivery properties of multivalent and branched delivery systems consisting of cell-penetrating peptides assembled onto a peptide scaffold using native chemical ligation. A trimeric delivery system presenting three copies of the prototypical cell-penetrating peptide TAT shows an endosomolytic activity much higher than its monomeric and dimeric counterparts. This novel reagent promotes the endosomal release of macromolecules internalized into cells by endocytosis, and as a result, it can be used to achieve cytosolic delivery of bioactive but cell-impermeable macromolecules in either cis (covalent conjugation) or trans (simple coincubation).     

Modular design of non-viral vectors with bioactive components

Inefficient gene delivery continues to limit gene therapy applications to both basic and applied sciences. Approaches for engineering vectors increasingly include bioactive components that bind cellular receptors, disrupt membranes, or enhance nuclear transport. Recently, a novel cationic lipid was developed by modifying the glucocorticoid dexamethasone. This cationic corticosteroid condenses plasmids for gene delivery, while also modulating inflammation. Modular vectors containing bioactive components that target various cellular processes can overcome the barriers limiting gene transfer.     

Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein.

The mechanisms that direct positive-stranded RNA virus replication complexes to plant and animal cellular membranes are poorly understood. We describe a specific interaction between a replication protein of an RNA plant virus and membranes in vitro and in live cells. The tobacco etch virus (TEV) 6 kDa protein associated with membranes as an integral protein via a central 19 amino acid hydrophobic domain. In the presence or absence of other viral proteins, fluorescent fusion proteins containing the 6 kDa protein associated with large vesicular compartments derived from the endoplasmic reticulum (ER). Infection by TEV was associated with a collapse of the ER network into a series of discrete aggregated structures. Viral RNA replication complexes from infected cells were also associated with ER-like membranes. Targeting of TEV RNA replication complexes to membranous sites of replication is proposed to involve post-translational interactions between the 6 kDa protein and the ER.     

The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity.

We have constructed two point mutants of Rous sarcoma virus in which the amino-terminal glycine residue of the transforming protein, p60src, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60src proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p60src proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60src proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60src was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p60src proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p60src apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur.     

Nonvesicular sterol transport: two protein families and a sterol sensor?

Sterols, essential components of eukaryotic membranes, are actively transported between cellular membranes. Although it is known that both vesicular and non-vesicular means are used to move sterols, the molecules and molecular mechanisms involved have yet to be identified. Recent studies point to a key role for oxysterol binding protein (OSBP) and its related proteins (ORPs) in nonvesicular sterol transport. Here, evidence that OSBP and ORPs are bona fide sterol carriers is discussed. In addition, I hypothesize that ATPases associated with various cellular activities regulate the recycling of soluble lipid carriers and that the Niemann Pick C1 protein facilitates the delivery of sterols from endosomal membranes to ORPs and/or the ensuing membrane dissociation of ORPs.