Mechanistic aspects of CPP-mediated intracellular drug delivery: Relevance of CPP self-assembly

Pujals, Sílvia; Fernández‐Carneado, Jimena; López‐Iglesias, Carmen; Kogan, Marcelo J.; Giralt, Ernest

doi:10.1016/j.bbamem.2006.01.006

Cited by 205 publications

(198 citation statements)

References 116 publications

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“…Analysis of the secondary structure of the entire ␥-zein protein by CD spectra in aqueous media showed that the whole protein has a high helical content but a low PPII conformation content, at 55 and 7%, respectively (57). In contrast, the synthetic peptide (PPPVHL) 8 , corresponding to the repeat region, has been shown to adopt an amphipathic polyproline II conformation (27,44) and has been linked to a new family of cell-penetrating peptides (58). We demonstrate here that the repeat region of Zera is required for Zera-ECFP assembly, suggesting that the amphipathic character of Zera facilitates proper molecular orientation in the aqueous environment of the ER to optimize interchain disulfide bond cross-linking.…”

Section: Discussionmentioning

confidence: 99%

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

Llop‐Tous

Madurga

Giralt

et al. 2010

Journal of Biological Chemistry

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The N-terminal proline-rich domain of ␥-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL) 8 of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4 -6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys 7 and Cys 9 ) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.The mechanisms by which prolamins, which lack the ER 3 retention (H/K)DEL motif, are retained and assembled in the ER-derived PBs are only partially understood. Different factors act as determinants of PB biogenesis, some of them derived from cis-cargo properties and others with a cellular trans-origin (1). It has been proposed that the physico-chemical properties of prolamins, such as hydrophobicity and disulfide bond formation, promote specific interactions resulting in the formation of large self-assemblies that are responsible for their retention in the ER and PB formation (2-4). These polymers could be excluded because of their size from being carried by the COP II vesicles that transport cargo proteins to the Golgi complex (5). However, the generic COPII vesicles are flexible enough for large cargo loading, including that of procollagen (more than 300 nm in size) (6) and the collagen VII trimer (900 kDa) (7).In maize, four distinct types of prolamins (␣-, ␤-, ␥-, and ␦-zein) coexist in the PBs (8) and play distinct roles in PB formation. Recently, maize storage protein mutants created through RNAi showed that ␥-RNAi maize mutant lines exhibited slightly altered protein body formation and that a more drastic effect was observed in the ␤-␥ combined mutant, where protein bodies showed an irregular shape, particularly in their periphery (9).Various studies on zein accumulation in heterologous expression systems suggest that ␥-zein and ␤-zein mediate t...

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Section: Discussionmentioning

confidence: 99%

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

Llop‐Tous

Madurga

Giralt

et al. 2010

Journal of Biological Chemistry

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“…Escape of some CPPs or viruses from early/late endosomes into the cytosol before its merging with lysosomes depends on acidification of endosomes. The acidic pH in the endosome alters the conformation of the CPPs or the N-terminal region of influenza virus hemagglutinin-2 (HA2) to induce destabilization of lipid membranes, facilitating the escape from the endosomes into the cytosol [35]. At this point, the exact mechanism of how 3D8 scFv escapes from caveolae/caveosome compartments into the cytosol, while maintaining its biochemical activity, is not clear.…”

Section: Discussionmentioning

confidence: 99%

A nucleic acid-hydrolyzing antibody penetrates into cells via caveolae-mediated endocytosis, localizes in the cytosol and exhibits cytotoxicity

Jang

Jeong

Jun

et al. 2009

Cell. Mol. Life Sci.

122

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Abstract. Many natural anti-DNA antibodies (Abs) have the ability to translocate across the plasma membrane and localize in the nucleus of mammalian cells, frequently leading to cytotoxicity to cells. Herein, we report detailed intracellular trafficking routes and cytotoxicity in HeLa cells for a single chain variable fragment (scFv) Ab, 3D8, which is an anti-DNA Ab capable of hydrolyzing both DNA and RNA. The intracellular penetration of 3D8 scFv occurred by caveolae/lipid raft endocytosis. The time-course chasing experiments revealed that 3D8 scFv escaped directly from the caveosome into the cytosol and remained in the cytosol without further trafficking into endosomes, lysosomes, endoplasmic reticulum, Golgi, or nucleus. The cytosolically localized 3D8 scFv maintained its nuclease activity to hydrolyze cellular RNAs, mainly mRNAs, eventually triggering apoptotic cell death. Our results demonstrate that 3D8 scFv has a unique intracellular trafficking route of localizing in the cytosol, thereby exhibiting cytotoxicity due to its nuclease activity.

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“…A nonendocytotic, receptor-free, energyindependent cellular process is another mechanism of the CPP translocation across biologic membranes, including formation of inverted micelles, direct translocation through the lipid bilayer, and pore formation on the membrane [46,47,55,56]. CPPs with high content in cationic residues are first absorbed at the cell surface to the numerous anionic moieties, such as sialic or phospholipidic acid or heparan sulfate proteoglycans [46,57]. Which of these mechanisms a CPP will use is dependent on such parameters as concentration, size (with cargo), temperature, cell type, and modifications of CPPs or their cargo [43,46,55].…”

Section: The Cytotoxicity Of the Synthesized Cpps And The Correspondimentioning

confidence: 99%

Synthesis and cellular characterization of various nano-assemblies of cell penetrating peptide-epirubicin-polyglutamate conjugates for the enhancement of antitumor activity

Mohammadi

Zakeri–Milani

Golkar

et al. 2017

Artificial Cells, Nanomedicine, and Biotechnology

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A new class of cell penetrating peptides (CPPs) named peptide amphiphile was designed to improve the intracellular uptake and antitumor activity of epirubicin (EPR). Various amphiphilic CPPs were synthesized by solid phase peptide synthesis method and were chemically conjugated to EPR. Their corresponding nanoparticles (CPPs-E4 and CPPs-E8)were prepared via non-covalent binding of the peptides and polyanions. Cytotoxicity and anti-proliferative activity were evaluated by MTT assay. Cellular uptake was examined by flow cytometry and fluorescence microscopy. The CPPs exhibited slight cytotoxicity.Binding of polyglutamate to CPPs (CPPs-E4 and CPPs-E8 nanoparticles) decreased their cytotoxicity. CPPs-E8 nanoparticles showed lower cytotoxicity than CPPs-E4 nanoparticles.Cellular uptake of K3W4K3-E8, K2W4K2-E8 and W3K4W3-E8 reached 100% with no difference between each of the mentioned CPPs and its nanoparticle at 50 µM. The antiproliferative activity of EPR was enhanced following conjugation to peptides and nanoparticles at 25 µM. CPPs-EPR-E4 and -E8 nanoparticles displayed higher antiproliferative activity than CPPs-EPR at 25 µM. CPPs-E8-EPR nanoparticles showed higher anti-proliferative activity than CPPs-E4-EPR. K3W4K3-E8-EPR nanoparticles exhibited the highest anti-proliferative activity at 25 µM. The synthesized peptide nanoparticles are proposed as suitable carriers for improving the intracellular delivery of EPR into tumor cells with low cytotoxicity and high antitumor activity.

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Mechanistic aspects of CPP-mediated intracellular drug delivery: Relevance of CPP self-assembly

Cited by 205 publications

References 116 publications

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

A nucleic acid-hydrolyzing antibody penetrates into cells via caveolae-mediated endocytosis, localizes in the cytosol and exhibits cytotoxicity

Synthesis and cellular characterization of various nano-assemblies of cell penetrating peptide-epirubicin-polyglutamate conjugates for the enhancement of antitumor activity

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