10 research outputs found
Fluorescence Correlation Spectroscopy Reveals Efficient Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins.
New methods for delivering proteins into the cytosol of mammalian cells are being reported at a rapid pace. Differentiating between these methods in a quantitative manner is difficult, however, as most assays for evaluating cytosolic protein delivery are qualitative and indirect and thus often misleading. Here we make use of fluorescence correlation spectroscopy (FCS) to determine with precision and accuracy the relative efficiencies with which seven different previously reported "cell-penetrating peptides" (CPPs) transport a model protein cargo-the self-labeling enzyme SNAP-tag-beyond endosomal membranes and into the cytosol. Using FCS, we discovered that the miniature protein ZF5.3 is an exceptional vehicle for delivering SNAP-tag to the cytosol. When delivered by ZF5.3, SNAP-tag can achieve a cytosolic concentration as high as 250 nM, generally at least 2-fold and as much as 6-fold higher than any other CPP evaluated. Additionally, we show that ZF5.3 can be fused to a second enzyme cargo-the engineered peroxidase APEX2-and reliably delivers the active enzyme to the cell interior. As FCS allows one to realistically assess the relative merits of protein transduction domains, we anticipate that it will greatly accelerate the identification, evaluation, and optimization of strategies to deliver large, intact proteins to intracellular locales
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Discovery and characterization of a peptide that enhances endosomal escape of delivered proteins in vitro and in vivo
The in efficient delivery of proteins into mammalian cells remains a major barrier to realizing the therapeutic potential of many proteins. We and others have previously shown that superpositively charged proteins are efficiently endocytosed and can bring associated proteins and nucleic acids into cells. The vast majority of cargo delivered in this manner, however, remains in endosomes and does not reach the cytosol. In this study we designed and implemented a screen to discover peptides that enhance the endosomal escape of proteins fused to superpositively charged GFP (+36 GFP). From a screen of peptides previously reported to disrupt microbial membranes without known mammalian cell toxicity, we discovered a 13-residue peptide, aurein 1.2, that substantially increases cytosolic protein delivery by up to ~5-fold in a cytosolic fractionation assay in cultured cells. Four additional independent assays for non-endosomal protein delivery collectively suggest that aurein 1.2 enhances endosomal escape of associated endocytosed protein cargo. Structure-function studies clarified peptide sequence and protein conjugation requirements for endosomal escape activity. When applied to the in vivo delivery of +36 GFP–Crerecombinase fusions into the inner ear of live mice, fusion with aurein 1.2 dramatically increased non-endosomal Crerecombinase delivery potency, resulting in up to 100% recombined inner hair cellsand 96% recombined outer hair cells, compared to 0-4% recombined hair cells from +36-GFP- Cre without aurein 1.2. Collectively, these findings describe a genetically encodable, endosome escape-enhancing peptide that can substantially increase the cytoplasmic delivery of cationic proteins
in vitro and in vivo.Chemistry and Chemical Biolog
HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins
Protein Cages: From Fundamentals to Advanced Applications
Proteins that self-assemble into polyhedral shell-like structures are useful molecular containers both in nature and in the laboratory. Here we review efforts to repurpose diverse protein cages, including viral capsids, ferritins, bacterial microcompartments, and designed capsules, as vaccines, drug delivery vehicles, targeted imaging agents, nanoreactors, templates for controlled materials synthesis, building blocks for higher-order architectures, and more. A deep understanding of the principles underlying the construction, function, and evolution of natural systems has been key to tailoring selective cargo encapsulation and interactions with both biological systems and synthetic materials through protein engineering and directed evolution. The ability to adapt and design increasingly sophisticated capsid structures and functions stands to benefit the fields of catalysis, materials science, and medicine.ISSN:0009-2665ISSN:1520-689
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Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent
Glycosylated natural products and synthetic glycopeptides represent a significant and growing source of biochemical probes and therapeutic agents. However, methods that enable the aqueous glycosylation of endogenous amino acid functionality in peptides without the use of protecting groups are scarce. Here, we report a transformation that facilitates the efficient aqueous O-glycosylation of phenolic functionality in a wide range of small molecules, unprotected tyrosine, and tyrosine residues embedded within a range of complex, fully unprotected peptides. The transformation, which uses glycosyl fluoride donors and is promoted by Ca(OH)2, proceeds rapidly at room temperature in water, with good yields and selective formation of unique anomeric products depending on the stereochemistry of the glycosyl donor. High functional group tolerance is observed, and the phenol glycosylation occurs selectively in the presence of virtually all side chains of the proteinogenic amino acids with the singular exception of Cys. This method offers a highly selective, efficient, and operationally simple approach for the protecting-group-free synthesis of O-aryl glycosides and Tyr-O-glycosylated peptides in water
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HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins
Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered a class of cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine (penta-Arg) motif that traffics readily to the cytosol and nucleus of mammalian cells with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs containing a penta-Arg motif are released efficiently from endosomes, while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using an RNA interference screen, fluorescence correlation spectroscopy, and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein-sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. CPMPs localize within the lumen of Rab7+ and Lamp1+ endosomes and their transport requires HOPS activity. Overall, our results identify Lamp1+ late endosomes and lysosomes as portals for passing proteins into the cytosol and suggest that this environment is prerequisite for endosomal escape
Fluorescence Correlation Spectroscopy Reveals Highly Efficient Cytosolic Delivery of Certain Penta-Arg Proteins and Stapled Peptides
We
used fluorescence correlation spectroscopy (FCS) to accurately
and precisely determine the relative efficiencies with which three
families of “cell-penetrating peptides” traffic to the
cytosol of mammalian cells. We find that certain molecules containing
a “penta-arg” motif reach the cytosol, intact, with
efficiencies greater than 50%. This value is at least 10-fold higher
than that observed for the widely studied cationic sequence derived
from HIV Tat or polyarginine Arg<sub>8</sub>, and equals that of hydrocarbon-stapled
peptides that are active in cells and animals. Moreover, we show that
the efficiency with which stapled peptides reach the cytosol, as determined
by FCS, correlates directly with their efficacy in cell-based assays.
We expect that these findings and the associated technology will aid
the design of peptides, proteins, and peptide mimetics that predictably
and efficiently reach the interior of mammalian cells
Tunable photochemical/redox properties of (phenylthio)ncorannulenes: application to a photovoltaic device
The additive substituent effect of phenylmercapto derivatives of corannulene are investigated by spectroscopic, electrochemical, and computational techniques. The per-substituted phenylmercaptocorannulene is incorporated as a replacement for [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) in a photovoltaic device