Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cytosolic Delivery of Proteins by Bioreversible Esterification.

ABSTRACT: Cloaking its carboxyl groups with a hydrophobic moiety is shown to enable a protein to enter the cytosol of a mammalian cell. Diazo compounds derived from (p-methylphenyl)glycine were screened for the ability to esterify the green fluorescent protein (GFP) in an aqueous environment. Esterification of GFP with 2-diazo-2-(p-methylphenyl)-N,N-dimethylacetamide was efficient. The esterified protein entered the cytosol by traversing the plasma membrane directly, like a small-molecule prodrug. As with prodrugs, the nascent esters are substrates for endogenous esterases, which regenerate native protein. Thus, esterification could provide a general means to deliver native proteins to the cytosol.

SUBMITTER: Mix KA

PROVIDER: S-EPMC5856659 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Cytosolic Delivery of Proteins by Bioreversible Esterification.

Mix Kalie A KA Lomax Jo E JE Raines Ronald T RT

Journal of the American Chemical Society 20171010 41

Cloaking its carboxyl groups with a hydrophobic moiety is shown to enable a protein to enter the cytosol of a mammalian cell. Diazo compounds derived from (p-methylphenyl)glycine were screened for the ability to esterify the green fluorescent protein (GFP) in an aqueous environment. Esterification of GFP with 2-diazo-2-(p-methylphenyl)-N,N-dimethylacetamide was efficient. The esterified protein entered the cytosol by traversing the plasma membrane directly, like a small-molecule prodrug. As with ...[more]

PMID: 28976737

Similar Datasets

Cytosolic delivery of proteins by cholesterol tagging.

Project description:Protein-based imaging agents and therapeutics are superior in structural and functional diversity compared to small molecules and are much easier to design or screen. Antibodies or antibody fragments can be easily raised against virtually any target. Despite these fundamental advantages, the power and impact of protein-based agents are substantially undermined, only acting on a limited number of extracellular targets because macrobiomolecules cannot spontaneously cross the cell membrane. Conventional protein delivery techniques fail to address this fundamental problem in that protein cargos are predominantly delivered inside cells via endocytosis, a remarkably effective cell defense mechanism developed by Mother Nature to prevent intact biomolecules from entering the cytoplasm. Here, we report a unique concept, noncovalent cholesterol tagging, enabling virtually any compact proteins to permeate through the cell membrane, completely bypassing endocytosis. This simple plug-and-play platform greatly expands the biological target space and has the potential to transform basic biology studies and drug discovery.

| S-EPMC7304968 | biostudies-literature

Direct Cytosolic Delivery of Proteins through Coengineering of Proteins and Polymeric Delivery Vehicles.

Project description:Nanocarrier-mediated protein delivery is a promising strategy for fundamental research and therapeutic applications. However, the efficacy of the current platforms for delivery into cells is limited by endosomal entrapment of delivered protein cargo with concomitantly inefficient access to the cytosol and other organelles, including the nucleus. We report here a robust, versatile polymeric-protein nanocomposite (PPNC) platform capable of efficient (≥90%) delivery of proteins to the cytosol. We synthesized a library of guanidinium-functionalized poly(oxanorborneneimide) (PONI) homopolymers with varying molecular weights to stabilize and deliver engineered proteins featuring terminal oligoglutamate "E-tags". The polymers were screened for cytosolic delivery efficiency using imaging flow cytometry with cytosolic delivery validated using confocal microscopy and activity of the delivered proteins demonstrated through functional assays. These studies indicate that the PPNC platform provides highly effective and tunable cytosolic delivery over a wide range of formulations, making them robust agents for therapeutic protein delivery.

| S-EPMC7392052 | biostudies-literature

Cytosolic delivery of large proteins using nanoparticle-stabilized nanocapsules.

Project description:We report an effective intracellular delivery strategy for proteins of high molecular weight using AuNP stabilized capsules. This strategy provides direct delivery to the cytosol, avoiding endosomal entrapment.

| S-EPMC5137506 | biostudies-literature

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Project description:Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

| S-EPMC5570165 | biostudies-literature

IgG-Engineered Protective Antigen for Cytosolic Delivery of Proteins into Cancer Cells.

Project description:Therapeutic immunotoxins composed of antibodies and bacterial toxins provide potent activity against malignant cells, but joining them with a defined covalent bond while maintaining the desired function is challenging. Here, we develop novel immunotoxins by dovetailing full-length immunoglobulin G (IgG) antibodies and nontoxic anthrax proteins, in which the C terminus of the IgG heavy chain is connected to the side chain of anthrax toxin protective antigen. This strategy enabled efficient conjugation of protective antigen variants to trastuzumab (Tmab) and cetuximab (Cmab) antibodies. The conjugates effectively perform intracellular delivery of edema factor and N terminus of lethal factor (LFN) fused with diphtheria toxin and Ras/Rap1-specific endopeptidase. Each conjugate shows high specificity for cells expressing human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), respectively, and potent activity across six Tmab- and Cmab-resistant cell lines. The conjugates also exhibit increased pharmacokinetics and pronounced in vivo safety, which shows promise for further therapeutic development.

| S-EPMC7908032 | biostudies-literature

Cytosolic Delivery of Functional Proteins <i>In Vitro</i> through Tunable Gigahertz Acoustics.

Project description:Intracellular delivery is essential to therapeutic applications such as genome engineering and disease diagnosis. Current methods lack simple, noninvasive strategies and are often hindered by long incubation time or high toxicity. Hydrodynamic approaches offer rapid and controllable delivery of small molecules, but thus far have not been demonstrated for delivering functional proteins. In this work, we developed a robust hydrodynamic approach based on gigahertz (GHz) acoustics to achieve rapid and noninvasive cytosolic delivery of biologically active proteins. With this method, GHz-based acoustic devices trigger oscillations through a liquid medium (acoustic streaming), generating shear stress on the cell membrane and inducing transient nanoporation. This mechanical effect enhances membrane permeability and enables cytosolic access to cationic proteins without disturbing their bioactivity. We evaluated the versatility of this approach through the delivery of cationic fluorescent proteins to a range of cell lines, all of which displayed equally efficient delivery speed (≤20 min). Delivery of multiple enzymatically active proteins with functionality related to apoptosis or genetic recombination further demonstrated the relevance of this method.

| S-EPMC7392053 | biostudies-literature

Fluoroalkylation promotes cytosolic peptide delivery.

Project description:Cytosolic delivery of peptides remains a challenging task owing to their susceptibility to enzymatic degradation and the existence of multiple intracellular barriers. Here, we report a new strategy to address these issues by decoration of a fluorous tag on the terminal of cargo peptides. The fluorous-tagged peptides were assembled into nanostructures, efficiently internalized by cells via several endocytic pathways and released into the cytosol after endosomal escape. They were relatively stable against enzymatic degradation and showed much higher efficiency than nonfluorinated analogs and cell penetrant peptide-conjugated ones. The proposed strategy also efficiently delivered a proapoptotic peptide into specific sites in the cells and restored the function of cargo peptide after cytosolic delivery. The fluorous-tagged proapoptotic peptide efficiently inhibited tumor growth in vivo. This study provides an efficient fluorination strategy to promote the cytosolic delivery of peptides.

| S-EPMC7423368 | biostudies-literature

Diazo compounds for the bioreversible esterification of proteins.

Project description:A diazo compound is shown to convert carboxylic acids to esters efficiently in an aqueous environment. The basicity of the diazo compound is critical: low basicity does not lead to a reaction but high basicity leads to hydrolysis. This reactivity extends to carboxylic acid groups in a protein. The ensuing esters are hydrolyzed by human cellular esterases to regenerate protein carboxyl groups. This new mode of chemical modification could enable the key advantages of prodrugs to be translated from small-molecules to proteins.

| S-EPMC4275067 | biostudies-literature

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins.

Project description: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.

| S-EPMC6329960 | biostudies-literature

Fluorescence Correlation Spectroscopy Reveals Efficient Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins.

Project description: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.

| S-EPMC6202653 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data