Browse
Submit Data
Databases
API
Help

Dataset Information

20 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

Direct Cytosolic Delivery of Citraconylated Proteins.

Project description:Current intracellular protein delivery strategies face the challenge of endosomal entrapment and consequent degradation of protein cargo. Methods to efficiently deliver proteins directly to the cytosol have the potential to overcome this hurdle. Here, we report the use of a straightforward approach of protein modification using citraconic anhydride to impart an overall negative charge on the proteins, enabling them to assemble with positively charged nano vectors. This strategy uses anhydride-modified proteins to electrostatically form polymer-protein nanocomposites with a cationic guanidinium-functionalized polymer. These supramolecular self-assemblies demonstrated the efficient cytosolic delivery of modified proteins through a membrane fusion-like mechanism. This approach was validated on five cell lines and seven proteins as cargo. Retention of protein function was confirmed through efficient cell killing via the intracellular enzymatic activity of RNase A. This platform provides a versatile, straightforward, and single-step method of protein modification and efficient direct cytosolic protein delivery.

| S-EPMC9861219 | biostudies-literature

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

Overcoming Cytosolic Delivery Barriers of Proteins Using Denatured Protein-Conjugated Mesoporous Silica Nanoparticles.

Project description:Intracellular delivery of therapeutic proteins has increased advantages over current small-molecule drugs and gene therapies, especially in therapeutic efficacies for a broad spectrum of diseases. Hence, developing the protein therapeutics approach provides a needed alternative. Here, we designed a mesoporous silica nanoparticle (MSN)-mediated protein delivery approach and demonstrated effective intracellular delivery of the denatured superoxide dismutase (SOD) protein, overcoming the delivery challenges and achieving higher enzymatic activity than native SOD-conjugated MSNs. The denatured SOD-conjugated MSN delivery strategy provides benefits of reduced size and steric hindrance, increased protein flexibility without distorting its secondary structure, exposure of the cell-penetrating peptide transactivator of transcription for enhanced efficient delivery, and a change in the corona protein composition, enabling cytosolic delivery. After delivery, SOD displayed a specific activity around threefold higher than in our previous reports. Furthermore, the in vivo biosafety and therapeutic potential for neuron therapy were evaluated, demonstrating the biocompatibility and the effective antioxidant effect in Neuro-2a cells that protected neurite outgrowth from paraquat-induced reactive oxygen species attack. This study offers an opportunity to realize the druggable possibility of cytosolic proteins using MSNs.

| S-EPMC9896485 | 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

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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