Arginine Topology Controls Escape of Minimally Cationic Proteins from Early Endosomes to the Cytoplasm

Appelbaum, Jacob; LaRochelle, Jonathan R.; Smith, Betsy A.; Balkin, Daniel M.; Holub, Justin M.; Schepartz, Alanna

doi:10.1016/j.chembiol.2012.05.022

Cited by 149 publications

(295 citation statements)

References 69 publications

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“…The poor cytosolic release from early endosomes were also observed for cell-penetrating cationic proteins, which showed about 1 to 5% cytosolic access efficiency from their endocytosed vesicles. 37 The specific interaction between KT4 and KRS in the cytosol was further confirmed by immunoprecipitation experiments for the lysates of HeLa cells that were pre-treated with KT4 for internalization prior to cell lysis. KRS co-precipitated with internalized KT4, but not internalized TMab4 (Fig.…”

Section: Cytotransmab Penetrates and Directly Binds To A Targeted Cytmentioning

confidence: 86%

“…11 Although cargo proteins that are internalized by clathrinmediated endocytosis often route to late endosomes and subsequently undergo lysosomal degradation (or routes to recycling endosomes), 7,11 this is not always the case, as exemplified by cytosolic release of some CPPs or toxins after cellular uptake by clathrin-mediated endocytosis. 9,37,44,45 Several lines of evidence, such as intracellular distribution and trafficking studies by confocal microscopy, direct access of KT4 to cytosolic KRS proteins, and calcein release assay, indicate that endocytosed cytotransmab is directly released to cytosol from early endosomes, passing over lysosome, ER, Golgi, and nucleus trafficking. However, the underlying molecular mechanism by which cytotransmab escapes from early endosomes into the cytosol is not clear.…”

Section: Discussionmentioning

confidence: 99%

“…(E) Colocalization of internalized cytotransmabs with endocytosis markers. HeLa cells were co-incubated with cytotransmabs (1 mM) and endocytosis markers including 10 mg/mL of Alexa Fluor 488-transferrin (TF, green), FITC-cholera toxin B (Ctx-B, green), and Oregon green-dextran (Dextran, green) for 30 min at 37 C. Subsequently, internalized cytotransmabs were stained with TRITC-conjugated anti-Human IgG Fc antibody (red). The regions in the white box were magnified for better imaging of co-localization.…”

Section: Endocytic Mechanisms Underlying Cytotransmab Internalizationmentioning

confidence: 99%

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A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

Choi¹,

Bae

Shin

et al. 2014

mAbs

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These authors contributed equally to this work.Keywords: cell-penetrating antibody, cytosolic protein targeting, cellular internalization, endosomal release, IgG antibody, intracellular trafficking, next-generation antibodyAbbreviations: IgG, immunoglobulin G; VL, light chain variable domain; VH, heavy chain variable domain; LC, light chain; HC, heavy chain; CDR, complementarity-determining region.Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were coexpressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira Ò ) and bevacizumab (Avastin Ò ), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.

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Section: Cytotransmab Penetrates and Directly Binds To A Targeted Cytmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Endocytic Mechanisms Underlying Cytotransmab Internalizationmentioning

confidence: 99%

See 1 more Smart Citation

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

Choi¹,

Bae

Shin

et al. 2014

mAbs

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“…For use as a therapeutic, the peptide would need to penetrate the cell membrane. While transfer across the cell membrane can be problematic for peptide therapeutics, this is an area of active research 26,27 and a focus of further study for our laboratory. …”

Section: Acs Medicinal Chemistry Lettersmentioning

confidence: 99%

Turning a Substrate Peptide into a Potent Inhibitor for the Histone Methyltransferase SETD8

Judge

Zhu

Upadhyay

et al. 2016

ACS Med. Chem. Lett.

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SETD8 is a histone H4−K20 methyltransferase that plays an essential role in the maintenance of genomic integrity during mitosis and in DNA damage repair, making it an intriguing target for cancer research. While some small molecule inhibitors for SETD8 have been reported, the structural binding modes for these inhibitors have not been revealed. Using the complex structure of the substrate peptide bound to SETD8 as a starting point, different natural and unnatural amino acid substitutions were tested, and a potent (K i 50 nM, IC 50 0.33 μM) and selective norleucine containing peptide inhibitor has been obtained.

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“…16,17 However, it has been recently shown that the cationic arginine topology dramatically influences important steps of the delivery process including endosomal escape. 18 The formation of dynamic bonds constitutes a powerful strategy for the preparation of amphiphilic membrane transporters. 14,[19][20][21][22][23] Dynamic bonds such as hydrazones, oximes and disulfides offer two fundamental advantages for the synthesis of membrane carriers.…”

mentioning

confidence: 99%

Tuning the Properties of Penetrating Peptides by Oxime Conjugation

Pazo

Fernández-Caro

Priegue

et al. 2017

Synlett

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We here describe the application of oxime bond formation between a peptide scaffold and different aldehydes to modify the transporting capabilities of penetrating peptides. We show that bonds such as oximes offer a great synthetic advantage for the modification of the properties of model penetrating peptides. We believe that this approach will allow the development of improved intracellular delivery vehicles.

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Arginine Topology Controls Escape of Minimally Cationic Proteins from Early Endosomes to the Cytoplasm

Cited by 149 publications

References 69 publications

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

Turning a Substrate Peptide into a Potent Inhibitor for the Histone Methyltransferase SETD8

Tuning the Properties of Penetrating Peptides by Oxime Conjugation

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