Discovery of a Macropinocytosis‐Inducing Peptide Potentiated by Medium‐Mediated Intramolecular Disulfide Formation

Arafiles, Jan Vincent V.; Hirai, Yusuke; Kuriyama, Masashi; Sakyiamah, Maxwell; Nomura, Wataru; Sonomura, Kazuhiro; Imanishi, Masahito; Otaka, Akira; Tamamura, Hirokazu; Futaki, Shiroh

doi:10.1002/anie.202016754

Cited by 13 publications

(28 citation statements)

References 45 publications

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“…4 Possible contributions from glutathione (GSH) pumped from the cytosol to the cell surface, and other extracellular thiols, should not be ignored either. 22,26,30,31 Besides these explicit early studies on thiol-mediated uptake, often supported by inhibition experiments, there is a remarkably rich literature that simply mentions the fact that uptake increases in the presence of chalcogenides, usually disulfides, without further explanations. 4,32−51 In gene transfection, disulfide polymers have been heavily explored but not with the intention to enable uptake but to degrade the transporters after uptake by reductive depolymerization in the cytosol.…”

Section: Chronologymentioning

confidence: 99%

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Thiol-Mediated Uptake

Laurent

Martinent

Lim

et al. 2021

JACS Au

112

208

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This Perspective focuses on thiol-mediated uptake, that is, the entry of substrates into cells enabled by oligochalcogenides or mimics, often disulfides, and inhibited by thiol-reactive agents. A short chronology from the initial observations in 1990 until today is followed by a summary of cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs) as privileged scaffolds in thiol-mediated uptake and inhibitors of thiol-mediated uptake as potential antivirals. In the spirit of a Perspective, the main part brings together topics that possibly could help to explain how thiol-mediated uptake really works. Extreme sulfur chemistry mostly related to COCs and their mimics, cyclic disulfides, thiosulfinates/-onates, diselenolanes, benzopolysulfanes, but also arsenics and Michael acceptors, is viewed in the context of acidity, ring tension, exchange cascades, adaptive networks, exchange affinity columns, molecular walkers, ring-opening polymerizations, and templated polymerizations. Micellar pores (or lipid ion channels) are considered, from cell-penetrating peptides and natural antibiotics to voltage sensors, and a concise gallery of membrane proteins, as possible targets of thiol-mediated uptake, is provided, including CLIC1, a thiol-reactive chloride channel; TMEM16F, a Ca-activated scramblase; EGFR, the epithelial growth factor receptor; and protein-disulfide isomerase, known from HIV entry or the transferrin receptor, a top hit in proteomics and recently identified in the cellular entry of SARS-CoV-2.

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Section: Chronologymentioning

confidence: 99%

“…This combination is beyond the term “thiol-mediated uptake”, which is however maintained for historical reasons and the lack of convincing alternatives . Possible contributions from glutathione (GSH) pumped from the cytosol to the cell surface, and other extracellular thiols, should not be ignored either. ,,, …”

Section: Chronologymentioning

confidence: 99%

Thiol-Mediated Uptake

Laurent

Martinent

Lim

et al. 2021

JACS Au

112

208

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“…This version of GFP is an engineered GFP with an unusually high net negative theoretical charge of −30 [22] . The nuclear localization signal (NLS) derived from the SV40 large T antigen (PKKKRKV) [23] was added to the (−30)GFP to generate NLS‐(−30)GFP [24] . Cytosolic translocation of NLS‐(−30)GFP should lead to nuclear accumulation of the protein, and thus internalization could be analyzed in terms of fluorescence signals localized in the nucleus.…”

Section: Resultsmentioning

confidence: 99%

Liquid Droplet Formation and Facile Cytosolic Translocation of IgG in the Presence of Attenuated Cationic Amphiphilic Lytic Peptides

et al. 2021

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Fc region binding peptide conjugated with attenuated cationic amphiphilic lytic peptide L17E trimer [FcB(L17E)3] was designed for immunoglobulin G (IgG) delivery into cells. Particle‐like liquid droplets were generated by mixing Alexa Fluor 488 labeled IgG (Alexa488‐IgG) with FcB(L17E)3. Droplet contact with the cellular membrane led to spontaneous influx and distribution of Alexa488‐IgG throughout cells in serum containing medium. Involvement of cellular machinery accompanied by actin polymerization and membrane ruffling was suggested for the translocation. Alexa488‐IgG negative charges were crucial in liquid droplet formation with positively charged FcB(L17E)3. Binding of IgG to FcB(L17E)3 may not be necessary. Successful intracellular delivery of Alexa Fluor 594‐labeled anti‐nuclear pore complex antibody and anti‐mCherry‐nanobody tagged with supernegatively charged green fluorescence protein allowed binding to cellular targets in the presence of FcB(L17E)3.

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“…The ability of a molecular tool to induce macropinocytosis plays a key role in efficient protein transduction. 13 , 14 …”

Section: Introductionmentioning

confidence: 99%

A Synthetic Multidomain Peptide That Drives a Macropinocytosis-Like Mechanism for Cytosolic Transport of Exogenous Proteins into Plants

Miyamoto

Toyooka

Chuah

et al. 2022

JACS Au

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Direct delivery of proteins into plants represents a promising alternative to conventional gene delivery for probing and modulating cellular functions without the risk of random integration of transgenes into the host genome. This remains challenging, however, because of the lack of a protein delivery tool applicable to diverse plant species and the limited information about the entry mechanisms of exogenous proteins in plant cells. Here, we present the synthetic multidomain peptide (named dTat-Sar-EED4) for cytosolic protein delivery in various plant species via simple peptide-protein coincubation. dTat-Sar-EED4 enabled the cytosolic delivery of an active enzyme with up to ∼20-fold greater efficiency than previously described cell-penetrating peptides in several model plant systems. Our analyses using pharmacological inhibitors and transmission electron microscopy revealed that dTat-Sar-EED4 triggered a unique endocytic mechanism for cargo protein internalization. This endocytic mechanism shares several features with macropinocytosis, including the dependency of actin polymerization, sensitivity to phosphatidylinositol-3 kinase activity, and formation of membrane protrusions and large intracellular vesicles (>200 nm in diameter), even though macropinocytosis has not been identified to date in plants. Our study thus presents a robust molecular tool that can induce a unique cellular uptake mechanism for the efficient transport of bioactive proteins into plants.

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Discovery of a Macropinocytosis‐Inducing Peptide Potentiated by Medium‐Mediated Intramolecular Disulfide Formation

Cited by 13 publications

References 45 publications

Thiol-Mediated Uptake

Thiol-Mediated Uptake

Liquid Droplet Formation and Facile Cytosolic Translocation of IgG in the Presence of Attenuated Cationic Amphiphilic Lytic Peptides

A Synthetic Multidomain Peptide That Drives a Macropinocytosis-Like Mechanism for Cytosolic Transport of Exogenous Proteins into Plants

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