A Protein Transduction Domain with Cell Uptake and Selectivity Profiles that Are Controlled by Multivalency Effects

DePorter, Sandra M.; Lui, Irene; Mohan, Utpal; McNaughton, Brian R.

doi:10.1016/j.chembiol.2013.01.015

Cited by 7 publications

(11 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Each of these elements was separated by a short, flexible linker that includes unique restriction sites in the encoding DNA for rapid element-shuttling, while an internal His tag was incorporated for protein purification and proved extremely successful. A range of canonical CPPs were selected, representing different CPP classes 25 26 : TAT 27 , R9 28 , Penetratin 29 (PEN), Penetratin-Arginine 30 (PenArg), SAP 31 , VP22 32 , PEP1 33 , hCT 34 , PTD4 35 , Ypep 36 , and Transportan 37 . In parallel we produced control proteins without an N-terminal CPP (TRX_S11) or where the “CPP” was a short peptide sequence with no cell-penetrating activity (PYC35 38 39 , fusion protein PYC35_TRX_S11).…”

Section: Resultsmentioning

confidence: 99%

GFP-complementation assay to detect functional CPP and protein delivery into living cells

Milech

Longville

Cunningham

et al. 2015

Sci Rep

View full text Add to dashboard Cite

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell’s membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease.

show abstract

Section: Resultsmentioning

confidence: 99%

GFP-complementation assay to detect functional CPP and protein delivery into living cells

Milech

Longville

Cunningham

et al. 2015

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Various screening and evolution-based techniques also permit relatively rapid analysis of large (≥10 6 ) protein libraries for binding to a target, and this effort is typically much higher-throughput and simpler than the analogous small-molecule discovery process. Finally, multiple technologies now exist – some developed in our own lab(21–23) - which enable functional protein delivery to the interior of mammalian cells and even specific subcellular environments(24). to the extent that multiple researchers have used exogenous natural or synthetic proteins as basic research tools or drug leads that act on intracellular targets.…”

mentioning

confidence: 99%

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription

Crawford¹,

Blakeley²,

Chen

et al. 2016

ACS Chem. Biol.

View full text Add to dashboard Cite

Potent and selective recognition and modulation of disease-relevant RNAs remains a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition, and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) Trans-Activation Response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here we use yeast display and saturation mutagenesis of established RNA binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA binding selectivity; it binds TAR with sub-nanomolar affinity (apparent dissociation constant ~0.5 nM), but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5′-untranslated region, inhibits the interaction between TAR RNA and an HIV Trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents – small molecule, nucleic acid, or protein - reported to date, and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition—and modulation—of a disease-relevant RNA.

show abstract

“…To determine uptake efficiency we fused each of our polycationic resurfaced nanobodies to GFP and measured uptake by flow cytometry. 3T3 cells were first treated with 10–500 nM polycationic resurfaced nanobody‐GFP fusion, then washed with a phosphate buffered saline solution containing 20 U/mL heparin sulfate—which has been previously shown to remove cell surface bound protein especially supercharged proteins . Following treatment with trypsin, which has also been shown to remove and/or degrade surface bound protein, intracellular levels of nanobody‐GFP was measured by flow cytometry.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, most current protein drugs and basic research tools target disease‐relevant receptors that reside on the surface of the cell or the extracellular matrix. Efforts to unlock the full potential of proteins in biomedical applications by enabling potent and functional cell penetration have been a major focus of modern biologics research . Incorporation of polycationic linkages—such as polyarginine—has previously been described as a means to enable cell penetration of various cargo, including proteins .…”

Section: Introductionmentioning

confidence: 99%

Resurfaced cell‐penetrating nanobodies: A potentially general scaffold for intracellularly targeted protein discovery

2016

View full text Add to dashboard Cite

By virtue of their size, functional group diversity, and complex structure, proteins can often recognize and modulate disease-relevant macromolecules that present a challenge to smallmolecule reagents. Additionally, high-throughput screening and evolution-based methods often make the discovery of new protein binders simpler than the analogous small-molecule discovery process. However, most proteins do not cross the lipid bilayer membrane of mammalian cells. This largely limits the scope of protein therapeutics and basic research tools to those targeting disease-relevant receptors on the cell surface or extracellular matrix. Previously, researchers have shown that cationic resurfacing of proteins can endow cell penetration. However, in our experience, many proteins are not amenable to such extensive mutagenesis. Here, we report that nanobodies-a small and stable protein that can be evolved to recognize virtually any disease-relevant receptor-are amenable to cationic resurfacing, which results in cell internalization. Once internalized, these nanobodies access the cytosol. Polycationic resurfacing does not appreciably alter the structure, expression, and function (target recognition) of a previously reported GFP-binding nanobody, and multiple nanobody scaffolds are amenable to polycationic resurfacing. Given this, we propose that polycationic resurfaced cell-penetrating nanobodies might represent a general scaffold for intracellularly targeted protein drug discovery.

show abstract

A Protein Transduction Domain with Cell Uptake and Selectivity Profiles that Are Controlled by Multivalency Effects

Cited by 7 publications

References 55 publications

GFP-complementation assay to detect functional CPP and protein delivery into living cells

GFP-complementation assay to detect functional CPP and protein delivery into living cells

An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription

Resurfaced cell‐penetrating nanobodies: A potentially general scaffold for intracellularly targeted protein discovery

Contact Info

Product

Resources

About