16 research outputs found
Fluorescence Correlation Spectroscopy Reveals Efficient Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins.
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
Labeling Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and SNAP-tags.
Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons-by orders of magnitude-than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging.This work was supported by a Wellcome Trust Foundation grant (095927/A/11/Z) and NIH grant (R01GM118486). R.S.E. was supported by an Advanced Postdoc Mobility Fellowship from the Swiss National Foundation
Fluorescence Correlation Spectroscopy Reveals Efficient Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins
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HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins
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
Synthesis and Biological Evaluation of an Indazole-Based Selective Protein Arginine Deiminase 4 (PAD4) Inhibitor
Protein arginine deiminase 4 (PAD4)
is a calcium-dependent enzyme that catalyzes the conversion of arginine
to citrulline within target proteins. Dysregulation of PAD4
has been implicated in a number of human diseases, including rheumatoid
arthritis and other inflammatory diseases as well as cancer. In this
study, we report on the design, synthesis, and evaluation of a new
class of haloacetamidine-based compounds as potential PAD4 inhibitors.
Specifically, we describe the identification of 4,5,6-trichloroindazole 24 as a highly potent PAD4 inhibitor that displays >10-fold
selectivity for PAD4 over PAD3 and >50-fold over PAD1 and PAD2.
The efficacy of this compound in cells was determined by measuring
the inhibition of PAD4-mediated H4 citrullination in HL-60 granulocytes
Native Chemical Ligation of Thioamide-Containing Peptides: Development and Application to the Synthesis of Labeled α-Synuclein for Misfolding Studies
Thioamide modifications of the peptide backbone are used
to perturb
secondary structure, to inhibit proteolysis, as photoswitches, and
as spectroscopic labels. Thus far, their incorporation has been confined
to single peptides synthesized on solid phase. We have generated thioamides
in C-terminal thioesters or N-terminal Cys fragments and examined
their compatibility with native chemical ligation conditions. Most
sequence variants can be coupled in good yields with either TCEP or
DTT as the reductant, though some byproducts are observed with prolonged
TCEP incubations. Furthermore, we find that thioamides are compatible
with thiazolidine protection of an N-terminal Cys, so that multiple
ligations can be used to construct larger proteins. Since the acid-lability
of the thioamide prohibits on-resin thioester synthesis using Boc
chemistry, we devised a method for the synthesis of thioamide peptides
with a masked C-terminal thioester that is revealed <i>in situ</i>. Finally, we have shown that thioamidous peptides can be coupled
to expressed protein fragments to generate large proteins with backbone
thioamide labels by synthesizing labeled versions of the amyloid protein
α-synuclein for protein folding studies. In a proof-of-principle
experiment, we demonstrated that quenching of fluorescence by thioamides
can be used to track conformational changes during aggregation of
labeled α-synuclein
HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins
Efficient Synthesis and In Vivo Incorporation of Acridon-2-ylalanine, a Fluorescent Amino Acid for Lifetime and Förster Resonance Energy Transfer/Luminescence Resonance Energy Transfer Studies
The
amino acid acridon-2-ylalanine (Acd) can be a valuable probe
of protein conformational change because it is a long lifetime, visible
wavelength fluorophore that is small enough to be incorporated during
ribosomal biosynthesis. Incorporation of Acd into proteins expressed
in <i>Escherichia coli</i> requires efficient chemical synthesis
to produce large quantities of the amino acid and the generation of
a mutant aminoacyl tRNA synthetase that can selectively charge the
amino acid onto a tRNA. Here, we report the synthesis of Acd in 87%
yield over five steps from Tyr and the identification of an Acd synthetase
by screening candidate enzymes previously evolved from <i>Methanococcus
janaschii</i> Tyr synthetase for unnatural amino acid incorporation.
Furthermore, we characterize the photophysical properties of Acd,
including quenching interactions with select natural amino acids and
Förster resonance energy transfer (FRET) interactions with
common fluorophores such as methoxycoumarin (Mcm). Finally, we demonstrate
the value of incorporation of Acd into proteins, using changes in
Acd fluorescence lifetimes, Mcm/Acd FRET, or energy transfer to Eu<sup>3+</sup> to monitor protein folding and binding interactions