Cytosolic Delivery of Argininosuccinate Synthetase Using a Cell-Permeant Miniature Protein.

Citrullinemia type I (CTLN-I) results from the absence or deficiency of argininosuccinate synthetase (AS), a 46 kDa enzyme that acts in the cytosol of hepatocytes to convert aspartic acid and citrulline into argininosuccinic acid. AS is an essential component of the urea cycle, and its absence or deficiency results in the harmful accumulation of ammonia in blood and cerebrospinal fluid. No disease-modifying treatment of CTLN-I exists. Here we report that the cell-permeant miniature protein (CPMP) ZF5.3 (ZF) can deliver AS to the cytosol of cells in culture and the livers of healthy mice. The fusion protein ZF-AS is catalytically active in vitro, stabilized in plasma, and traffics successfully to the cytosol of cultured Saos-2 and SK-HEP-1 cells, achieving cytosolic concentrations greater than 100 nM. This value is 3-10-fold higher than the concentration of endogenous AS (11 ± 1 to 44 ± 5 nM). When injected into healthy C57BL/6 mice, ZF-AS reaches the mouse liver to establish concentrations almost 200 nM above baseline. These studies demonstrate that ZF5.3 can deliver a complex enzyme to the cytosol at therapeutically relevant concentrations and support its application as an improved delivery vehicle for therapeutic proteins that function in the cytosol, including enzyme replacement therapies.

Quantification of protein delivery in live cells using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a quantitative single-molecule method that measures the concentration and rate of diffusion of fluorophore-tagged molecules, both large and small, in vitro and within live cells, and even within discrete cellular compartments. FCS is exceptionally well-suited to directly quantify the efficiency of intracellular protein delivery-specifically, how well different "cell-penetrating" proteins and peptides guide proteinaceous materials into the cytosol and nuclei of live mammalian cells. This article provides an overview of the procedures necessary to execute robust FCS experiments and evaluate endosomal escape efficiencies: preparation of fluorophore-tagged proteins, incubation with mammalian cells and preparation of FCS samples, setup and execution of an FCS experiment, and a detailed discussion of and custom MATLAB® script for analyzing the resulting autocorrelation curves in the context of appropriate diffusion models.

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.

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.