The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to 3-galactosidase, horseradish peroxidase, RNase A, and domain Ill ofPseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-f-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages.Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.The potential for intracellular therapeutic use of proteins, peptides, and oligonucleotides has been limited by the impermeable nature of the cell membrane to these compounds. A wide variety of methods have been proposed for the delivery of proteins and other macromolecules into living cells for either experimental or therapeutic uses, including microinjection, scrape loading, electroporation, liposomes (1-7), bacterial toxins (8)(9)(10), and receptor-mediated endocytosis (11-16). Most of these methods are either inefficient or time-consuming, cause appreciable cell death, or result in uptake into intracellular vesicles without efficient cytoplasmic delivery. Several approaches (15-18) rely on binding of macromolecules to the cell surface, followed by internalization via the endocytic route. However, since proteins that have entered this pathway remain enclosed within lipid vesicles, they do not have access to the cell cytoplasm, most typically the target environment. It seems reasonable to assume that the escape from endocytic vesicles is the ratelimiting step in achieving true cellular delivery, yet many assays fail to measure this.Recently the Tat protein from human immunodeficiency virus 1 (HIV-1) was shown to enter cells when added exogenously (19,20). Tat protein can simply be added to medium at concentrations as low as 1 nM, and biological responses can be detected. Since the assay for this process was the transactivation of a transfected reporter gene, activity reflects those molecules that had been targeted to the nucleus, presumably after cytoplasmic delivery. The mechanism by which Tat traverses a membrane and the precise intracellular location of this event remain unclear. However, Tat binds efficiently to cells, with >107 sites per cell and then is internalized by an adsorptive endocytosis process (20). In characterizing the uptake process using iodinated Tat, Mann and Frankel (20) observed that only 3% of the Tat becameThe publication costs of this article were...
The role of the zinc site in the N-terminal fragment of human Sonic hedgehog (ShhN) was explored by comparing the biophysical and functional properties of wild-type ShhN with those of mutants in which the zinc-coordinating residues H140, D147, and H182, or E176 which interacts with the metal ion via a bridging water molecule, were mutated to alanine. The wild-type and E176A mutant proteins retained 1 mol of zinc/mol of protein after extensive dialysis, whereas the H140A and D147A mutants retained only 0.03 and 0.05 mol of zinc/mol of protein, respectively. Assay of the wild-type and mutant proteins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were significantly less active. These assays also indicated that the H140A and D147A mutants were susceptible to proteolysis. CD, fluorescence, and (1)H NMR spectra of the H140A, D147A, and E176A mutants measured at 20 or 25 degrees C were very similar to those observed for wild-type ShhN. However, CD measurements at 37 degrees C showed evidence of some structural differences in the H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation studies revealed that the loss of zinc from the H140A and D147A mutants destabilized the folded proteins by approximately 3.5 kcal/mol, comparable to the effect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal melting curves of wild-type ShhN gave a single unfolding transition with a midpoint T(m) of approximately 59 degrees C, whereas both the H140A and D147A mutants displayed two distinct transitions with T(m) values of 37-38 and 52-54 degrees C, similar to that observed for EDTA-treated wild-type ShhN. Addition of zinc to the H140A and D147A mutants resulted in a partial restoration of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assay that indicated that they bound zinc with K(d) values of approximately 1.6 and approximately 15 nM, respectively, as compared to a value of =100 pM for wild-type ShhN. The properties of the E176A mutant were indistinguishable from those of wild-type ShhN in all biophysical and functional assays, indicating that this residue does not contribute significantly to stabilization of the zinc-binding site and that ShhN does not require hydrolase activity for in vitro biological function.
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