Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATP-and temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolindependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. Whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. Our results suggest that unconjugated peptide might follow endocytic pathways different from those utilized by TAT peptide conjugated to different proteins.Recent advances in the identification of new molecular therapy targets and disease-relevant proteins, accelerated by the completion of the human genome project, emphasized an importance of high molecular weight information-rich biomolecules, such as peptides, proteins, antisense DNA, and small interfering RNA, for molecular therapy. However, the delivery of proteins and nucleic acids into cells is greatly hampered by the low permeability of the cell plasma membrane to polar molecules. Not surprisingly, the discovery that a number of cationic peptides known as protein transduction domains (PTDs) 1 can facilitate cytoplasmic and nuclear delivery of a conjugated cargo has attracted a lot of interest (1-3). Up to date, a wide range of cargo molecules, including low molecular weight drugs (4), oligonucleotides (5), peptides (6) and even full-length proteins (7-10), have been successfully delivered into cells using PTDs and, most importantly, the functional activity of the delivered cargo has been observed (7-10).Despite significant progress in the cytoplasmic and nuclear delivery of various cargo molecules using PTDs, the underlying mechanisms remain under active debate. Until recently, it was widely assumed that the internalization of cationic PTDs is an energy-and receptor-independent process based on direct transport through the lipid bilayer (11-15). On the other hand, there have been indications that uptake of full-length TAT protein, from which one of the most commonly used PTDs referred to as TAT peptide is derived, occurs via endocytosis and depends on cell surface heparan sulfate receptors (16). Moreover, the validity of some of the important data, supporting a direct transport model for synthetic TAT peptide, has been questioned in several recent ...

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The Menkes protein (MNK) is a copper-transporting P-type ATPase, which has six highly conserved metalbinding sites, GMTCXXC, at the N terminus. The metalbinding sites may be involved in MNK trafficking and/or copper-translocating activity. In this study, we report the detailed functional analysis in mammalian cells of recombinant human MNK and its mutants with various metal-binding sites altered by site-directed mutagenesis. The results of the study, both in vitro and in vivo, provide evidence that the metal-binding sites of MNK are not essential for the ATP-dependent copper-translocating activity of MNK. Moreover, metal-binding site mutations, which resulted in a loss of ability of MNK to traffick to the plasma membrane, produced a copper hyperaccumulating phenotype. Using an in vitro vesicle assay, we demonstrated that the apparent K m and V max values for the wild type MNK and its mutants were not significantly different. The results of this study suggest that copper-translocating activity of MNK and its copper-induced relocalization to the plasma membrane represent a well coordinated copper homeostasis system. It is proposed that mutations in MNK which alter either its catalytic activity or/and ability to traffick can be the cause of Menkes disease.

Menkes' disease is a fatal, X-linked, copper deficiency disorder that results from defective copper efflux from intestinal cells and inadequate copper delivery to other tissues, leading to deficiencies of critical copper-dependent enzymes. Wilson's disease is an autosomally inherited, copper toxicosis disorder resulting from defective biliary excretion of copper, which leads to copper accumulation in the liver. The ATP7A and ATP7B genes that are defective in patients with Menkes' and Wilson's diseases, respectively, encode transmembrane, P-type ATPase proteins (ATP7A or MNK and ATP7B or WND, respectively) that function to translocate copper across cellular membranes. In this study, the cDNAs derived from a normal human ATP7A gene and the murine ATP7B homologue, Atp7b, were separately transfected into an immortalized fibroblast cell line obtained from a Menkes' disease patient. Both MNK and WND expressed from plasmid constructs were able to correct the copper accumulation and copper retention phenotype of these cells. However, the two proteins responded differently to elevated extracellular copper levels. Although MNK showed copper-induced trafficking from the trans-Golgi network to the plasma membrane, in the same cell line the intracellular location of WND did not appear to be affected by elevated copper.