Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4–12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.

We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solutionphase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately two orders of magnitude less active than the other three PTPs. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, is highly active towards multiply phosphorylated peptides, but disfavors basic residues at any position, a Gly at the pY−1 position, or a Pro at the pY+1 position. By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of the pY residue. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the Nterminal side and one or more acidic residues on the C-terminal side of pY, but no basic residues. Subtle differences exist between SHP-1 and SHP-2 in that SHP-1 has a stronger preference for acidic residues at the pY−1 and pY+1 positions and the two SHPs prefer acidic residues at different positions N-terminal to pY. A survey of the known protein substrates of PTP1B, SHP-1, and SHP-2 shows an excellent agreement between the in vivo dephosphorylation pattern and the in vitro specificity profiles derived from library screening. These results suggest that different PTPs have distinct sequence specificity profiles and the intrinsic activity/specificity of the PTP domain is an important determinant of the enzyme's in vivo substrate specificity. † This work was supported by grants from the National Institutes of Health (CA132855, GM062820, CA49132, and CA49152). L.R.was supported by a predoctoral fellowship from China Scholarship Council affiliated with the Ministry of Education of P. R. China. T.M.M. was supported by a predoctoral fellowship from the NIH Chemistry-Biology Interface Training Program (T32GM08512). * To whom correspondence should be addressed: Department of Chemistry, The Ohio State University, 100 West 18 th Avenue, Columbus, OH 43210. Telephone: (614) Protein-tyrosine phosphatases (PTPs) are a large family of enzymes (the human genome encodes 107 PTPs) that catalyze the hydrolysis of phosphotyrosine (pY) in proteins to tyrosine and inorganic phosphate (1). Once thought to be promiscuous "housekeeping" enzymes, PTPs are now known to be actively involved in cell signaling and have been described as having "exquisite substrate specificity" in vivo. However, in contrast to their well-established catalytic mechanism, their physiological substrates and functions remain poorly defined. Substantial evidence suggests that the substrate specificity of PTPs is controlled by both the intrinsic sequence specificity of the catalytic domain and the presence of targeting domains, which dire...

N-substituted glycine oligomers, or peptoids, have emerged as an important class of foldamers for the study of biomolecular interactions and for potential use as therapeutic agents. However, the design of peptoids with well-defined conformations a priori remains a formidable challenge. New approaches are required to address this problem, and the systematic study of the role of individual monomer units in the global peptoid folding process represents one strategy. Here, we report our efforts toward this approach through the design, synthesis, and characterization of peptoids containing nitroaromatic monomer units. This work required the synthesis of a new chiral amine building block, (S)-1-(2-nitrophenyl)ethanamine (s2ne), which could be readily installed into peptoids using standard solid-phase peptoid synthesis techniques. We designed a series of peptoid nonamers that allowed us to probe the effects of this relatively electron-deficient and sterically encumbered α-chiral side chain on peptoid structure, namely, the peptoid threaded loop and helix. Circular dichroism (CD) spectroscopy of the peptoids revealed that the nitroaromatic monomer has a significant effect on peptoid secondary structure. Specifically, the threaded loop structure was disrupted in a nonamer containing alternating N-(S)-1-phenylethylglycine (Nspe) and Ns2ne monomers, and the major conformation was helical instead. Indeed, placement of a single Ns2ne at the N-terminal position of (Nspe)9 resulted in a destabilized form of the threaded loop structure relative to the homononamer (Nspe)9. Conversely, we observed that incorporation of N-(S)-1-(4-nitrophenyl)ethylglycine (Nsnp, a para-nitro monomer) at the N-terminal position stabilized the threaded loop structure relative to (Nspe)9. Additional experiments revealed that nitroaromatic side chains can influence peptoid nonamer folding by modulating the strength of key intramolecular hydrogen bonds in the peptoid threaded loop structure. Steric interactions were also implicated for the Ns2ne monomer. Overall, this study provides further evidence that aromatic side chain structure, even if perturbed in a single monomer unit, can strongly influence local peptoid backbone conformation.

The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences, but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >105-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >105-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3–18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A co-crystal structure of PTP1B bound with a nephrin pY1193 peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic activities with respect to PTP in vivo substrate specificity and biological functions are discussed.

Background:Factors that determine the in vivo substrate specificity of dual specificity phosphatases are currently unknown. Results: Specificity profiling of VHR through peptide library screening identified two distinct classes of peptide substrates, which bind to VHR in opposite orientations. Conclusion: VHR may act on a previously unrecognized class of protein substrates. Significance: The results should help identify new VHR substrates and elucidate its biological function.