Understanding the cellular uptake of phosphopeptides.

Phosphopeptide-cellular uptake has been studied with a unique combination of tools designed to quantitate this phenomena and to understand properties that contribute to transmembrane penetration. High-affinity src-homology domain (SH2) hexapeptides for the phosphatidyl inositol 3-kinase system were used to judge cell penetration using red blood cells--a model system for the study of transmembrane cellular uptake. Hexapeptides without phosphate groups and devoid of charged residues poorly entered cells. N-terminal modification with bulky hydrophobic groups enhanced partitioning into octanol, an index of hydrophobicity, and allowed certain non-phosphorylated peptides to pass into red cells. However, tyrosine phosphorylation of hexapeptides markedly decreased octanol-water partitioning and completely eliminated cellular uptake. Inclusion of ion-pairing agents that masked the phosphate hydrophilic character enabled partitioning of phosphopeptides into octanol and achieved cellular uptake. This effect was demonstrated using fluorescent derivatives of phosphopeptides and CV1 cells in culture. The results validate the concept of facilitating cell entry by charge masking and open the way to future refinements of this principle. Various penetration techniques are compared and discussed in the context of maximizing cellular viability.

Characterization of SH2-ligand interactions via library affinity selection with mass spectrometric detection.

Synthetic combinatorial libraries have proven to be a valuable source of diverse structures useful for large-scale biochemical screening. Their use has greatly facilitated the study of protein-protein interactions. We have developed a practical technique for screening such libraries by integrating affinity chromatography selection with electrospray ionization mass spectrometric detection, referred to as library affinity selection-mass spectrometry (LAS-MS). The process allows for rapid and efficient screening of solution phase libraries and provides detailed information such as the relative affinities of substrates. The method is generally applicable to include nonpeptide libraries; moreover, electrospray tandem mass spectrometry (ES-MS/MS) yields sequence-specific identification of individual components without the need for chemical tags. This technique is demonstrated using the Src homology 2 (SH2) domain of phosphatidylinositol 3-kinase (PI 3-kinase). The critical importance of methionine in the position +3 (relative to the phosphotyrosine position) is demonstrated in a library built with a phosphotyrosine mimic, (phosphonodifluoromethyl)phenylalanine. The described method has broad applicability to combinatorial library screening.

Characterization of the S3 subsite specificity of cathepsin B.

Five synthetic substrates containing different amino acid residues at the P3 position (acetyl-X-Arg-Arg-AMC, where X is Gly, Glu, Arg, Val, and Tyr and where AMC represents 7-amindo-4-methylcoumarin) were used to investigate the S3 subsite specificity of cathepsin B. At pH 6.0, the specificity constant, kcat/Km, for tripeptide substrate hydrolysis was observed to increase in the order Glu < Gly < Arg < Val < Tyr. Molecular modeling studies of substrates containing a P3 Glu, Arg, or Tyr covalently bound as the tetrahedral intermediate to the enzyme suggest that the specificity for a P3 Tyr is because of a favorable aromatic-aromatic interaction with Tyr75 on the enzyme as well as a possible H bond between the P3 Tyr hydroxyl and the side chain carboxyl of Asp69.

Identification of a receptor-binding region in the core segment of the human anaphylatoxin C5a.

In order to identify regions of C5a that contribute to receptor binding and functional activity of the anaphylatoxin, a series of peptides was synthesized in which core segments have been attached to C-terminal segments via native peptidic or disulfide bonds. It has been found that residues Arg40 and Arg46 in the loop-3 region of the core induce a 1000-fold increase in the affinity of the disordered C-terminal segment of C5a. The results obtained from this work lead to the conclusion that the loop-3 region is most likely the core binding site of C5a.