Peptide synthesis using unprotected peptides through orthogonal coupling methods.

We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C alpha-thiocarboxylic acid reacting with a beta-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled to aqueous solution to give free peptides up to 54 residues long with high efficiency.

Structural basis for major histocompatibility complex (MHC)-linked susceptibility to autoimmunity: charged residues of a single MHC binding pocket confer selective presentation of self-peptides in pemphigus vulgaris.

Human T-cell-mediated autoimmune diseases are genetically linked to particular alleles of MHC class II genes. Susceptibility to pemphigus vulgaris (PV), an autoimmune disease of the skin, is linked to a rare subtype of HLA-DR4 (DRB1*0402, 1 of 22 known DR4 subtypes). The PV-linked DR4 subtype differs from a rheumatoid arthritis-associated DR4 subtype (DRB1*0404) only at three residues (DR beta 67, 70, and 71). The disease is caused by autoantibodies against desmoglein 3 (DG), and T cells are thought to trigger the autoantibody production against this keratinocyte adhesion molecule. Based on the DRB1*0402 binding motif, seven candidate peptides of the DG autoantigen were identified. T cells from four PV patients with active disease responded to one of these DG peptides (residues 190-204); two patients also responded to DG-(206-220). T-cell clones specific for DG-(190-204) secreted high levels of interleukins 4 and 10, indicating that they may be important in triggering the production of DG-specific autoantibodies. The DG-(190-204) peptide was presented by the disease-linked DRB1*0402 molecule but not by other DR4 subtypes. Site-directed mutagenesis of DRB1*0402 demonstrated that selective presentation of DG-(190-204), which carries a positive charge at the P4 position, was due to the negatively charged residues of the P4 pocket (DR beta 70 and 71). DR beta 71 has a negative charge in DRB1*0402 but a positive charge in other DR4 subtypes, including the DR4 subtypes linked to rheumatoid arthritis. The charge of the P4 pocket in the DR4 peptide binding site therefore appears to be a critical determinant of MHC-linked susceptibility to PV and rheumatoid arthritis.

Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform.

Conversion of the cellular isoform of prion protein (PrPC) into the scrapie isoform (PrPSc) involves an increase in the beta-sheet content, diminished solubility, and resistance to proteolytic digestion. Transgenetic studies argue that PrPC and PrPSc form a complex during PrPSc formation; thus, synthetic PrP peptides, which mimic the conformational pluralism of PrP, were mixed with PrPC to determine whether its properties were altered. Peptides encompassing two alpha-helical domains of PrP when mixed with PrPC produced a complex that displayed many properties of PrPSc. The PrPC-peptide complex formed fibrous aggregates and up to 65% of complexed PrPC sedimented at 100,000 x g for 1 h, whereas PrPC alone did not. These complexes were resistant to proteolytic digestion and displayed a high beta-sheet content. Unexpectedly, the peptide in a beta-sheet conformation did not form the complex, whereas the random coil did. Addition of 2% Sarkosyl disrupted the complex and rendered PrPC sensitive to protease digestion. While the pathogenic A117V mutation increased the efficacy of complex formation, anti-PrP monoclonal antibody prevented interaction between PrPC and peptides. Our findings in concert with transgenetic investigations argue that PrPC interacts with PrPSc through a domain that contains the first two putative alpha-helices. Whether PrPC-peptide complexes possess prion infectivity as determined by bioassays remains to be established.

Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity.

To explore the possible involvement of STAT factors ("signal transducers and activators of transcription") in the interleukin 2 receptor (IL-2R) signaling cascade, murine HT-2 cells expressing chimeric receptors composed of the extracellular domain of the erythropoietin receptor fused to the cytoplasmic domains of the IL-2R beta or -gamma c chains were prepared. Erythropoietin or IL-2 activation of these cells resulted in rapid nuclear expression of a DNA-binding activity that reacted with select STAT response elements. Based on reactivity with specific anti-STAT antibodies, this DNA-binding activity was identified as a murine homologue of STAT-5. Induction of nuclear expression of this STAT-5-like factor was blocked by the addition of herbimycin A, a tyrosine kinase inhibitor, but not by rapamycin, an immunophilin-binding antagonist of IL-2-induced proliferation. The IL-2R beta chain appeared critical for IL-2-induced activation of STAT-5, since a mutant beta chain lacking all cytoplasmic tyrosine residues was incapable of inducing this DNA binding. In contrast, a gamma c mutant lacking all of its cytoplasmic tyrosine residues proved fully competent for the induction of STAT-5. Physical binding of STAT-5 to functionally important tyrosine residues within IL-2R beta was supported by the finding that phosphorylated, but not nonphosphorylated, peptides corresponding to sequences spanning Y392 and Y510 of the IL-2R beta tail specifically inhibited STAT-5 DNA binding.

Periodicity of polar and nonpolar amino acids is the major determinant of secondary structure in self-assembling oligomeric peptides.

The tendency of a polypeptide chain to form alpha-helical or beta-strand secondary structure depends upon local and nonlocal effects. Local effects reflect the intrinsic propensities of the amino acid residues for particular secondary structures, while nonlocal effects reflect the positioning of the individual residues in the context of the entire amino acid sequence. In particular, the periodicity of polar and nonpolar residues specifies whether a given sequence is consistent with amphiphilic alpha-helices or beta-strands. The importance of intrinsic propensities was compared to that of polar/nonpolar periodicity by a direct competition. Synthetic peptides were designed using residues with intrinsic propensities that favored one or the other type of secondary structure. The polar/nonpolar periodicities of the peptides were designed either to be consistent with the secondary structure favored by the intrinsic propensities of the component residues or in other cases to oppose these intrinsic propensities. Characterization of the synthetic peptides demonstrated that in all cases the observed secondary structure correlates with the periodicity of the peptide sequence--even when this secondary structure differs from that predicted from the intrinsic propensities of the component amino acids. The observed secondary structures are concentration dependent, indicating that oligomerization of the amphiphilic peptides is responsible for the observed secondary structures. Thus, for self-assembling oligomeric peptides, the polar/nonpolar periodicity can overwhelm the intrinsic propensities of the amino acid residues and serves as the major determinant of peptide secondary structure.

Early Alzheimer disease-like histopathology increases in frequency with age in mice transgenic for beta-APP751.

beta-Amyloid deposition and neurofibrillary tangle formation are two histopathological features of Alzheimer disease. We have previously reported that beta-amyloid immunoreactive deposits form in the brains of transgenic mice programmed for neuronal expression of the 751-amino acid isoform of human beta-amyloid precursor protein (beta-APP751) and now describe that these animals also display Alz50 intraneuronal immunoreactivity similar to that seen in early Alzheimer disease. This suggests that abnormal beta-APP expression and/or beta-amyloid deposition promotes pathogenic alterations in tau protein. The frequency of both beta-amyloid deposition and Alz50-positive neurons was twice as prevalent in brains from old (22 months) as compared to young (2-3 months) beta-APP751 transgenic mice. This increase in histopathology with age in beta-APP751 transgenic mice parallels the time-dependent progression seen in the human disease.