Mark A. Jarosinski

Parkinson's Disease-associated α-Synuclein Is More Fibrillogenic than β- and γ-Synuclein and Cannot Cross-seed Its Homologs

Parkinsons disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in α-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes β- and γ-synuclein. α-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for β- or γ-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between α- and β-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of α-, β-, and γ- synuclein show the same natively unfolded structure. While over time α-synuclein forms the previously described fibrils, no fibrils could be detected for β- and γ-synuclein under the same conditions. Most importantly, β- and γ-synuclein could not be cross-seeded with α-synuclein fibrils. However, under conditions that drastically accelerate aggregation, γ-synuclein can form fibrils with a lag phase roughly three times longer than α-synuclein. These results indicate that β- and γ-synuclein are intrinsically less fibrillogenic than α-synuclein and cannot form mixed fibrils with α-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to α-synuclein and are also abundant in brain.

Structure of a human DNA repair protein UBA domain that interacts with HIV-1 Vpr.

The HIV-1 protein Vpr is critical for a number of viral functions including a unique ability to arrest T-cells at a G2/M checkpoint and induce subsequent apoptosis. It has been shown to interact specifically with the second UBA (ubiquitin associated) domain found in the DNA repair protein HHR23A, a highly evolutionarily conserved protein. This domain is a commonly occurring sequence motif in some members of the ubiquitination pathway, UV excision repair proteins, and certain protein kinases. The three dimensional structure of the UBA domain, determined by NMR spectroscopy, is presented. The protein domain forms a compact three-helix bundle. One side of the protein has a hydrophobic surface that is the most likely Vpr target site.

ENHANCED RESOLUTION IN MAS NMR FOR COMBINATORIAL CHEMISTRY

Abstract We have demonstrated the ability to obtain high resolution NMR spectra from samples on resin using J-resolved 2D NMR. Projection of the non-tilted spectrum onto the chemical shift dimension provides enhanced resolution and virtually complete disappearance of the polymer resonances.

Proteinase-activated receptor-2 mediates hyperresponsiveness in isolated guinea pig bronchi.

The mast cell serine protease tryptase has been implicated as a critical mediator of airway hyperresponsiveness in vitro and in vivo. We have previously demonstrated that tryptase promotes hyperresponsiveness in isolated guinea pig bronchi. In this study, we have investigated the potential role of tryptase-mediated activation of proteinase-activated receptor-2 (PAR-2) in promoting airway hyperresponsiveness. Ex vivo exposure of guinea pig bronchi to the PAR-2 agonists H(2)N-Ser-Leu-Ile-Gly-Arg-Leu-CONH(2) (SLIGRL) and t-cinnamoyl-H(2)N-Leu-Ile-Gly-Arg-Leu-O-CONH(2) (t-c-LIGRLO) (0.1-10 microM) induced a concentration-dependent increase of contractile response to histamine. Treatment with 10 microM SLIGRL or t-c LIGRLO for 45 min increased subsequent responsiveness to histamine (0.3mM) by 54+/-3% and 69+/-5%, respectively (P<0.05 vs. control). In contrast, the PAR-1 agonist peptide H(2)N-Ser-Phe-Leu-Leu-Arg-Asn-CONH(2) (SFLLRN) did not promote significant changes in the airway. Effects of the peptides were observed following at least a 30-min preincubation with the tissue. Coincubation with indomethacin or removal of epithelial cells is required for PAR-2-mediated hyperreactivity. The inactive analogue H(2)N-Leu-Ser-Ile-Gly-Arg-Leu-CONH(2) (LISGRL; 10 microM) failed to promote hyperresponsiveness. Neuropeptide antagonists blocked the effect of the PAR-2 agonists. Selective antagonists of NK1 (L-703,606), NK2 (L-659,877), and CGRP (alphaCGRP 8-37) provided additive inhibition of PAR-2-mediated hyperreactivity. Pretreatment of bronchi with capsaicin (0.8 microM) also prevented the effects of SLIGRL. These results demonstrate the potential involvement of tryptase-mediated activation of PAR-2 in promoting airway hyperresponsiveness. These results further demonstrate that the PAR-2-mediated response involves a neurogenic mechanism involving neuropeptide release.

Design of a new peptidomimetic agonist for the melanocortin receptors based on the solution structure of the peptide ligand, Ac-Nle-cyclo[Asp-Pro-dPhe-Arg-Trp-Lys]-NH2

The solution structure of a potent melanocortin receptor agonist, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2) (1) was calculated using distance restraints determined from 1H NMR spectroscopy. Eight of the lowest energy conformations from this study were used to identify non-peptide cores that mimic the spatial arrangement of the critical tripeptide region, DPhe-Arg-Trp, found in 1. From these studies, compound 2a, containing the cis-cyclohexyl core, was identified as a functional agonist of the melanocortin-4 receptor (MC4R) with an IC(50) and EC(50) below 10 nM. Compound 2a also showed 36- and 7-fold selectivity over MC3R and MC1R, respectively, in the binding assays. Subtle changes in cyclohexane stereochemistry and removal of functional groups led to analogues with lower affinity for the MC receptors.

Solid-phase synthesis via 5-oxazolidinones. Ring opening reactions with amines and reaction monitoring by single bead FT-IR microspectroscopy

Until recently, large compound libraries produced by solid-phase synthesis have been restricted to those of linear peptides and oligonucleotides, but the need for development and synthesis of libraries of small organic molecules using this method is growing rapidly.1 We are interested in developing strategies and chemistries that would allow entry to combinatorial libraries of peptidomimetic structures on solid support. Our approach to the synthesis of peptide mimetics involves attachment of an essential amino acid residue via its side-chain functionality to the solid support. A similar strategy has been employed by Ellman in creating a peptidomimetic library using the hydroxyethylamino isostere of phenylalanine found in most HIV protease inhibitors anchored via the secondary alcohol.2 Anchoring the amino acid side chain to polymeric supports is now widely used to prepare head-to-tail cyclic peptides via solid-phase-mediated cyclization.3 Typically, Asp or Glu are attached via their ω-carboxyls to hydroxymethyl or aminomethyl resins, with the expectation that the corresponding peptides containing Asp/Glu or Asn/Gln, respectively, will be obtained after final cleavage from the resin. The most widely practiced strategy to obtain head-to-tail cyclic peptides uses the three-dimensional Fmoc/t-Bu/allyl orthogonal protection scheme. Kates et al. developed the use of R-allyl-protected aspartic acid for automated continuous flow synthesis of cyclic peptides containing Asp or Asn residues via side-chain anchoring to the resin followed by on-resin cyclization,4 which is applicable to Gluand Gln-containing sequences. Also, the Fmoc/allyl ester protection scheme was applied to anchor lysine via its -amino group.3e Additionally, side-chain attachment to Merrifield (chloromethylated polystyrene) resin has been applied to cysteine in its unprotected form; this has allowed both Nand C-terminal derivatization.5 In the latter case, C-terminal derivatization to amides and esters required carboxylic acid activation with BOP reagent.

Preparation of phosphohistidyl peptides via oxidative coupling of H-phosphonates

Post-translational phosphorylation of proteins is an important event in signal transduction. Extensive research has focused on Ser, Thr, and Tyr phosphorylation events. This research has been facilitated by the availability of efficient synthetic methods to prepare peptides carrying O-phosphorylated residues. N-Phosphorylation events on His, Lys, & Arg residues are of current interest, because of their emerging role in signal transduction. However, efficient synthetic methods to prepare peptide reagents carrying N-phosphorylated residues are less developed. Here we report an expedient strategy to obtain N-phosphohistidyl peptides via oxidative coupling to an appropriately protected H-phosphonate.

SAR of the novel neuropeptides orexin-A and B

Detailed functional evaluation and structure analysis of these Ox-A and Ox-B analogs are currently underway. Future analog design aims to identify peptides with greater selectivity ratios at the Ox-1R and Ox-2R receptors. We are also interested in identifying antagonists molecules to aid in understanding the novel pharmacologyassociated with these new receptors.