Isolation of antagonists of antigen-specific autoimmune T cell proliferation.

Antigen-specific T cells play a major role in mediating the pathogenesis of a variety of autoimmune conditions as well as other diseases. In the context of experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis, we present here a general approach to the discovery of highly specific ligands for autoreactive cells. These ligands are obtained from a combinatorial library of hundreds of thousands of synthetic peptoids that is screened simultaneously against two populations of CD4+ T cells.

The hydrophobic patch of ubiquitin is required to protect transactivator-promoter complexes from destabilization by the proteasomal ATPases.

Mono-ubiquitylation of a transactivator is known to promote transcriptional activation of certain transactivator proteins. For the Sacchromyces cerevisiae transactivator, GAL4, attachment of mono-ubiquitin prevents destabilization of the DNA-transactivator complex by the ATPases of the 26S proteasome. This inhibition of destabilization depends on the arrangement of ubiquitin; a chain of ubiquitin tetramers linked through lysine 48 did not display the same protective effect as mono-ubiquitin.

Physical and functional interactions of monoubiquitylated transactivators with the proteasome.

Destabilization of activator-DNA complexes by the proteasomal ATPases can inhibit transcription by limiting activator interaction with DNA. Modification of the activator by monoubiquitylation protects the activator from this destabilization activity. In this study, we probe the mechanism of this protective effect of monoubiquitylation.

Activation domain-dependent monoubiquitylation of Gal4 protein is essential for promoter binding in vivo.

The Saccharomyces cerevisiae Gal4 protein is a paradigmatic transcriptional activator containing a C-terminal acidic activation domain (AD) of 34 amino acids. A mutation that results in the truncation of about two-thirds of the Gal4AD (gal4D) results in a crippled protein with only 3% the activity of the wild-type activator. We show here that although the Gal4D protein is not intrinsically deficient in DNA binding, it is nonetheless unable to stably occupy GAL promoters in vivo.

Rapid identification of the pharmacophore in a peptoid inhibitor of the proteasome regulatory particle.

Here we report a simple and effective method to identify the minimal pharmacophore in the first peptoid inhibitor of the 19S proteasome regulatory particle, which has led to the development of a derivative that exhibits improved cellular activity, presumably due to a reduction in mass of about two-fold and the elimination of positively charged lysine-like residues.

Periodate-triggered cross-linking reveals Sug2/Rpt4 as the molecular target of a peptoid inhibitor of the 19S proteasome regulatory particle.

This study describes the identification of the protein target of the first chemical inhibitor (RIP-1) of the 19S regulatory particle (RP) of the 26S proteasome. Periodate-triggered chemical cross-linking of DOPA-conjugated RIP-1 and the 26S proteasome identified Sug2/Rpt4, one of the six ATPases in the 19S RP as the molecular target of RIP-1. The specificity of RIP-1 for Sug2/Rpt4 was demonstrated by examining cross-linking reactions with each ATPase of the 19S RP.

Label transfer chemistry for the characterization of protein-protein interactions.

A new label transfer method is presented that overcomes most of the limitations of current systems. A protein of interest is tagged with tetra-cysteine sequence (FlAsH Receptor Peptide (FRP)) that binds tightly and specifically to a chimeric molecule 3,4-dihydroxyphenylalanine – biotin – 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (DOPA-biotin-FlAsH). Upon brief periodate oxidation, the DOPA moiety is cross-linked to nearby surface-exposed nucleophiles.

Identification of a peptoid inhibitor of the proteasome 19S regulatory particle.

The first chemical inhibitor of the 19S Regulatory Particle (RP) of the proteasome is described. The molecule was identified by screening a library of nucleoside-capped peptoids for binding to the yeast 26S proteasome in a crude extract. The hit was resynthesized and shown to block 19S RP-mediated protein unfolding in vitro and proteasome-mediated turnover of p27 in HeLa cells.

Identification of Gal4 activation domain-binding proteins in the 26S proteasome by periodate-triggered cross-linking.

A common occurrence in biology is that a regulatory peptide, protein, or small molecule regulates the activity of a large multi-protein complex through direct interactions with a protein(s) in that complex. To characterize the direct receptor of the regulatory molecule, one would ideally like to study the native system. We report here that periodate-triggered cross-linking of catechol-containing regulatory factors, followed by two-dimensional electrophoresis and Western blotting, is an effective method for the characterization of regulatory factor--protein interactions in the context of large multi-protein complexes.