No abstract

Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immuneprivileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Mü ller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis. Molecular & Cellular Proteomics 9: 2292-2305, 2010.

A minimalist Cys2His2 zinc finger peptide, Lys-Tyr-Ala-Cys-Ala-Ala-Cys-Ala-Ala-Ala-Phe-Ala-Ala-LysAla-Ala-Leu-Ala-Ala-His-Ala-Ala-Ala-His-Ala-Lys, has been synthesized. Metal binding studies using Co2+ as a probe indicated that this peptide forms a 1:1 peptide/metal complex with a dissociation constant comparable to that observed for other zinc finger peptides. At high peptide concentrations, a 2:1 peptide/metal complex also forms, with four cysteinates coordinated to Co2+. Additional studies with sequence variants in which the canonical hydrophobic residues were changed to alanine, or in which one of the residues between the cysteines and the histidines was deleted, revealed an even more pronounced formation of the 2:1 complex over the 1:1 complex. In addition, the absorption spectra of the 1:1 peptide/Co2+ complexes of the variant peptides are significantly different from those seen for complexes of the parent peptide or those of more typical zinc finger peptides. NMR studies revealed that the parent peptide folds in the presence of Zn2+ to a structure very similar to that observed for other zinc finger peptides of this class. Taken together, these results suggest that the metalbinding and canonical hydrophobic residues are necessary and sufficient to determine the structure of this class of zinc finger peptides.The amino acid sequence of a protein contains the information sufficient to determine its three-dimensional structure (1). A variety of experiments have demonstrated that this information is not distributed uniformly but that a smaller set of residues are crucial for correct structure formation. For example, the elegant work of Kaiser and Kezdy (2) showed that simplified (or minimalist) versions of naturally occurring peptides could be designed and synthesized, retaining many of the structural (and, in many cases, functional) properties of the native materials. This approach has been extended to more complex oligomeric structures by DeGrado et al. (3). The uneven distribution of folding information has also been demonstrated by Sauer and coworkers (4) using combinatorial mutagenesis. Here, a protein sequence is randomized in a number of positions, functional molecules are selected, and their amino acid sequences are determined. It has been found that some positions are quite tolerant ofa wide range ofamino acids whereas others have very stringent restrictions. These observations suggest that a large number of sequences that have only a small subset of residues fixed may fold in functionally equivalent manners. The Cys2His2 zinc finger sequences typified by transcription factor TFIIIA may represent a naturally occurring set of sequences of this type. After the determination of the sequence of a cDNA clone of TFIIIA (5), it was noted that the deduced amino acid sequence contained nine tandem sequences that shared the consensus (Phe,Tyr)-Xaa-Cys-Xaa2,4-Cys-Xaa3-Phe-Xaa5-Leu-Xaa2-His-Xaa3,4-His-Xaa2-6 where Xaa represents relatively variable amino acids (6,7). Subsequently, a large number of other ...

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites.

Nup475 is a nuclear zinc-binding protein of unknown function that is induced in mammalian cells by growth factor mitogens. For structural studies by nuclear magnetic resonance spectroscopy, we used a more soluble two-domain peptide that had a single amino acid substitution in a nonconserved amino acid residue in the second Cys 3 His repeat. The mutant peptide unexpectedly showed loss of one of its metal binding sites and displayed ordered structure for only the first Cys 3 His sequence. On the basis of the nuclear magnetic resonance data, we propose a structure for the Nup475 metal-binding domain in which the zinc ion is coordinated by the conserved cysteines and histidine, and the conserved YKTEL motif forms a parallel sheet-like structure with the C terminus of this domain. This structure is unlike that of any previously described class of metal binding domain.