Detection and identification of virulence factors in Yersinia pestis using SELDI ProteinChip system.

A rapid method for the detection, purification, and identification of proteins in bacterial extracts was developed using surface enhanced laser desorption/ionization (SELDI) ProteinChip technology. The effectiveness of this technique for monitoring the expression and identification of temperature- and calcium-regulated virulence factors of Yersinia pestis, the bacterium that causes human plague, is demonstrated. Y. pestis infection of its mammalian host is thought to be accompanied by rapid up-regulation of a number of genes following a shift from 26 degrees C (the temperature of the flea vector) to 37 degrees C (the temperature of the mammalian host). To model this process, Y. pestis cells were grown at 26 degrees C and 37 degrees C in a Ca(2+)-deficient medium. Through an initial protein profiling of the crude bacterial extract on strong anion exchange and copper affinity, ProteinChip arrays detected five proteins that were up-regulated and three proteins that were down-regulated at 37 degrees C. Two of the proteins predominately expressed at 37 degrees C were semi-purified in less than two days. The two proteins were identified as catalase-peroxidase and Antigen 4. Aside from its speed, a salient feature of the SELDI technique is the microgram amounts of crude sample required for analysis.

cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome.

The primary structure of PI31, a protein inhibitor of the 20 S proteasome, was deduced by cDNA cloning and sequencing. The human protein has a calculated molecular weight of 29,792, a value in excellent accord with 31,000, as estimated by SDS-polyacrylamide gel electrophoresis for purified bovine PI31, and is not similar to any other protein in current data bases. PI31 is a proline-rich protein, particularly within its carboxyl-terminal half where 26% of the amino acids are proline. Wild-type PI31 and various truncation mutants were expressed in Escherichia coli and purified to homogeneity. Recombinant wild-type PI31 displayed structural and functional properties similar to those of PI31 purified from bovine red blood cells and inhibited the hydrolysis of protein and peptide substrates by the 20 S proteasome. Analysis of truncation mutants demonstrated that proteasome inhibition was conferred by the carboxyl-terminal proline-rich domain of PI31, which appears to have an extended secondary structure. Inhibition of the 20 S proteasome by PI31 involved formation a proteasome-PI31 complex. In addition to its direct inhibition of the 20 S proteasome, PI31 inhibited the activation of the proteasome by each of two proteasome regulatory proteins, PA700 and PA28. These results suggest that PI31 plays an important role in control of proteasome function, including that in ubiquitin-dependent pathways of protein degradation.

Domain mapping of the DNA binding, endonuclease, and ERCC1 binding properties of the human DNA repair protein XPF.

During nucleotide excision repair, one of the two incisions necessary for removal of a broad spectrum of DNA adducts is made by the human XPF/ERCC1 protein complex. To characterize the biochemical function of XPF, we have expressed and purified the independent 104 kDa recombinant XPF protein from E. coli and determined that it is an endonuclease and can bind DNA in the absence of the ERCC1 subunit. Endonuclease activity was also identified in a stable 70 kDa proteolysis fragment of XPF obtained during protein expression, indicating an N-terminal catalytic domain. Sequence homology and secondary structure predictions indicated a second functional domain at the C-terminus of XPF. To investigate the significance of the two predicted domains, a series of XPF deletion fragments spanning the entire protein were designed and examined for DNA binding, endonuclease activity, and ERCC1 subunit binding. Our results indicate that the N-terminal 378 amino acids of XPF are capable of binding and hydrolyzing DNA, while the C-terminal 214 residues are capable of binding specifically to ERCC1. We propose that the N-terminal domain of XPF contributes to the junction-specific endonuclease activity observed during DNA repair and recombination events. In addition, evidence presented here suggests that the C-terminal domain of XPF is responsible for XPF/ERCC1 complex formation. A working model for the XPF protein is presented illustrating the function of XPF in the nucleotide excision pathway and depicting the two functional domains interacting with DNA and ERCC1.

Characterization of the mouse Xpf DNA repair gene and differential expression during spermatogenesis.

The human XPF protein, an endonuclease subunit essential for DNA excision repair, may also function in homologous recombination. To investigate a possible link between mammalian XPF and recombination that occurs during meiosis, we isolated, characterized, and determined an expression profile for the mouse Xpf gene. The predicted mouse XPF protein, encoded by a 3.4-kb cDNA, contains 917 amino acids and is 86% identical to human XPF. Appreciable similarity also exists between mouse XPF and homologous proteins in budding yeast (Rad1), fission yeast (Rad16), and fruit fly (Mei-9), all of which have dual functions in excision repair and recombination. Sequence analysis of the 38.3-kb Xpf gene, localized to a region in proximal mouse chromosome 16, revealed greater than 72% identity to human XPF in 16 regions. Of these conserved elements, 11 were exons and 5 were noncoding sequence within introns. Xpf transcript and protein levels were specifically elevated in adult mouse testis. Moreover, increased levels of Xpf and Ercc1 mRNAs correlated with meiotic and early postmeiotic spermatogenic cells. These results support a distinct role for the XPF/ERCC1 junction-specific endonuclease during meiosis, most likely in the resolution of heteroduplex intermediates that arise during recombination.