Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.     

Deoxycholic acid induces intracellular signaling through membrane perturbations

Secondary bile acids have long been postulated to be tumor promoters in the colon; however, their mechanism of action remains unclear. In this study, we examined the actions of bile acids at the cell membrane and found that they can perturb membrane structure by alteration of membrane microdomains. Depletion of membrane cholesterol by treating with methyl-beta-cyclodextrin suppressed deoxycholic acid (DCA)-induced apoptosis, and staining for cholesterol with filipin showed that DCA caused a marked rearrangement of this lipid in the membrane. Likewise, DCA was found to affect membrane distribution of caveolin-1, a marker protein that is enriched in caveolae membrane microdomains. Additionally, fluorescence anisotropy revealed that DCA causes a decrease in membrane fluidity consistent with the increase in membrane cholesterol content observed after 4 h of DCA treatment of HCT116 cells. Significantly, by using radiolabeled bile acids, we found that bile acids are able to interact with and localize to microdomains differently depending on their physicochemical properties. DCA was also found to induce tyrosine phosphorylation and activate the receptor tyrosine kinase epidermal growth factor receptor in a ligand-independent manner. In contrast, ursodeoxycholic acid did not exhibit any of these effects even though it interacted significantly with the microdomains. Collectively, these data suggest that bile acid-induced signaling is initiated through alterations of the plasma membrane structure and the redistribution of cholesterol.     

Antibody responses to mycobacterial and self heat shock protein 65 in autoimmune arthritis: epitope specificity and implication in pathogenesis

Many autoimmune diseases are believed to involve primarily T cell-mediated effector mechanisms. There is increasing realization, however, that Abs may also play a vital role in the propagation of T cell-driven disorders. In this study, on the rat adjuvant-induced arthritis (AA) model of human rheumatoid arthritis, we examined the characteristics of serum Ab response to mycobacterial heat shock protein (hsp) 65 (Bhsp65), self (rat) hsp65 (Rhsp65), and linear peptides spanning these two molecules. The AA-resistant WKY (RT.1(l)) rat responded to the heat-killed Mycobacterium tuberculosis immunization with a rapid burst of Abs to both Bhsp65 and Rhsp65. These Abs reacted with numerous peptide epitopes; however, this response was reduced to a few epitopes with time. On the contrary, the susceptible Lewis (RT.1(l)) rat developed a relatively lower Ab response to Bhsp65, and Abs to Rhsp65 did not appear until the recovery from the disease. The Ab response in Lewis rats diversified with progression of AA, and there was an intriguing overlap between the repertoire of Bhsp65-reactive B and T cells during the recovery phase of AA. Nonetheless, subsets of the repertoire of the late Abs in both rat strains became focused on the same epitope regions of Bhsp65 and Rhsp65. The functional relevance of these Abs was evident from the results showing that sera from recovery phase Lewis or WKY rats, but not that of naive rats, afforded protection against subsequent AA. These results are of significance in further understanding of the role of humoral immunity in the pathogenesis of autoimmune arthritis.     

The novel cytokine p43 induces IL-12 production in macrophages via NF-kappaB activation, leading to enhanced IFN-gamma production in CD4+ T cells

Recently, we determined that p43, an auxiliary factor of mammalian multiaminoacyl-tRNA synthetases, is secreted, and functions as a novel pleiotropic cytokine. In this study, we have attempted to characterize the effects of p43 on the generation of IL-12 in mouse macrophages. p43 was determined to induce significant IL-12 production from mouse macrophages in a dose-dependent manner. The stimulatory effect of p43 on the activation of IL-12p40 promoter was mapped to a region harboring an NF-kappaB binding site. The nuclear extracts from the p43-stimulated macrophages exhibited profound NF-kappaB DNA-binding activity, as determined by the EMSA. In addition, the p43-stimulated IL-12 induction and NF-kappaB DNA-binding activity were significantly suppressed by caffeic acid phenethyl ester and BAY11-7082, both inhibitors of NF-kappaB activation, indicating that p43 induced the production of IL-12 in macrophages mainly via the activation of NF-kappaB. Importantly, p43 increased the level of IFN-gamma production in the Ag-primed lymph node cells, but had no effect on IL-4 levels. The addition of a neutralizing anti-IL-12p40 mAb to the cell cultures resulted in a decrease of the production of p43-enhanced IFN-gamma by the keyhole limpet hemocyanin-primed lymph node cells. Furthermore, coincubation with p43-pretreated macrophages enhanced the production of IFN-gamma by the keyhole limpet hemocyanin-primed CD4+ T cells, thereby indicating that p43 may enhance IFN-gamma expression in CD4+ T cells via the induction of IL-12 production in macrophages. These results indicate that p43 may play an essential role in the development of the Th1 immune responses associated with cancer immunotherapy and protective immunity against intracellular pathogens.     

RecJ exonuclease: substrates, products and interaction with SSB

The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5′–3′ direction and participates in homologous recombination and mismatch repair. The experiments described here address RecJ's substrate requirements and reaction products. RecJ complexes on a variety of 5′ single-strand tailed substrates were analyzed by electrophoretic mobility shift in the absence of Mg²⁺ ion required for substrate degradation. RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg²⁺ confirmed that substrates with 5′ tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading ~1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease was equally potent on 5′ phosphorylated and unphosphorylated ends. Finally, DNA binding and nuclease activity of RecJ was specifically enhanced by the pre-addition of ssDNA-binding protein and we propose that this specific interaction may aid recruitment of RecJ.     

Src SH2 arginine 175 is required for cell motility: specific focal adhesion kinase targeting and focal adhesion assembly function

Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.