Timothy S. Vincent

Evidence of covalent binding of the dietary flavonoid quercetin to DNA and protein in human intestinal and hepatic cells

Quercetin-rich foods have the potential to prevent human disease. However, knowledge of its biological fate and mechanism of action is limited. This study extends previous observations of the oxidation of quercetin by peroxidases to quinone/quinone methide intermediates and, for the first time, demonstrates covalent binding of [14C]quercetin to macromolecules. This was first demonstrated using horseradish peroxidase and hydrogen peroxide with human liver microsomal protein to trap the intermediates. To extend this observation to the cellular level, human intestinal Caco-2 cells and hepatic Hep G2 cells were incubated for up to 2hr with [14C]quercetin, and cellular DNA and protein were isolated. The cellular uptake of [14C]quercetin was rapid, and the covalent binding of [14C]quercetin to DNA and protein was determined by liquid scintillation spectrometry after extensive purification. Both cell types demonstrated DNA binding with a maximum level of 5-15pmol/mg DNA. The level of covalent binding to protein was considerably higher in both cell types, 75-125pmol/mg protein. To determine potential specificity in the protein binding, Hep G2 cells were treated with [14C]quercetin, and the cell lysate was subjected to SDS-PAGE followed by staining and autoradiography. Several distinct radiolabeled protein bands did not correspond to the major Coomassie blue stained cellular proteins. We propose that this specific binding may mediate part of the antiproliferative and other cellular actions of quercetin.

Drosophila Pin1 prolyl isomerase Dodo is a MAP kinase signal responder during oogenesis

The mammalian cis–trans prolyl isomerase Pin1 and its yeast orthologue Ess1/Ptf1 have been implicated in cell cycle control but a correlation between biochemical and physiological functions has not been established conclusively. Pin1 targets the proline residue carboxy-terminal to the phosphorylated threonine or serine residue, which constitutes part of the phosphorylated mitogen-activated protein kinase (MAPK) site PXpT/SP. Here we show that the Drosophila Pin1 homologue, the Dodo protein, is involved in dorsoventral patterning of the follicular epithelium in the egg chamber. Its function is to facilitate the degradation of transcription factor CF2, which requires, a priori, activated epidermal growth factor receptor–MAPK signalling.

Pseudomonas aeruginosa Exoenzyme S Disrupts Ras-mediated Signal Transduction by Inhibiting Guanine Nucleotide Exchange Factor-catalyzed Nucleotide Exchange

Pseudomonas aeruginosa exoenzyme S double ADP-ribosylates Ras at Arg41 and Arg128. Since Arg41 is adjacent to the switch 1 region of Ras, ADP-ribosylation could interfere with Ras-mediated signal transduction via several mechanisms, including interaction with Raf, or guanine nucleotide exchange factor-stimulated or intrinsic nucleotide exchange. Initial experiments showed that ADP-ribosylated Ras (ADP-r-Ras) and unmodified Ras (Ras) interacted with Raf with equal efficiencies, indicating that ADP-ribosylation did not interfere with Ras-Raf interactions. While ADP-r-Ras and Ras possessed equivalent intrinsic nucleotide exchange rates, guanine nucleotide exchange factor (Cdc25) stimulated the nucleotide exchange of ADP-r-Ras at a 3-fold slower rate than Ras. ADP-r-Ras did not affect the nucleotide exchange of Ras, indicating that the ADP-ribosylation of Ras was not a dominant negative phenotype. Ras-R41K and ADP-r-Ras R41K possessed similar exchange rates as Ras, indicating that ADP-ribosylation at Arg128 did not inhibit Cdc25-stimulated nucleotide exchange. Consistent with the slower nucleotide exchange rate of ADP-r-Ras as compared with Ras, ADP-r-Ras bound its guanine nucleotide exchange factor (Cdc25) less efficiently than Ras in direct binding experiments. Together, these data indicate that ADP-ribosylation of Ras at Arg41 disrupts Ras-Cdc25 interactions, which inhibits the rate-limiting step in Ras signal transduction, the activation of Ras by its guanine nucleotide exchange factor.

Inhibition of Growth-factor Signaling Pathways By Lovastatin

Human fibroblasts treated with the antihypercholesterolaemic drug, lovastatin, displayed a diminished signaling response to epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). Supplementing the culture medium with mevalonic acid restored the signaling response. Not all growth factor signaling pathways were impaired, however, as platelet-derived growth factor (PDGF-BB) and basic fibroblast growth factor (bFGF) responses were refractory to lovastatin treatment. These results suggest the involvement of product(s) of mevalonate metabolism (e.g., prenylated proteins such as p21ras or G proteins) in the signal transduction of EGF, insulin and IGF-I. The inhibition of cell growth by lovastatin may be caused by the inability of the cell to enter the S phase of the cell cycle due to obstruction of the signaling of progression factors.

ADP‐ribosylation of oncogenic Ras proteins by Pseudomonas aeruginosa exoenzyme S in vivo

The exoenzyme S (ExoS)‐producing Pseudomonas aeruginosa strain, 388, and corresponding ExoS knock‐out strain, 388ΔexoS, were used in a bacterial and mammalian co‐culture system as a model for the contact‐dependent delivery of ExoS into host cells. Examination of DNA synthesis and Ras ADP‐ribosylation in tumour cell lines expressing normal and mutant Ras revealed a decrease in DNA synthesis concomitant with ADP‐ribosylation of Ras proteins after exposure to ExoS‐producing bacteria, but not after exposure to non‐ExoS‐producing bacteria. Examination of normal H‐Ras, K‐Ras and N‐Ras by two‐dimensional electrophoresis after exposure to bacteria revealed differences in the degree of ADP‐ribosylation by ExoS, with H‐Ras being modified most extensively. ADP‐ribosylation of oncogenic forms of Ras was examined in vivo using cancer lines expressing mutant forms of H‐, N‐ or K‐Ras. The mutant Ras proteins were modified in a manner qualitatively similar to their normal counterparts. Using Ras/Raf‐1 co‐immunoprecipitation after co‐culture, it was found that exposure to ExoS‐producing bacteria caused a decrease in the amount of Raf‐1 associated with EGF‐activated Ras and oncogenic Ras. The results from this study indicate that ExoS ADP‐ribosylates both normal and mutant Ras proteins in vivo and inhibits signalling through Ras.

Comparison of the exoS Gene and Protein Expression in Soil and Clinical Isolates of Pseudomonas aeruginosa

ABSTRACT Exoenzyme S (ExoS) is translocated into eukaryotic cells by the type III secretory process and has been hypothesized to function in conjunction with other virulence factors in the pathogenesis ofPseudomonas aeruginosa. To gain further understanding of how ExoS might contribute to P. aeruginosa survival and virulence, ExoS expression and the structural gene sequence were determined in P. aeruginosa soil isolates and compared with ExoS of clinical isolates. Significantly higher levels of ExoS ADP-ribosyltransferase (ADPRT) activity were detected in culture supernatants of soil isolates compared to those of clinical isolates. The higher levels of ADPRT activity of soil isolates reflected both the increased production of ExoS and the production of ExoS having a higher specific activity. ExoS structural gene sequence comparisons found the gene to be highly conserved among soil and clinical isolates, with the greatest number of nonsynonymous substitutions occurring within the region of ExoS encoding GAP function. The lack of amino acid changes in the ADPRT region in association with a higher specific activity implies that other factors produced by P. aeruginosa or residues outside the ADPRT region are affecting ExoS ADPRT activity. The data are consistent with ExoS being integral to P. aeruginosa survival in the soil and suggest that, in the transition of P. aeruginosa from the soil to certain clinical settings, the loss of ExoS expression is favored.

Independent and Coordinate Effects of ADP-Ribosyltransferase and GTPase-Activating Activities of Exoenzyme S on HT-29 Epithelial Cell Function

ABSTRACT Type III-mediated translocation of exoenzyme S (ExoS) into HT-29 epithelial cells by Pseudomonas aeruginosa causes complex alterations in cell function, including inhibition of DNA synthesis, altered cytoskeletal structure, loss of readherence, microvillus effacement, and interruption of signal transduction. ExoS is a bifunctional protein having both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) functional domains. Comparisons of alterations in HT-29 cell function caused by P. aeruginosastrains that translocate ExoS having GAP or ADPRT mutations allowed the independent and coordinate functions of the two activities to be assessed. An E381A ADPRT mutation revealed that ExoS ADPRT activity was required for effects of ExoS on DNA synthesis and long-term cell rounding. Conversely, the R146A GAP mutation appeared to have little impact on the cellular effects of ExoS. While transient cell rounding was detected following exposure to the E381A mutant, this rounding was eliminated by an E379A-E381A ADPRT double mutation, implying that residual ADPRT activity, rather than GAP activity, was effecting transient cell rounding by the E381A mutant. To explore this possibility, E381A and R146A-E381A mutants were examined for their ability to ADP-ribosylate Ras in vitro or in vivo. While no ADP-ribosylation of Ras was detected by either mutant in vitro, both mutants were able to modify Ras when translocated by the bacteria, with the R146A-E381A mutant causing more efficient modification than the E381A mutant, in association with increased inhibition of DNA synthesis. Comparisons of Ras ADP-ribosylation by wild-type and E381A mutant ExoS by two-dimensional electrophoresis found the former to ADP-ribosylate Ras at two sites, while the latter modified Ras only once. These studies draw attention to the key role of ExoS ADPRT activity in causing the effects of bacterially translocated ExoS on DNA synthesis and cell rounding. In addition, the studies provide insight into the enhancement of ExoS ADPRT activity within the eukaryotic cell microenvironment and into possible modulatory roles that the GAP and ADPRT domains might have on the function of each other.

Identification of functional epitopes of Pseudomonas aeruginosa exotoxin A using synthetic peptides and subclone products

The structure-function relationship of P. aeruginosa exotoxin A (ETA) was examined using synthetic peptides and genetically engineered ETA deletion mutants. Antibodies directed against synthetic peptides have allowed the identification of three ETA epitopes, two within domain I and one within the last 33 amino acids of domain III. In addition two distinct neutralizing determinants have been identified by antibodies directed against subclone products. One was associated with the amino-terminal half of ETA, the proposed receptor binding region. The second was associated with the carboxy-terminal half of ETA, a region previously not associated with receptor-binding. The amino-terminal subclone also offers potential as an ETA vaccine, since it produces a stable, non-enzymatically active product, effective in inducing ETA neutralizing antibodies. Data derived from these studies were used in a re-evaluation of structure-function relationships between ETA and diphtheria toxin.