Molecular heterogeneity of a type III cytotoxin, Pseudomonas aeruginosa exoenzyme S

Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin. The N-terminus (residues 1-232) is a Rho GTPase activating protein (GAP) domain, while the C-terminus (residues 233-453) is a FAS-dependent ADP-ribosyltransferase domain that targets Ras and Ras-like GTPases. A membrane localization domain (residues 51-72) localizes ExoS to a perinuclear region within eukaryotic cells. Recent studies observed that ExoS is auto-ADP-ribosylated upon delivery into eukaryotic cells. Auto-ADP-ribosylated ExoS analyzed from eukaryotic cells displayed pI heterogeneity and prompted an analysis of this heterogeneity. Bacterial-associated ExoS and ExoS that had been secreted by P. aeruginosa also showed pI heterogeneity with five charge forms ranging in pI from 5.1 to 5.9. The pI heterogeneity of ExoS was independent of a mass change and thus represented molecular charge conformers. Urea was not required to observe the pI conformers of ExoS; it enhanced the resolution and formation of pI conformers during the focusing component of the analysis. ExoS(E381D), a mutant deficient in ADP-ribosyltransferase activity, isolated from cultured cells showed charge forms that migrated to a more acidic pI than type III secreted ExoS but more basic than auto-ADP-ribosylated ExoS. Incubation of cell lysates with Mn(2+) shifted the pI of ExoS(E381D) to a pI identical to secreted ExoS. This indicates that within the mammalian cells ExoS undergoes a negatively charged modification, in addition to auto-ADP-ribosylation observed for wild-type ExoS. ExoT, ExoU, and YopE also focus into multiple pI forms, suggesting that this is a common property of type III cytotoxins.     

Memory and cellular immunity induced by a DNA vaccine encoding self antigen TPD52 administered with soluble GM-CSF

Tumor protein D52 (TPD52) is involved in cellular transformation, proliferation and metastasis. TPD52 over expression has been demonstrated in several cancers including prostate, breast, and ovarian carcinomas. Murine TPD52 (mD52) has been shown to induce anchorage independent growth in vitro and metastasis in vivo, and mirrors the function and normal tissue expression patterns of the human orthologue of TPD52. We believe TPD52 represents a self, non-mutated tumor associated antigen (TAA) important for maintaining a transformed and metastatic cellular phenotype. The transgenic adeno-carcinoma of the mouse prostate (TRAMP) model was employed to study mD52 as a vaccine antigen. Naïve mice were immunized with either recombinant mD52 protein or plasmid DNA encoding the full-length cDNA of mD52. Following immunization, mice were challenged with a subcutaneous, tumorigenic dose of mD52 positive, autochthonous TRAMP-C1 tumor cells. Sixty percent of mice were tumor free 85 days post challenge with TRAMP-C1 when immunized with mD52 as a DNA-based vaccine admixed with soluble granulocyte-macrophage colony stimulating factor (GM-CSF). Survivors of the initial tumor challenge rejected a second tumor challenge given in the opposite flank approximately 150 days after the first challenge, and remained tumor free for more than an additional 100 days. The T cell cytokine secretion patterns from tumor challenge survivors indicated that a T_H1-type cellular immune response was involved in tumor protection. These data suggest that mD52 vaccination induced a memory, cellular immune response that resulted in protection from murine prostate tumors that naturally over express mD52 protein.     

Altered EGFR localization and degradation in human breast cancer cells with an amphiregulin/EGFR autocrine loop

The epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AR) have been shown to be co-over expressed in breast cancer. We have previously shown that an AR/EGFR autocrine loop is required for SUM149 human breast cancer cell proliferation, motility and invasion. We also demonstrated that AR can induce these altered phenotypes when expressed in the normal mammary epithelial cell line MCF10A, or by exposure of these cells to AR in the medium. In the present studies, we demonstrate that SUM149 cells and immortalized human mammary epithelial MCF10A cells that over express AR (MCF10A AR) or are cultured in the presence of exogenous AR, express higher levels of EGFR protein than MCF10A cells cultured in EGF. Pulse-chase analysis showed that EGFR protein remained stable in the presence of AR, yet was degraded in the presence of EGF. Consistent with this observation, tyrosine 1045 on the EGFR, the c-cbl binding site, exhibited less phosphorylation following stimulation with AR than following stimulation with EGF. Ubiquitination of the receptor was also dramatically less following stimulation with AR than following stimulation with EGF. Flow cytometry analysis showed that EGFR remained on the cell surface following stimulation with AR but was rapidly internalized following stimulation with EGF. Immunofluorescence and confocal microscopy confirmed the flow cytometry results. EGFR in MCF10A cells cultured in the presence of EGF exhibited a predominantly intracellular, punctate localization. In stark contrast, SUM149 cells and MCF10A cells growing in the presence of AR expressed EGFR predominantly on the membrane and at cell-cell junctions. We propose that AR alters EGFR internalization and degradation in a way that favors accumulation of EGFR at the cell surface and ultimately leads to changes in EGFR signaling.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Structural and functional differentiation of two clinally distributed glucosephosphate isomerase allelic isozymes from the teleost Fundulus heteroclitus

The teleost Fundulus heteroclitus (L.) possesses two loci, Gpi-A and Gpi-B, for the glycolytic enzyme, glucose-phosphate isomerase (GPI; D- glucose-6-phosphate ketol-isomerase; E.C. 5.3.1.9). The Gpi-B locus is polymorphic in Fundulus, with two common alleles, Gpi-Bb and Gpi-Bc, distributed in a clinal manner in populations along the east coast of North America. Since this clinal distribution is strongly correlated with a temperature gradient, we asked whether the GPI-B2 allozymes were functionally adapted to the thermal environment in which a given phenotype predominated. The two major GPI-B2 allozymes were purified to homogeneity and were characterized as to molecular weight, isoelectric pH, thermal denaturation, and kinetic parameters. Both GPI-Bb2 and GPI- Bc2 allozymes have molecular masses of 110 kD, and they have isoelectric pHs of 6.4 and 6.6, respectively. The GPI-Bb2 allozyme was more stable to thermal denaturation than was the GPI-Bc2 enzyme. Kinetic properties of the allelic isozymes were investigated both as a function of pH and as a function of temperature. At 25 degrees C, over the pH range considered, there were no significant differences between allozymes, either in Km for fructose-6-phosphate or in Ki for 6- phosphogluconate, but apparent Vmax values differed between pH 7.5 and pH 8.5. All steady-state kinetic parameters showed strong temperature dependence, but the allozymes differed only in the Ki for 6- phosphogluconate at temperatures greater than 30 degrees C. On the basis of the observed structural and functional differences alluded to above, the hypothesis that the major allelic isozymes of the Gpi-B locus were functionally equivalent was rejected. However, it is not yet known whether these structural and functional differences have any significance at higher levels of biological organization.