Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

Transdominant genetic analysis of a growth control pathway

Genetic selections that use proteinaceous transdominant inhibitors encoded by DNA libraries to cause mutant phenocopies may facilitate genetic analysis in traditionally nongenetic organisms. We performed a selection for random short peptides and larger protein fragments (collectively termed “perturbagens”) that inhibit the yeast pheromone response pathway. Peptide and protein fragment perturbagens that permit cell division in the presence of pheromone were recovered. Two perturbagens were derived from proteins required for pheromone response, and an additional two were derived from proteins that may negatively influence the pheromone response pathway. Furthermore, three known components of the pathway were identified as probable perturbagen targets based on physical interaction assays. Thus, by selection for transdominant inhibitors of pheromone response, multiple pathway components were identified either directly as gene fragments or indirectly as the likely targets of specific perturbagens. These results, combined with the results of previous work [Holzmayer, T. A., Pestov, D. G. & Roninson, I. B. (1992) Nucl. Acids. Res. 20, 711–717; Whiteway, M., Dignard, D. & Thomas, D. Y. (1992) Proc. Natl. Acad. Sci. USA 89, 9410–9414; and Gudkov, A. V., Kazarov, A. R., Thimmapaya, R., Axenovich, S. A., Mazo, I. A. & Roninson, I. B. (1994) Proc. Natl. Acad. Sci. USA 91, 3744–3748], suggest that transdominant genetic analysis of the type described here will be broadly applicable.

Dynamical principles in biological processes: A model of charge migration in proteins and DNA

The generalized master equations (GMEs) that contain multiple time scales have been derived quantum mechanically. The GME method has then been applied to a model of charge migration in proteins that invokes the hole hopping between local amino acid sites driven by the torsional motions of the floppy backbones. This model is then applied to analyze the experimental results for sequence-dependent long-range hole transport in DNA reported by Meggers et al. [Meggers, E., Michel-Beyerle, M. E., & Giese, B. (1998) J. Am. Chem. Soc. 120, 12950–12955]. The model has also been applied to analyze the experimental results of femtosecond dynamics of DNA-mediated electron transfer reported by Zewail and co-workers [Wan, C., Fiebig, T., Kelley, S. O., Treadway, C. R., Barton, J. K. & Zewail, A. H. (1999) Proc. Natl. Acad. Sci. USA 96, 6014–6019]. The initial events in the dynamics of protein folding have begun to attract attention. The GME obtained in this paper will be applicable to this problem.

Effects of inefficient cleavage of the signal sequence of HIV-1 gp 120 on its association with calnexin, folding, and intracellular transport.

The HIV-1 envelope glycoprotein gp120 displays inefficient intracellular transport, which is caused by its retention in the endoplasmic reticulum. Coexpression in insect cells (Sf9) of HIV-1 gp120 with calnexin has shown that their interaction was modulated by the signal sequence of HIV-1 gp120. gp120, with its natural signal sequence, showed a prolonged association with calnexin with a t1/2 of greater than 20 min. Replacement of the natural signal sequence with the signal sequence from mellitin led to a decreased time of association of gp120 with calnexin (t1/2 < 10 min). These different times of calnexin association coincided both with the folding of gp120 as measured by the ability of bind CD4 and with endoplasmic reticulum to Golgi transport as analyzed by the acquisition of partial endoglycosidase H resistance. Using a monospecific antibody to the HIV-1 gp120 natural signal peptide, we showed that calnexin associated with N-glycosylated but uncleaved gp120. Only after dissociation from calnexin was gp120 cleaved, but very inefficiently. Only the small proportion of signal-cleaved gp120 molecules acquired transport competence and were secreted. This is the first report demonstrating the effect of the signal sequence on calnexin association.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Differential effects of staphylococcal toxic shock syndrome toxin-1 on B cell apoptosis.

Superantigens, such as toxic shock syndrome toxin 1 (TSST-1), have been implicated in the pathogenesis of several autoimmune and allergic diseases associated with polyclonal B cell activation. In this report, we studied the in vitro effects of TSST-1 on B cell activation. We show herein that TSST-1 produced antagonistic effects on Ig synthesis by peripheral blood mononuclear cells (PBMC) from normal subjects, depending on the concentration used; Ig production was inhibited at 1000 pg/ml (P < 0.01) and enhanced at 1 and 0.01 pg/ml (P < 0.01) of toxin. Cultures of PBMC were then examined for morphologic features and DNA fragmentation characteristic for apoptosis. B cells exhibited a significantly higher (P < 0.01) incidence of apoptosis after stimulation with 1000 pg/ml of TSST-1 compared with 1 or 0.01 pg/ml of toxin or medium alone. Abundant expression of Fas, a cell surface protein that mediates apoptosis, was detected on B cells after stimulation with 1000 pg/ml of TSST-1 and was significantly higher on B cells undergoing apoptosis than on live cells (P = 0.01). Additionally, increased Fas expression and B cell death occurred at concentrations of TSST-1 inducing the production of high amounts of gamma interferon (IFN-gamma), and both events could be blocked by neutralizing anti-IFN-gamma antibody. These findings suggest that high concentrations of TSST-1 can induce IFN-gamma-dependent B cell apoptosis, whereas at low concentrations it stimulates Ig synthesis by PBMC from normal subjects. These findings support the concept that staphylococcal toxins have a role in B cell hyperactivity in autoimmunity and allergy.

Nuclear/cytoplasmic localization of the von Hippel-Lindau tumor suppressor gene product is determined by cell density.

The product of the von Hippel-Lindau (VHL) tumor suppressor gene, the gene inactivated in VHL disease and in sporadic clear-cell renal carcinomas, has recently been shown to have as a functional target the transcription elongation complex, elongin (also called SIII). Here it is shown that there is a tightly regulated, cell-density-dependent transport of VHL into and/or out of the nucleus. In densely grown cells, the VHL protein is predominantly in the cytoplasm, whereas in sparse cultures, most of the protein can be detected in the nucleus. We have identified a putative nuclear localization signal in the first 60 and first 28 amino acids of the human and rat VHL protein, respectively. Sequences in the C-terminal region of the VHL protein may also be required for localization to the cytosol. These findings provide the initial indication of a novel cell density-dependent pathway that is responsible for the regulation of VHL cellular localization.

Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss.

We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid.

Characterization of the VHL tumor suppressor gene product: localization, complex formation, and the effect of natural inactivating mutations.

The human VHL tumor suppressor gene has been implicated in the inherited disorder von Hippel-Lindau disease and in sporadic renal carcinoma. The homologous rat gene encodes a 185-amino acid protein that is 88% sequence identical to the aligned 213-amino acid human VHL gene product. When expressed in COS-7 cells, both the human and the rat VHL proteins showed predominant nuclear, nuclear and cytosolic, or predominant cytosolic VHL staining by immunofluorescence. A complicated pattern of cellular proteins was seen that could be specifically coimmunoprecipitated with the introduced VHL protein. A complex containing VHL and proteins of apparent molecular masses 16 and 9 kDa was the most consistently observed. Certain naturally occurring VHL missense mutations demonstrated either complete or partial loss of the p16-p9 complex. Thus, the VHL tumor suppressor gene product is a nuclear protein, perhaps capable of specifically translocating between the nucleus and the cytosol. It is likely that VHL executes its functions via formation of specific multiprotein complexes. Identification of these VHL-associated proteins will likely clarify the physiology of this tumor suppressor gene.