Designing conditions for in vitro formation of amyloid protofilaments and fibrils

We have been able to convert a small α/β protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30–50 Å in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive β-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions.

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson–Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.

Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial growth factor isoforms VEGF121 and VEGF165 but not VEGF189

The vascular endothelial growth factor (VEGF) has been shown to be a significant mediator of angiogenesis during a variety of normal and pathological processes, including tumor development. Human U87MG glioblastoma cells express the three VEGF isoforms: VEGF121, VEGF165, and VEGF189. Here, we have investigated whether these three isoforms have distinct roles in glioblastoma angiogenesis. Clones that overexpressed each isoform were derived and inoculated into mouse brains. Mice that received VEGF121- and VEGF165-overexpressing cells developed intracerebral hemorrhages after 60–90 hr. In contrast, mice implanted with VEGF189-overexpressing cells had only slightly larger tumors than those caused by parental cells and little evidence of hemorrhage at these early times after implantation, whereas, after longer periods of growth, enhanced angiogenicity and tumorigenicity were apparent. There was rapid blood vessel growth and breakdown around the tumors caused by cells overexpressing VEGF121 and VEGF165, whereas there was similar vascularization but no eruption in the vicinity of those tumors caused by cells overexpressing VEGF189, and none on the border of the tumors caused by the parental cells. Thus, by introducing VEGF-overexpressing glioblastoma cells into the brain, we have established a reproducible and predictable in vivo model of tumor-associated intracerebral hemorrhage caused by the enhanced expression of single molecular species. Such a model should be useful for uncovering the role of VEGF isoforms in the mechanisms of angiogenesis and for investigating intracerebral hemorrhage due to ischemic stroke or congenital malformations.

A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas

Molting or ecdysis is the most fundamentally important process in arthropod life history, because shedding of the exoskeleton is an absolute prerequisite for growth and metamorphosis. Although the hormonal mechanisms driving ecdysis in insects have been studied extensively, nothing is known about these processes in crustaceans. During late premolt and during ecdysis in the crab Carcinus maenas, we observed a precise and reproducible surge in hemolymph hyperglycemic hormone (CHH) levels, which was over 100-fold greater than levels seen in intermolt animals. The source of this hormone surge was not from the eyestalk neurosecretory tissues but from previously undescribed endocrine cells (paraneurons), in defined areas of the foregut and hindgut. During premolt (the only time when CHH is expressed by these tissues), the gut is the largest endocrine tissue in the crab. The CHH surge, which is a result of an unusual, almost complete discharge of the contents of the gut endocrine cell, regulates water and ion uptake during molting, thus allowing the swelling necessary for successful ecdysis and the subsequent increase in size during postmolt. This study defines an endocrine brain/gut axis in the arthropods. We propose that the ionoregulatory process controlled by CHH may be common to arthropods, in that, for insects, a similar mechanism seems to be involved in antidiuresis. It also seems likely that a cascade of very precisely coordinated release of (neuro) hormones controls ecdysis.

Gi regulation of secretory vesicle swelling examined by atomic force microscopy

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the α subunit of a heterotrimeric GTP binding protein Gαi3 and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15–25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that Gαi3 protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain

Deletions of all or part of chromosome 10 are the most common genetic alterations in high-grade gliomas. The PTEN gene (also called MMAC1 and TEP1) maps to chromosome region 10q23 and has been implicated as a target of alteration in gliomas and also in other cancers such as those of the breast, prostate, and kidney. Here we sought to provide a functional test of its candidacy as a growth suppressor in glioma cells. We used a combination of Northern blot analysis, protein truncation assays, and sequence analysis to determine the types and frequency of PTEN mutations in glioma cell lines so that we could define appropriate recipients to assess the growth suppressive function of PTEN by gene transfer. Introduction of wild-type PTEN into glioma cells containing endogenous mutant alleles caused growth suppression, but was without effect in cells containing endogenous wild-type PTEN. The ectopic expression of PTEN alleles, which carried mutations found in primary tumors and have been shown or are expected to inactivate its phosphatase activity, caused little growth suppression. These data strongly suggest that PTEN is a protein phosphatase that exhibits functional and specific growth-suppressing activity.

A peptide derived from the non-receptor-binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of Å6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. Å6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells in vitro. In contrast, Å6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth in vivo by 48% and 53%, respectively, and, more strikingly, the combination of Å6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or Å6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving Å6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.

Embryonic expression of the tumor-associated PAX3-FKHR fusion protein interferes with the developmental functions of Pax3

A unique chromosomal translocation involving the genes PAX3 and FKHR is characteristic of most human alveolar rhabdomyosarcomas. The resultant chimeric protein fuses the PAX3 DNA-binding domains to the transactivation domain of FKHR, suggesting that PAX3-FKHR exerts its role in alveolar rhabdomyosarcomas through dysregulation of PAX3-specific target genes. Here, we have produced transgenic mice in which PAX3-FKHR expression was driven by mouse Pax3 promoter/enhancer sequences. Five independent lines expressed PAX3-FKHR in the dorsal neural tube and lateral dermomyotome. Each line exhibited phenotypes that correlated with PAX3-FKHR expression levels and predominantly involved pigmentary disturbances of the abdomen, hindpaws, and tail, with additional neurological related alterations. Phenotypic severity could be increased by reducing Pax3 levels through matings with Pax3-defective Splotch mice, and interference between PAX3 and PAX3-FKHR was apparent in transcription reporter assays. These data suggest that the tumor-associated PAX3-FKHR fusion protein interferes with normal Pax3 developmental functions as a prelude to transformation.

A role for estrogen receptor β in the regulation of growth of the ventral prostate

In normal rats and mice, immunostaining with specific antibodies revealed that nuclei of most prostatic epithelial cells harbor estrogen receptor β (ERβ). In rat ventral prostate, 530- and 549-aa isoforms of the receptor were identified. These sediment in the 4S region of low-salt sucrose gradients, indicating that prostatic ERβ does not contain the same protein chaperones that are associated with ERα. Estradiol (E2) binding and ERβ immunoreactivity coincide on the gradient, with no indication of ERα. In prostates from mice in which the ERβ gene has been inactivated (BERKO), androgen receptor (AR) levels are elevated, and the tissue contains multiple hyperplastic foci. Most epithelial cells express the proliferation antigen Ki-67. In contrast, prostatic epithelium from wild-type littermates is single layered with no hyperplasia, and very few cells express Ki-67. Rat ventral prostate contains an estrogenic component, which comigrates on HPLC with the testosterone metabolite 5α-androstane-3β,17β-diol (3βAdiol). This compound, which competes with E2 for binding to ERβ and elicits an estrogenic response in the aorta but not in the pituitary, decreases the AR content in prostates of wild-type mice but does not affect the elevated levels seen in ERβ knockout (BERKO) mice. Thus ERβ, probably as a complex with 3βAdiol, is involved in regulating the AR content of the rodent prostate and in restraining epithelial growth. These findings suggest that ligands specific for ERβ may be useful in the prevention and/or clinical management of prostatic hyperplasia and neoplasia.

Proinflammatory cytokines regulate human glucocorticoid receptor gene expression and lead to the accumulation of the dominant negative β isoform: A mechanism for the generation of glucocorticoid resistance

Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-κB and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GRα), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GRβ), which does not bind known ligands and attenuates GRα action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-κB DNA binding site 5′ to the hGR promoter that leads to a 1.5-fold increase in GRα mRNA and a 2.0-fold increase in GRβ mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-α treatment disproportionately increased the steady-state levels of the GRβ protein isoform over GRα, making GRβ the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-α or IL-1. The increase in GRβ protein expression correlated with the development of glucocorticoid resistance.

Induction of Acclimative Proteolysis of the Light-Harvesting Chlorophyll a/b Protein of Photosystem II in Response to Elevated Light Intensities1

Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine or cysteine type and is associated with the outer membrane surface of the stroma-exposed thylakoid regions. The identity of the protease is not known, but it does not correspond to the recently identified chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to bacterial enzymes. The acclimative response shows a delay of 2 d after transfer of the leaves to high light. This lag period was shown to be attributed to expression or activation of the responsible protease. Furthermore, the LHCII degradation was found to be regulated at the substrate level. The degradation process involves lateral migration of LHCII from the appressed to the nonappressed thylakoid regions, which is the location for the responsible protease. Phosphorylated LHCII was found to be a poor substrate for degradation in comparison with the unphosphorylated form of the protein. The relationship between LHCII degradation and other regulatory proteolytic processes in the thylakoid membrane, such as D1-protein degradation, is discussed.

Constitutive receptor activation by Crouzon syndrome mutations in fibroblast growth factor receptor (FGFR)2 and FGFR2/Neu chimeras.

Crouzon syndrome is an autosomal dominant condition primarily characterized by craniosynostosis. This syndrome has been associated with a variety of amino acid point mutations in the extracellular domain of fibroblast growth factor receptor 2 (FGFR2). FGFR2/Neu chimeras were generated by substituting the extracellular domain of Neu with that of FGFR2 containing the following Crouzon mutations: Tyr-340-->His; Cys-342-->Tyr; Cys-342-->Arg; Cys-342-->Ser; Ser-354-->Cys: and delta17 (deletion of amino acids 345-361). Each of the mutant chimeric FGFR2/Neu constructs stimulated focus formation in NIH 3T3 cells, indicating that Crouzon mutations can stimulate signal transduction through a heterologous receptor tyrosine kinase. In vitro kinase assay results indicate that FGFR2 receptors containing Crouzon mutations have increased tyrosine kinase activity and, when analyzed under nonreducing conditions, exhibited disulfide-bonded dimers. Thus the human developmental abnormality Crouzon syndrome arises from constitutive activation of FGFR2 due to aberrant intermolecular disulfide-bonding. These results together with our earlier observation that achondroplasia results from constitutive activation of the related receptor FGFR3, leads to the prediction that other malformation syndromes attributed to FGFRs, such as Pfeiffer syndrome and Thanatophoric dysplasia, also arise from constitutive receptor activation.