N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state.

Scrapie and related transmissible spongiform encephalopathies result in the accumulation of a protease-resistant form of an endogenous brain protein called PrP. As an approach to understanding the scrapie-associated modification of PrP, we have studied the processing and sedimentation properties of protease-resistant PrP (PrP-res) in scrapie-infected mouse neuroblastoma cells. Like brain-derived PrP-res, the neuroblastoma cell PrP-res aggregated in detergent lysates, providing evidence that the tendency to aggregate is an intrinsic property of PrP-res and not merely a secondary consequence of degenerative brain pathology. The PrP-res species had lower apparent molecular masses than the normal, protease-sensitive PrP species and were not affected by moderate treatments with proteinase K. This suggested that the PrP-res species were partially proteolyzed by the neuroblastoma cells. Immunoblot analysis of PrP-res with a panel of monospecific anti-PrP peptide sera confirmed that the PrP-res species were quantitatively truncated at the N terminus. The metabolic labeling of PrP-res in serum-free medium did not prevent the proteolysis of PrP-res, showing that the protease(s) involved was cellular rather than serum-derived. The PrP-res truncation was inhibited in intact cells by leupeptin and NH4Cl. This provided evidence that a lysosomal protease(s) was involved, and therefore, that PrP-res was translocated to lysosomes. When considered with other studies, these results imply that the conversion of PrP to the protease-resistant state occurs in the plasma membrane or along an endocytic pathway before PrP-res is exposed to endosomal and lysosomal proteases.     

The protein component of scrapie-associated fibrils is a glycosylated low molecular weight protein.

Scrapie-associated fibril protein (SAF-protein) extracted from infectious scrapie-associated fibrils (SAF) isolated from scrapie hamster brains is not infectious. SAF-protein is composed of various mol. wt. species of glycoproteins differing in carbohydrate content rather than amino acid composition. The N-linked carbohydrate chains represent approximately 40-60% of the mol. wt. of SAF-protein. The deglycosylated SAF-protein has a surprisingly low mol. wt. of approximately 7 kd, representing approximately 55 amino acid residues. This size and chemical analyses indicate that SAF-protein is an amyloid-type of protein. The simplest explanation for the available data is that SAF-polypeptide is very likely not to be part of the scrapie agent but that it is, like other amyloid proteins, derived from host-encoded proteins and not infectious. It is suggested that the infectivity of fractions rich in SAF is due to co-purification of scrapie virus and SAF caused by the high carbohydrate content of SAF-protein.     

Segregation of cells that differentiated without cell movement from a single precursor population

Previous studies have demonstrated that limb cartilage and limb muscle arise from the mesenchyme cells of the early limb bud without any detectable cell movement. In the present study, binary mixtures of these three cell types have been made in order to determine whether cells gain the ability to segregate as part of the differentiative process. Limb cartilage, limb muscle, and limb mesenchyme can each segregate from the other two cell types. Thus, the ability to segregate is, in this case, a differentiative characteristic.     

HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae.

We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S. cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S. Kasahara, H. Yamada, T. Mio, Y. Shiratori, C. Miyamoto, T. Yabe, T. Nakajima, E. Ichishima, and Y. Furuichi, J. Bacteriol. 176:1488-1499, 1994). HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain. Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells. This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells. Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele. Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface. The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells. These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S. cerevisiae.     

Evidence that a 77-kilodalton protein from the starch of pea embryos is an isoform of starch synthase that is both soluble and granule bound.

In this paper we provide further evidence about the nature of a 77-kD starch synthase (SSII) that is both soluble and bound to the starch granules in developing pea (Pisum sativum L.) embryos. Mature SSII gives rise to starch synthase activity when expressed in a strain of Escherichia coli lacking glycogen synthase. In transgenic potatoes (Solanum tuberosum L.) expressing SSII, the protein is both soluble and bound to the starch granules. These results confirm that SSII is a starch synthase and indicate that partitioning between the soluble and granule-bound fraction of storage organs is an intrinsic property of the protein. A 60-kD isoform of starch synthase found both in the soluble and granule-bound fraction of the pea embryos is probably derived by the processing of SSII and is a different gene product from GBSSI, the exclusively granule-bound 59-kD isoform of starch synthase that is similar to starch synthases encoded by the waxy genes of cereals and the amf gene of potatoes. Consistent with this, expression in E. coli of an N-terminally truncated version of SSII gives rise to starch synthase activity.     

Herpesvirus-induced cell fusion that is dependent on cell surface heparan sulfate or soluble heparin.

The entry of enveloped viruses into animal cells and the cell-to-cell spread of infection via cell fusion require the membrane-fusing activity of viral glycoproteins. This activity can be dependent on variable cell factors or triggered by environmental factors. Here we show that cell fusion induced by herpes simplex virus glycoproteins is dependent on the presence of cell surface glycosaminoglycans, principally heparan sulfate, or on the addition of heparin to the medium. The role of the glycosaminoglycan is probably to alter the conformation of a viral heparin-binding glycoprotein required for the fusion.     

Nervous and nonnervous cell transduction by recombinant adenoviruses that inducibly express the human PrP.

The study of the prion protein (PrP) physiological functions or its specific role in transmissible spongiform encephalopathies (TSE) requires new tools, particularly those able to induce PrP overexpression in a large range of cells, in vivo as well as in vitro. Here we describe the construction of two recombinant adenoviruses encoding the human PrP either with a valine at position 129 (AdTRVal) or a methionine (AdTRMet). Both genes were put under the control of the tetracycline-responsive promoter, allowing tight regulation of PrP expression. AdTRVal and AdTRMet induced high expression of the human PrP in CHO-KI cells and in organotypic brain slices in culture. The proteins expressed from these viruses exhibited a glycosylphosphatidyl inositol (GPI) anchor, proper glycosylation and sensitivity to proteinase K digestion. AdTRVal and AdTRMet will allow future studies on the human PrP and on the role of the codon 129 polyphormism in human TSE.     

The generation of neurons and oligodendrocytes from a common precursor cell

We have used a recombinant retrovirus carrying the lacZ gene to study the developmental potential of precursor cells from the embryonic rat cerebral cortex in dissociated cell culture. Virus was used to label a small number of cultured cells genetically so that their fate could be determined. Infected clones were detected with an anti-beta-galactosidase serum, and the labeled cells were identified using monoclonal antibodies. The results revealed that most precursor cells generated a single cell type, the majority being either neurons or oligodendrocytes. However, a proportion of the neuronal clones also included oligodendrocytes. This proportion increased until embryonic day 16 when 18% of the neuronal clones were of this type. This suggests that during neurogenesis in the cerebral cortex there exists a cell with the potential to generate these two quite different neural cell types.     

Monoclonal antibody DH12 reacts with a cell surface and a precursor form of the beta subunit of the human fibronectin receptor

Monoclonal and polyclonal antibodies were raised against a placenta plasma membrane protein preparation, which was obtained by fractionation on Blue B dye matrix and by HPLC-anionexchange, and which was shown to contain fibronectin receptors. Immunochemical and functional evidence showed that monoclonal antibody DH12 recognized the beta subunit of the human fibronectin receptor on fibroblasts. This monoclonal antibody reacted with two proteins in Western blots and in double immune precipitations of whole cell preparations. Only the higher Mr protein became labeled by surface iodination of intact fibroblasts. The lower Mr protein is thought to be an intracellular precursor of the beta subunit of the fibronectin receptor.     

The acylated precursor form of the colicin A lysis protein is a natural substrate of the DegP protease.

The acylated precursor form of the colicin A lysis protein (pCalm) is specifically cleaved by the DegP protease into two acylated fragments of 6 and 4.5 kilodaltons (kDa). This cleavage was observed after globomycin treatment, which inhibits the processing of pCalm into mature colicin A lysis protein (Cal) and the signal peptide. The cleavage took place in lpp, pldA, and wild-type strans carrying plasmids which express the lysis protein following SOS induction and also in cells containing a plasmid which expresses it under the control of the tac promoter. Furthermore, the DegP protease was responsible for the production of two acylated Cal fragments of 3 and 2.5 kDa in cells carrying plasmids which overproduce the Cal protein, without treatment with globomycin. DegP could also cleave the acylated precursor form of a mutant Cal protein containing a substitution in he amino-terminal portion of the protein, but not that of a mutant Cal containing a frameshift mutation in its carboxyl-terminal end. The functions of Cal in causing protein release, quasi-lysis, and lethality were increased in degP41 cells, suggesting that mature Cal was produced in higher amounts in the mutant than in the wild type. These effects were limited in cells deficient in phospholipase A. Interactions between the DegP protease and phospholipase A were suggested by the characteristics of degP pldA double mutants.     

Cross-induction of cell types in Dictyostelium: evidence that DIF-1 is made by prespore cells.

To investigate how cell type proportions are regulated during Dictyostelium development, we have attempted to find out which cell type produces DIF-1, a diffusible signal molecule inducing the differentiation of prestalk-O cells. DIF-1 is a chlorinated alkyl phenone that is synthesized from a C12 polyketide precursor by chlorination and methylation, with the final step catalysed by the dmtA methyltransferase. All our evidence points to the prespore cells as the major source of DIF-1. (1) dmtA mRNA and enzyme activity are greatly enriched in prespore compared with prestalk cells. The chlorinating activity is also somewhat prespore-enriched. (2) Expression of dmtA is induced by cyclic-AMP and this induction is inhibited by DIF-1. This regulatory behaviour is characteristic of prespore products. (3) Short-term labelling experiments, using the polyketide precursor, show that purified prespore cells produce DIF-1 at more than 20 times the rate of prestalk cells. (4) Although DIF-1 has little effect on its own synthesis in short-term labelling experiments, in long-term experiments, using 36Cl(-) as label, it is strongly inhibitory (IC(50) about 5 nM), presumably because it represses expression of dmtA; this is again consistent with DIF-1 production by prespore cells. Inhibition takes about 1 hour to become effective. We propose that prespore cells cross-induce the differentiation of prestalk-O cells by making DIF-1, and that this is one of the regulatory loops that sets the proportion of prespore-to-prestalk cells in the aggregate.     

Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin

Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. Here, we demonstrate that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. We speculate that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity.     

Characterization of a large form of DNA polymerase delta from HeLa cells that is insensitive to proliferating cell nuclear antigen

A large form of DNA polymerase delta from HeLa cells was recently purified in this laboratory as a factor required for conservative DNA synthesis in a reconstituted system utilizing UV-irradiated permeabilized human diploid fibroblasts (Nishida, C., Reinhard, P., and Linn, S. (1988) J. Biol. Chem. 263, 501-510). We have now purified this form of the enzyme utilizing its polymerase activity and further characterized it. The enzyme activity sediments at 11.1 S in low salt and 6.8 S in high salt. In both cases, activity cosediments with the major visible peptide displayed by sodium dodecyl sulfate-polyacrylamide gels which has an Mr of 215,000. This value is consistent with the molecular mass calculated from the sedimentation coefficient and gel filtration behavior in high salt. In low salt the apparent molecular mass was approximately double. The enzyme prefers poly(dA).oligo(dT) as template/primer in low salt, with which it has a processivity of several thousand nucleotides in 1 mM MgCl2. At isotonic KCl or potassium phosphate concentrations, the preferred template/primer is activated DNA. Proliferating cell nuclear antigen, also characterized as a DNA polymerase delta auxiliary protein, does not increase the activity of this preparation of the enzyme. An antibody to the proliferating cell nuclear antigen has no inhibitory effect, nor is it able to recognize any peptides in immunoblots of purified enzyme fractions. Under polymerizing conditions, the enzyme removes mismatched, but not matched nucleotides from the 3' terminus of oligo(dT) annealed to poly(dA) suggesting a proofreading function. The properties of this form of DNA polymerase delta distinguish it from other preparations reported in the literature.     

Planar bilayer membranes made from phospholipid monolayers form by a thinning process.

We investigated the manner in which planar phospholipid membranes form when monolayers are sequentially raised. Simultaneous electrical and optical recordings showed that initially a thick film forms, and the capacitance of the film increases with the same time course as the observed thinning. The diameter of fully thinned membranes varies from membrane to membrane and a torus is readily observed. The frequency-dependent admittance of the membrane was measured using a wide-bandwidth voltage clamp whose frequency response is essentially independent of capacitative load. The membrane capacitance dominates the total admittance and the membrane dielectric is not lossy. The specific capacitance of membranes of several mixtures was measured. A schematic diagram of the formation of these membranes is presented.     

Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms.

We have previously reported the identification of a novel mast cell growth factor (MGF) that was shown to be a ligand for c-kit and is encoded by a gene that maps near the steel locus on mouse chromosome 10. We now report the cloning of cDNAs encoding the MGF protein. The MGF protein encoded by this cDNA can be expressed in a biologically active form as either a membrane bound protein or as a soluble factor. The soluble protein promotes the proliferation of MGF-responsive cell lines and, in the presence of erythropoietin, stimulates the formation of macroscopic [corrected] erythroid and multilineage hematopoietic colonies.     

Processing of the human heparanase precursor and evidence that the active enzyme is a heterodimer.

Human platelet heparanase has been purified to homogeneity and shown to consist of two, non-covalently associated polypeptide chains of molecular masses 50 and 8 kDa. Protein sequencing provided the basis for determination of the full-length cDNA for this novel protein. Based upon this information and results from protein analysis and mass spectrometry, we propose a scheme to define the structural organization of heparanase in relation to its precursor forms, proheparanase and pre-proheparanase. The 8- and 50-kDa chains which make up the active enzyme reside, respectively, at the NH(2)- and COOH-terminal regions of the inactive precursor, proheparanase. The heparanase heterodimer is produced by excision and loss of an internal linking segment. This paper is the first to suggest that human heparanase is a two-chain enzyme.     

The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.     

The AGAAAAGA palindrome in PrP is required to generate a productive PrPSc-PrPC complex that leads to prion propagation

The molecular hallmark of prion disease is the conversion of normal prion protein (PrPC) to an insoluble, proteinase K-resistant, pathogenic isoform (PrPSc). Once generated, PrPSc propagates by complexing with, and transferring its pathogenic conformation onto, PrPC. Defining the specific nature of this PrPSc-PrPC interaction is critical to understanding prion genesis. To begin to approach this question, we employed a prion-infected neuroblastoma cell line (ScN2a) combined with a heterologous yeast expression system to independently model PrPSc generation and propagation. We additionally applied fluorescence resonance energy transfer analysis to the latter to specifically study PrP-PrP interactions. In this report we focus on an N-terminal hydrophobic palindrome of PrP (112-AGAAAAGA-119) thought to feature intimately in prion generation via an unclear mechanism. We found that, in contrast to wild type (wt) PrP, PrP lacking the palindrome (PrPDelta112-119) neither converted to PrPSc when expressed in ScN2a cells nor generated proteinase K-resistant PrP when expressed in yeast. Furthermore, PrPDelta112-119 was a dominant-negative inhibitor of wtPrP in ScN2a cells. Both wtPrP and PrPDelta112-119 were highly insoluble when expressed in yeast and produced distinct cytosolic aggregates when expressed as fluorescent fusion proteins (PrP::YFP). Although self-aggregation was evident, fluorescence resonance energy transfer studies in live yeast co-expressing PrPSc-like protein and PrPDelta112-119 indicated altered interaction properties. These results suggest that the palindrome is required, not only for the attainment of the PrPSc conformation but also to facilitate the proper association of PrPSc with PrPC to effect prion propagation.