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Biomedical subjects

D A Harris

Publications and source records attributed to D A Harris.

At least 55 records · Page 3Linked to original sources
PubMed1998

Neurological illness in transgenic mice expressing a prion protein with an insertional mutation.

Familial prion diseases are caused by mutations in the gene encoding the prion protein (PrP). We have produced transgenic mice that express the mouse homolog of a mutant human PrP containing a nine octapeptide insertion associated with prion dementia. These mice exhibit a slowly progressive neurological disorder characterized clinically by ataxia and neuropathologically by cerebellar atrophy and granule cell loss, gliosis, and PrP deposition that is most prominent in the cerebellum and hippocampus. Mutant PrP molecules expressed in the brains of these mice are resistant to digestion by low concentrations of proteinase K and display several other biochemical properties reminiscent of PrP(Sc), the pathogenic isoform of PrP. These results establish a new transgenic animal model of an inherited human prion disorder.

Animals↗
PubMed1997

A wild-type prion protein does not acquire properties of the scrapie isoform when coexpressed with a mutant prion protein in cultured cells.

Inherited prion diseases are linked to autosomal dominant mutations in the gene that encodes the prion protein (PrP). These mutations are thought to induce PrP to undergo a conformational alteration that converts it to a pathogenic form designated PrP(Sc). In patients who are heterozygous for PrP mutations, the protein encoded by the wild-type allele might influence the conversion of the mutant protein to the PrP(Sc) state, and might itself be converted into PrP(Sc). To test these possibilities, we have constructed stably transfected lines of CHO cells that express both wild-type mouse PrP and mouse PrP carrying an insertional mutation that is homologous to one associated with familial Creutzfeldt-Jakob disease. We find that wild-type PrP in these cells does not acquire any of four different biochemical properties characteristic of PrP(Sc) that we have previously documented in mutant PrPs expressed in CHO cells. We also observe that conversion of the mutant protein to a PrP(Sc)-like state is not impaired by coexpression of the wild-type protein. These results are consistent with the idea that sequence homology between PrP molecules has an important influence on PrP(Sc) generation, and they provide insight into the metabolism of PrP in patients who are heterozygous at the PrP locus.

Animals↗
PubMed1997

Blockade of glycosylation promotes acquisition of scrapie-like properties by the prion protein in cultured cells.

The conformational conversion of the prion protein, a sialoglycoprotein containing two N-linked oligosaccharide chains, from its normal form (PrPC) to a pathogenic form (PrPSc) is the central causative event in prion diseases. Although PrPSc can be generated in the absence of glycosylation, there is evidence that oligosaccharide chains may modulate the efficiency of the conversion process, and may also serve as molecular markers of diverse prion strains. In addition, mutational inactivation of one of the N-glycosylation sites has recently been associated with a familial spongiform encephalopathy. To investigate the role of N-glycans in determining the properties of PrP, we have expressed in transfected Chinese hamster ovary cells mouse PrP molecules in which N-glycosylation has been blocked either by treatment with the drug tunicamycin, or by substitution of alanine for threonine at one or both of the N-X-T consensus sites. We report that PrP molecules mutated at Thr182 alone or at both Thr182 and Thr198 [corrected] fail to reach the cell surface after synthesis, but that those mutated at Thr198 [corrected] or synthesized in the presence of tunicamycin can be detected on the plasma membrane. We also find that all three mutant PrPs, and to a limited extent wild-type PrP synthesized in the presence of inhibitor, acquire biochemical attributes reminiscent of PrPSc. We suggest that the PrP molecule has an intrinsic tendency to acquire some PrPSc-like properties, and that N-glycan chains protect against this change. However, pathogenic mutations, or presumably contact with exogenous prions, are necessary to fully convert the protein to a PrPSc state.

Animals↗
PubMed1997

Functional regions of the H(+)-ATPase inhibitory protein from ox heart mitochondria.

Derivatives of the inhibitor protein (IF1) of the mitochondrial H(+)-ATP synthase, bearing deletions at the N- or C-terminal ends, were tested for their abilities (a) to bind to the synthase, (b) to inhibit its ATPase activity and (c) to respond to energisation of the mitochondrial membrane. Deletion of nine residues from its N-terminus, or ten from its C-terminus had little effect on any of these three properties of IF1. Further deletions from the N-terminus (up to residue 17) led to an increase in binding affinity but a reduced ability to inhibit ATPase activity and to form a stable ATPase-IF1 complex. Removal of five more residues from the N-terminus (up to residue 22) reduced these abilities further, but also decreased binding affinity by an order of magnitude. It was concluded that residues 10-17 of IF1 interact with F1 in a way which modulates the stability and function of the interaction between F1 and IF1.

Animals↗
PubMed1997

Evidence for a six-transmembrane domain structure of presenilin 1.

Mutations in genes encoding presenilin 1 and presenilin 2 account for the majority of cases of early-onset familial Alzheimer's disease. The presenilins have been localized to the endoplasmic reticulum and Golgi, but which of the multiple hydrophobic segments of the polypeptide chain span the lipid bilayer is unclear. To address this question, we have constructed a series of chimeric molecules in which a topologically neutral reporter protein (a C-terminal fragment of prolactin) containing three artificial glycosylation sites is fused to presenilin 1 following each of the 10 potential transmembrane domains identified in hydrophobicity plots. We have expressed these chimeras by translation in reticulocyte lysate containing canine pancreatic microsomes and by synthesis in transfected COS cells. Based on utilization of the glycosylation sites and sensitivity of the reporter to protease digestion, we provide evidence that presenilin 1 has six transmembrane segments with the N and C termini in the cytoplasm. This model provides important clues to the potential functions of different parts of the presenilin molecule and how these might relate to the pathogenesis of Alzheimer's disease.

Alternative Splicing↗
PubMed1997

Identification of intermediate steps in the conversion of a mutant prion protein to a scrapie-like form in cultured cells.

The central causative event in infectious, familial, and sporadic forms of prion disease is thought to be a conformational change that converts the cellular isoform of the prion protein (PrPC) into the scrapie isoform (PrPSc) that is the primary constituent of infectious prion particles. To provide a model system for analyzing the mechanistic details of this critical transformation, we have previously prepared cultured Chinese hamster ovary cells that stably express mouse PrP molecules carrying mutations homologous to those seen in familial prion diseases of humans. In the present work, we have analyzed the kinetics with which a PrP molecule containing an insertional mutation associated with Creutzfeldt-Jakob disease acquires several biochemical properties characteristic of PrPSc. Within 10 min of pulse labeling, the mutant protein undergoes a molecular alteration that is detectable by a change in Triton X-114 phase partitioning and phenyl-Sepharose binding. After 30 min of labeling, a detergent-insoluble and protease-sensitive form of the protein appears. After a chase period of several hours, the protein becomes protease-resistant. Incubation of cells at 18 degrees C or treatment with brefeldin A inhibits acquisition of detergent insolubility and protease resistance but does not affect Triton X-114 partitioning and phenyl-Sepharose binding. Our results support a model in which conversion of mutant PrPs to a PrPSc-like state proceeds in a stepwise fashion via a series of identifiable biochemical intermediates, with the earliest step occurring during or very soon after synthesis of the polypeptide in the endoplasmic reticulum.

Animals↗
PubMed1997

Properties of the phosphorylation reaction catalyzed by SpoIIAB that help to regulate sporulation of Bacillus subtilis.

Phosphorylation of SpoIIAA on Ser-58 catalyzed by SpoIIAB is important in the regulation of sporulation of Bacillus subtilis. Nucleotide binding experiments showed that the affinity of SpoIIAB for ATP was greatly increased in the presence of SpoIIAA or a mutant SpoIIAA in which Ser-58 had been changed to alanine. Study of the phosphorylation reaction showed that the Km for ATP and the Ki for ADP were both about 1 microM. The kinetics of phosphorylation of SpoIIAA by SpoIIAB were biphasic, comprising a rapid phase (leading to phosphorylation of 1 mol of SpoIIAA/mol of SpoIIAB) followed by a slower, steady-state phase. In the steady state, the rate-determining step proved to be the dissociation of a SpoIIAB-ADP complex. The rate of this dissociation was not affected significantly by changes in the concentration of ATP.

Adenosine Diphosphate↗
PubMed1997

The use of an automated microsampling system for the characterization of growth hormone pulsatility in newborn babies.

To overcome the difficulties of studying hormone pulsatility in the newborn, we have developed an automated microsampling system that permits the measurement of hormones in small prediluted samples of blood (40 microL) taken at 10-min intervals over 12 h. The system has been validated in adult volunteers, and the error attributable to the dilution was <4%. Using this method in 10 preterm babies, we have been able to describe pulsatile changes in GH and have demonstrated a clear postprandial elevation in GH levels peaking 60 min after a feed. Fourier transform analysis indicated a pulse periodicity of 180 min in babies who were appropriate for gestational age (n = 6), but faster, co-dominant pulse periodicities of 90-100 and 140 min in babies who were small for gestational age (weight and length below the 10th centile) (n = 4). There was no significant difference between mean, peak, and baseline GH levels between the two groups.

Adult↗
PubMed1996

Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform.

Prion diseases are a group of fatal neurodegenerative disorders that are unique in being infectious, genetic, and sporadic in origin. Infectious cases are caused by prions, which are composed primarily of PrPSc, a posttranslationally modified isoform of the normal cellular prion protein PrPC. Inherited cases are linked to insertional or point mutations in the host gene encoding PrPC. To investigate the molecular mechanisms underlying inherited prion diseases, we have constructed stably transfected Chinese hamster ovary cells that express mouse PrPs homologous to two human PrPs associated with familial Creutzfeldt-Jakob disease. One mouse PrP molecule carries a Glu-->Lys substitution at codon 199, and the other carries an insertion of six additional octapeptide repeats between codons 51 and 90. We find that both of these mutant PrPs display several biochemical hallmarks of PrPSc when synthesized in cell culture. Unlike wild-type PrP, the mutant proteins are detergent insoluble and are relatively resistant to digestion by proteinase K, yielding an N-terminally truncated core fragment of 27-30 kDa. Pulse-chase labeling experiments demonstrate that these properties are acquired posttranslationally, and are accompanied by increased metabolic stability of the protein. Our results provide the first evidence that a molecule with properties reminiscent of PrPSc can be generated de novo in cultured cells.

Amino Acid Sequence↗
PubMed1996

Mutant and infectious prion proteins display common biochemical properties in cultured cells.

Prion diseases are unusual neurodegenerative disorders that can be both infectious and inherited. Both forms are hypothesized to result from a posttranslational structural alteration in the cell surface glycoprotein PrPc (cellular isoform of the prion protein) that converts it into the protease-resistant isoform PrPSc (scrapie isoform of the prion protein). However, a direct comparison of molecular events underlying these two manifestations of prion diseases has not been possible, because there has been no cell culture model for the familial forms. We report here that when mutant prion proteins associated with three different inherited prion disorders of humans are expressed as their murine homologues in cultured Chinese hamster ovary cells, the proteins are protease-resistant and detergent-insoluble, two biochemical properties characteristic of infectious PrPSc. In addition, each mutant protein remains tightly associated with the plasma membrane after enzymatic cleavage of its glycosylphosphatidylinositol anchor, a property that we now show is also typical of infectious PrPSc. The cell culture system described here is the first in vitro model for familial prion diseases and provides compelling evidence that infectious and genetic cases share common molecular features.

Amino Acid Sequence↗
PubMed1996

The SpoIIAA protein of Bacillus subtilis has GTP-binding properties.

SpoIIAA is the first protein of the spoIIA operon. Here we show that SpoIIAA can bind and hydrolyze GTP. The protein also accepts ATP, but with lower affinity. GDP competes poorly for binding of GTP. The GTPase activity of SpoIIAA is within the range found for other GTP-binding proteins.

Bacillus subtilis↗
PubMed1995

Sulfated glycans stimulate endocytosis of the cellular isoform of the prion protein, PrPC, in cultured cells.

There is currently no effective therapy for human prion diseases. However, several polyanionic glycans, including pentosan sulfate and dextran sulfate, prolong the incubation time of scrapie in rodents, and inhibit the production of the scrapie isoform of the prion protein (PrPSc), the major component of infectious prions, in cultured neuroblastoma cells. We report here that pentosan sulfate and related compounds rapidly and dramatically reduce the amount of PrPC, the non-infectious precursor of PrPSc, present on the cell surface. This effect results primarily from the ability of these agents to stimulate endocytosis of PrPC, thereby causing a redistribution of the protein from the plasma membrane to the cell interior. Pentosan sulfate also causes a change in the ultrastructural localization of PrPC, such that a portion of the protein molecules are shifted into late endosomes and/or lysosomes. In addition, we demonstrate, using PrP-containing bacterial fusion proteins, that cultured cells express saturable and specific surface binding sites for PrP, many of which are glycosaminoglycan molecules. Our results raise the possibility that sulfated glycans inhibit prion production by altering the cellular localization of PrPC precursor, and they indicate that endogenous proteoglycans are likely to play an important role in the cellular metabolism of both PrPC and PrPSc.

Animals↗
PubMed1995

A mutant prion protein displays an aberrant membrane association when expressed in cultured cells.

Inherited forms of prion disease have been linked to mutations in the gene encoding PrP, a neuronal and glial protein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. One familial form of Creutzfeldt-Jakob disease is associated with a mutant PrP containing six additional octapeptide repeats. We report here our analysis of cultured Chinese hamster ovary cells expressing a murine homologue of this mutant PrP. We find that, like wild-type PrP, the mutant protein is glycosylated, GPI-anchored, and expressed on the cell surface. Surprisingly, however, cleavage of the GPI anchor using phosphatidylinositol-specific phospholipase C fails to release the mutant PrP from the surface of intact cells, suggesting that it has an additional mode of membrane attachment. The phospholipase-treated protein is hydrophobic, since it partitions into the detergent phase of Triton X-114 lysates; and it is tightly membrane-associated, since it is not extractable in carbonate buffer at pH 11.5. Whether membrane attachment of the mutant PrP involves integration of the polypeptide into the lipid bilayer, self-association, or binding to other membrane proteins remains to be determined. Our results suggest that alterations in the membrane association of PrP may be an important feature of prion diseases.

Amino Acid Sequence↗
PubMed1995

The N-terminal domain of a glycolipid-anchored prion protein is essential for its endocytosis via clathrin-coated pits.

The cellular prion protein (PrPC) is a glycolipid-anchored protein that is involved in the pathogenesis of fatal spongiform encephalopathies. We have shown previously that, in contrast to several other glycolipid-anchored proteins, chPrP, the chicken homologue of mammalian PrPC, is endocytosed via clathrin-coated pits in cultured neuroblastoma cells, as well as in embryonic neurons and glia (Shyng, S.-L., Heuser, J. E., and Harris, D. A. (1994) J. Cell Biol. 125, 1239-1250). In this study, we have determined that the N-terminal half of the chPrP polypeptide chain is essential for its endocytosis. Deletions within this region reduce the amount of chPrP internalized, as measured by surface iodination or biotinylation, and decrease its concentration in clathrin-coated pits, as determined by quantitative electron microscopic immunogold labeling. Mouse PrP, as well as two mouse PrP/chPrP chimeras, are internalized as efficiently as chPrP, suggesting that conserved features of secondary and tertiary structure are involved in interaction with the endocytic machinery. Our results indicate that the ectodomain of a protein can contain endocytic targeting information, and they strongly support a model in which the polypeptide chain of PrPC binds to the extracellular domain of a transmembrane protein that contains a coated pit localization signal in its cytoplasmic tail.

Amino Acid Sequence↗
PubMed1995

Primary structure and properties of the inhibitory protein of the mitochondrial ATPase (H(+)-ATP synthase) from potato.

The primary structure of the inhibitory protein (IF1) of the potato mitochondrial ATPase has been determined by protein sequencing, and its molecular weight determined by electrospray mass spectrometry. Both are consistent with a 56-residue protein of molecular weight 6697. This protein shows only weak homology with IF1 sequences from mammals and yeasts, and significant deletions are present compared to these sequences. Homology is strongest in the region between residues 22 and 46 (ox heart numbering), where 5 identities and 6 conserved residues are observed across all five IF1 species. In addition, this region shows homology with protein inhibitors from ATPases other than mitochondrial F1. It is suggested that this region might constitute an ATPase 'inhibitory motif'. Functional studies show that, unlike IF1 from mammals or yeasts, potato IF1 binds only poorly to ox heart F1, and does not show the ability to exist in 2 (alternate) stable conformations.

Amino Acid Sequence↗
PubMed1995

Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin.

It has been known for a number of years that glycosyl-phosphatidylinositol (GPI)-anchored proteins, in contrast to many transmembrane proteins, are insoluble at 4 degrees C in nonionic detergents such as Triton X-100. Recently, it has been proposed that this behavior reflects the incorporation of GPI-linked proteins into large aggregates that are rich in sphingolipids and cholesterol, as well as in cytoplasmic signaling molecules such as heterotrimeric G proteins and src-family tyrosine kinases. It has been suggested that these lipid-protein complexes are derived from caveolae, non-clathrin-coated invaginations of the plasmalemma that are abundant in endothelial cells, smooth muscle, and lung. Caveolin, a proposed coat protein of caveolae, has been hypothesized to be essential for formation of the complexes. To further investigate the relationship between the detergent-resistant complexes and caveolae, we have characterized the behavior of GPI-anchored proteins in lysates of N2a neuroblastoma cells, which lack morphologically identifiable caveolae, and which do not express caveolin (Shyng, S.-L., J. E. Heuser, and D. A. Harris. 1994. J. Cell Biol. 125:1239-1250). We report here that the complexes prepared from N2a cells display the large size and low buoyant density characteristic of complexes isolated from sources that are rich in caveolae, and contain the same major constituents, including multiple GPI-anchored proteins, alpha and beta subunits of heterotrimeric G proteins, and the tyrosine kinases fyn and yes. Our results argue strongly that detergent-resistant complexes are not equivalent to caveolae in all cell types, and that in neuronal cells caveolin is not essential for the integrity of these complexes.

Animals↗