Yeast prions and human prion-like proteins: sequence features and prediction methods

Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis. This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms.     

Structural features of prions explored by sequence analysis. II. A PrP<Superscript>Sc</Superscript> model

Prion diseases are neurodegenerative disorders associated with a conformational conversion of the prion PrP protein, in which the β-strand content increases and that of the α helix decreases. However, the structure of the pathogenous form PrP^Sc, occurring after conformational conversion of the normal cellular form PrP^C, is not yet known. From sequence analysis, we have previously proposed that helix H2 of the prion PrP^C structure might be a key region for this structural conversion. More recently, we identified the TATA box-binding protein fold as a putative scaffold that may locally satisfy the predicted secondary-structure organisation of PrP^Sc. In the present analysis, we detail the schematic construction of PrP^Sc monomeric and dimeric models, based on this hypothesis. These models are globally compatible with available data and therefore may provide further insights into the structurally and functionally elusive PrP protein. Some comments are also devoted to a comparison of the yeast Ure2p prion and animal prions. Received 29 July 2002; received after revision 24 October 2002; accepted 24 October 2002 RID="*" ID="*"Corresponding author.     

On the synthesis of serine and homoserine samples asymmetrically labelled with tritium and deuterium in the hydroxymethylene group.

(1R) [1-3H, 2H1] 3-Phenylpropanol, the key intermediate in the synthesis of (4R) [4-3H, 2H1] D,L-homoserine and of the (4S)-isomer, is obtained from (1S) [1-2H1] 3-phenylpropanol and (1RS) [1-3H] ethanol upon incubation with yeast alcohol dehydrogenase and NAD+; under similar conditions 2-phenylethanol undergoes very small exchange with [1-2H2] ethanol.     

Human platelet 5-hydroxytryptamine receptors: binding of [3H]-lysergic acid diethylamide (LSD). Effects of chronic neuroleptic and antidepressant drug administration

Chronic treatment with phenothiazines and thioxanthenes has been found to enhance 5-HT-induced aggregation of human platelets. A method has been developed to study 5-HT2 receptor binding sites on platelets utilising [3H]-LSD and more recently 125I/LSD. Results are presented which suggest that the LSD binding site is indeed the 5-HT2 binding site and that the LSD binding characterises the specific receptor responsible for 5-HT-induced shape change and aggregation. In a group of patients receiving phenothiazines or thioxanthenes, the Bmax of LSD binding was increased. The mean binding affinity was decreased possibly due to a persistence of neuroleptic in the platelet membrane preparation. Analysis showed that this was not the reason why the mean binding capacity was increased. The results show that chronic phenothiazine and thioxanthene delta treatment 'up-regulates' platelet 5-HT2 binding sites and that this may be accompanied by increased sensitivity to platelet aggregation by 5-HT. In normal subjects desipramine treatment increased the Bmax of platelet LSD binding and this was accompanied by an increased prolactin response to tryptophan which is thought to be mediated by central 5-HT function.     

On the synthesis of serine and homoserine samples asymmetrically labelled with tritium and deuterium in the hydroxymethylene group

Summary (1 R) [1-³H,²H₁] 3-Phenylpropanol, the key intermediate in the synthesis of (4 R) [4-³H,²H₁] D, L-homoserine and of the (4 S)-isomer, is obtained from (1 S) [1-²H₁] 3-phenylpropanol and (1 RS) [1-³H] ethanol upon incubation with yeast alcohol dehydrogenase and NAD⁺; under similar conditions 2-phenylethanol undergoes very small exchange with [1-²H₂] ethanol.     

The β6/β7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI ⁺] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the β6 and β7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the β6/β7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes.     

Nucleotide sequence of the gene for the mitochondrial 15S ribosomal RNA of yeast

We have determined the nucleotide sequence of a DNA segment carrying the entire 15S ribosomal RNA gene of yeast mitochondrial genome. Many stretches of sequence are present which are homologous to the E. coli 16S ribosomal RNA gene. The gene sequence can be folded into a secondary structure according to the [1] model on bacterial ribosomal RNAs. The structure reveals a striking similarity between the two RNAs despite the large difference in their base compositions. In the middle of the gene, we found a guanine-cytosine rich sequence that is also present in several other regions of the mitochondrial genome.     

NLRP3 inflammasome activation in macrophage cell lines by prion protein fibrils as the source of IL-1β and neuronal toxicity

Prion diseases are fatal transmissible neurodegenerative diseases, characterized by aggregation of the pathological form of prion protein, spongiform degeneration, and neuronal loss, and activation of astrocytes and microglia. Microglia can clear prion plaques, but on the other hand cause neuronal death via release of neurotoxic species. Elevated expression of the proinflammatory cytokine IL-1β has been observed in brains affected by several prion diseases, and IL-1R-deficiency significantly prolonged the onset of the neurodegeneration in mice. We show that microglial cells stimulated by prion protein (PrP) fibrils induced neuronal toxicity. Microglia and macrophages release IL-1β upon stimulation by PrP fibrils, which depends on the NLRP3 inflammasome. Activation of NLRP3 inflammasome by PrP fibrils requires depletion of intracellular K⁺, and requires phagocytosis of PrP fibrils and consecutive lysosome destabilization. Among the well-defined molecular forms of PrP, the strongest NLRP3 activation was observed by fibrils, followed by aggregates, while neither native monomeric nor oligomeric PrP were able to activate the NLRP3 inflammasome. Our results together with previous studies on IL-1R-deficient mice suggest the IL-1 signaling pathway as the perspective target for the therapy of prion disease.     

Inhibition of protein misfolding and aggregation by natural phenolic compounds

Protein misfolding and aggregation into fibrillar deposits is a common feature of a large group of degenerative diseases affecting the central nervous system or peripheral organs, termed protein misfolding disorders (PMDs). Despite their established toxic nature, clinical trials aiming to reduce misfolded aggregates have been unsuccessful in treating or curing PMDs. An interesting possibility for disease intervention is the regular intake of natural food or herbal extracts, which contain active molecules that inhibit aggregation or induce the disassembly of misfolded aggregates. Among natural compounds, phenolic molecules are of particular interest, since most have dual activity as amyloid aggregation inhibitors and antioxidants. In this article, we review many phenolic natural compounds which have been reported in diverse model systems to have the potential to delay or prevent the development of various PMDs, including Alzheimer’s and Parkinson’s diseases, prion diseases, amyotrophic lateral sclerosis, systemic amyloidosis, and type 2 diabetes. The lower toxicity of natural compounds compared to synthetic chemical molecules suggest that they could serve as a good starting point to discover protein misfolding inhibitors that might be useful for the treatment of various incurable diseases.     

Investigation of the effects of copper ions on protein aggregation using a model system

Protein aggregation is a notable feature of various human disorders, including Parkinsons disease, Alzheimers disease and many others systemic amyloidoses. An increasing number of observations in vitro suggest that transition metals are able to accelerate the aggregation process of several proteins found in pathological deposits, e.g. -synuclein, amyloid (A) peptide, ₂-microglobulin and fragments of the prion protein. Here we report the effects of metal ions on the aggregation rate of human muscle acylphosphatase, a suitable model system for aggregation studies in vitro. Among the different species tested, Cu²⁺ produced the most remarkable acceleration of aggregation, the rate of the process being 2.5-fold higher in the presence of 0.1 mM metal concentration. Data reported in the literature suggest the possible role played by histidine residues or negatively charged clusters present in the amino acid sequence in Cu²⁺-mediated aggregation of pathological proteins. Acylphosphatase does not contain histidine residues and is a basic protein. A number of histidine-containing mutational variants of acylphosphatase were produced to evaluate the importance of histidine in the aggregation process. The Cu²⁺-induced acceleration of aggregation was not significantly altered in the protein variants. The different aggregation rates shown by each variant were entirely explained by the changes of hydrophobicity or propensity to form a structure introduced by the point mutation. The effect of Cu²⁺ on acylphosphatase aggregation cannot therefore be attributed to the specific factors usually invoked in the aggregation of pathological proteins. The effect, rather, seems to be a general related to the chemistry of the polypeptide backbone and could represent an additional deleterious factor resulting from the alteration of the homeostasis of metal ions in cells.Received 4 December 2003; received after revision 7 January 2004; accepted 3 February 2004     

Protein misfolding and disease: the case of prion disorders

Recent findings strongly support the hypothesis that diverse human disorders, including the most common neurodegenerative diseases, arise from misfolding and aggregation of an underlying protein. Despite the good evidence for the involvement of protein misfolding in disease pathogenesis, the mechanism by which protein conformational changes participate in the disease is still unclear. Among the best-studied diseases of this group are the transmissible spongiform encephalopathies or prion-related disorders, in which misfolding of the normal prion protein plays a key role in the disease. In this article we review recent data on the link between prion protein misfolding and the pathogensis of spongiform encephalopathies. Received 15 July 2002; received after revision 19 August 2002; accepted 23 August 2002 RID="*" ID="*"Corresponding author.     

Biosynthesis of 1-aminocyclopropanecarboxylic acid: Steric course of the reaction at the C-4 position

Summary Under the action of the appropriate synthase from ripe tomatoes a 11 mixture of (3S, 4R)-[3,4-²H₂] and (3R, 4S)-[3,4-²H₂]-(2S)-adenosylmethionine is transformed into a 11 mixture of the two meso forms of [²H₂]-1-aminocyclopropanecarboxylic acid, a result which proves the operation of an inversion mechanism and which is consistent with direct nucleophilic displacement of the leaving group in the substrate.     

The effect of the phytoalexins, lubimin, (-)-maackiain, pinosylvin, and the related compounds dehydroloroglossol and hordatine M on human lymphoblastoid cell lines

We have tested the effect of the phytoalexins lubimin, (-)-maackiain and pinosylvin and the related compounds dehydroloroglossol and hordatine M on the growth of the human lymphoblastoid cell lines Molt and Raji. (-)-maackiain, pinosylvin and dehydroloroglossol showed significant growth inhibitory action on the cells. Suppression of [3H] thymidine and [3H] leucine uptake was tested and noted in pinosylvin and dehydroloroglossol. The phytoalexins and related compounds are widespread in plants and provide a potential source of antineoplastic substances.     

Dérivés trifluorométhylés des [1]benzothiopyrano[4,3-b]indoles et des 6H-[1]benzothiopyrano-[4,3-b]quinoléines

Summary Some fluoro derivatives of [1]benzothiopyrano[4,3-b]indoles and 6H[1]benzothiopyrano[4,3-b] quinolines have shown carciuogenic activities in regard to the fluorine position; we thought it interesting to prepare some trifluoromethyl analogs of there compounds to study the biological activities and to compare with other compounds of the same family. In the present work, we report the synthesis of 7-trifluoromethyl and 8-trifluoromethyl [1]thiochroman-4-one and some [1]benzothiopyrano[4,3-b]indoles and quinolines substitued in 3 or 4 position which have been obtained from these two ketones.     

Formation of 4,4′-methylene-bis(2-chloroaniline)-DNA adducts in yeast expressing recombinant cytochrome P450s

N-Oxidation of 4,4-methylene-bis(2-chloroaniline) (MBOCA) may lead to formation of DNA adducts. To determine if cytochrome P450s are involved in the formation of MBOCA derived-DNA adducts, yeast strains expressing rodent P450s were exposed to MBOCA, and³²P-postlabelling of nucleotides from yeast genomic DNA was done. Chromatographic analysis on PEI cellulose showed that, upon exposure to MBOCA for 1 h, nine DNA adducts were formed in yeast expressing phenobarbital-inducible rabbit P450 2B5. With a 4-h-exposure, all adducts increased in parallel. In cell-free experiments, the incubation of MBOCA with phenobarbital-induced rat microsomal fraction followed by incubation with thymus DNA, led to the formation of more than ten DNA adducts. When yeast expressing 3-methylcholanthrene-inducible rat P450 1A1 was exposed to MBOCA, one major and two minor adducts were formed. No adducts were detected in control yeast. These results show that recombinant rabbit P450 2B5 exhibits a potential activation of MBOCA and that rat P450 1A1 has some effect. The use of yeast expressing recombinant P450s and the technique of³²P-postlabelling facilitates a simple search for chemicals with carcinogenic potential.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

The metabolic fate of [35S]-diallyl disulphide in mice

Summary Diallyl disulphide (DADS) is a major constituent of garlic oil. Uptake of [³⁵S]-labelled diallyl disulphide by mouse liver is highest at 90 min after treatment. 2h after treatment with [³⁵S]-DADS, 70% of the radioactivity is present in the liver cytosol of which 80% is metabolized to sulphate.     

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm,Manduca sexta,and the corn earworm,Helicoverpa zea

The tritium-labeled bis-norleucine analog ofHelicoverpa zea pheromone biosynthesis-activating neuropeptide ([³H]NLPBAN) was incubated in vitro with hemolymph fromManduca sexta orH. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [³H]NLPBAN concentration of 0.9 μM the degradation proceeded at a very slow but physiologically plausible rate (2–10 fmol/min/μl hemolymph). The primary [³H]NLPBAN degradation reaction inM. sexta hemolymph was not inhibited by 20 μM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph ofM. sexta andH. zea contains peptidase(s) capable of inactivating circulating PBAN.