A Promiscuous Prion: Efficient Induction of [URE3] Prion Formation by Heterologous Prion Domains

The [URE3] and [PSI⁺] prions are the infections amyloid forms of the Saccharomyces cerevisiae proteins Ure2p and Sup35p, respectively. Randomizing the order of the amino acids in the Ure2 and Sup35 prion domains while retaining amino acid composition does not block prion formation, indicating that amino acid composition, not primary sequence, is the predominant feature driving [URE3] and [PSI⁺] formation. Here we show that Ure2p promiscuously interacts with various compositionally similar proteins to influence [URE3] levels. Overexpression of scrambled Ure2p prion domains efficiently increases de novo formation of wild-type [URE3] in vivo. In vitro, amyloid aggregates of the scrambled prion domains efficiently seed wild-type Ure2p amyloid formation, suggesting that the wild-type and scrambled prion domains can directly interact to seed prion formation. To test whether interactions between Ure2p and naturally occurring yeast proteins could similarly affect [URE3] formation, we identified yeast proteins with domains that are compositionally similar to the Ure2p prion domain. Remarkably, all but one of these domains were also able to efficiently increase [URE3] formation. These results suggest that a wide variety of proteins could potentially affect [URE3] formation.AMYLOID fibril formation is associated with numerous human diseases, including Alzheimer''s disease, type II diabetes, and the transmissible spongiform encephalopathies. Yeast prions provide a powerful model system for examining amyloid fibril formation in vivo. [URE3] and [PSI⁺] are the prion forms of the Saccharomyces cerevisiae proteins Ure2p and Sup35p, respectively (Wickner 1994). In both cases, prion formation is thought to result from conversion of the native protein into an inactive amyloid form (Glover et al. 1997; King et al. 1997; Taylor et al. 1999). Both proteins contain an N-terminal glutamine/asparagine (Q/N)-rich prion-forming domain (PFD) and a C-terminal functional domain (Ter-Avanesyan et al. 1993; Ter-Avanesyan et al. 1994; Masison and Wickner 1995; Liebman and Derkatch 1999; Maddelein and Wickner 1999). Sup35p contains an additional highly charged middle domain (M) that is not required either for prion formation or for normal protein function, but stabilizes [PSI⁺] aggregates (Liu et al. 2002).Amyloid fibril formation is thought to occur through a seeded polymerization mechanism. In vitro, amyloid fibril formation from native proteins is generally characterized by a significant lag time, thought to result from the slow rate of formation of amyloid nuclei; addition of a small amount of preformed amyloid aggregates (seeds) eliminates the lag time, resulting in rapid polymerization (Glover et al. 1997; Taylor et al. 1999; Serio et al. 2000).Despite considerable study, the mechanism by which amyloid seeds initially form is unclear. At least some of the amyloid proteins involved in human disease can interact with unrelated amyloidogenic proteins, resulting in cross-seeding and modulation of toxicity. Injecting mice with amyloid-like fibrils formed by a variety of short synthetic peptides promotes amyloid formation by amyloid protein A, a protein whose deposition is found in systemic AA amyloidosis (Johan et al. 1998). In yeast, [PSI⁺] and [PIN⁺], the prion form of the protein Rnq1p (Sondheimer and Lindquist 2000; Derkatch et al. 2001), both promote the aggregation of and increase toxicity of expanded polyglutamine tracts, like those seen in Huntington''s disease (Osherovich and Weissman 2001; Meriin et al. 2002; Derkatch et al. 2004; Gokhale et al. 2005; Duennwald et al. 2006); however, in Drosophila, [PSI⁺] aggregates reduce polyglutamine toxicity (Li et al. 2007). Thus, interactions between heterologous amyloidogenic proteins can influence amyloid formation both positively and negatively in vivo.A variety of interactions have been observed among the yeast prions. Under normal cellular conditions, efficient formation, but not maintenance, of [PSI⁺] requires the presence of [PIN⁺] (Derkatch et al. 2000). Overexpression of various Q/N-rich proteins can effectively substitute for [PIN⁺], allowing [PSI⁺] formation in cells lacking [PIN⁺] (Derkatch et al. 2001; Osherovich and Weissman 2001). In vitro and in vivo evidence suggest that the ability of [PIN⁺] to facilitate [PSI⁺] formation is the result of a direct interaction between Rnq1p aggregates and Sup35p (Derkatch et al. 2004; Bardill and True 2009; Choe et al. 2009). [PIN⁺] also increases the frequency of [URE3] formation, while [PSI⁺] inhibits [URE3] formation (Bradley et al. 2002; Schwimmer and Masison 2002).It is unclear whether the ability of Ure2p, Sup35p, and Rnq1p to cross-react is an intrinsic feature of all similar amyloidogenic proteins, or whether it has specifically evolved to regulate prion formation. There is debate as to whether yeast prion formation is a beneficial phenomenon, allowing for regulation of the activity of the prion protein (True and Lindquist 2000; True et al. 2004), or a deleterious event analogous to human amyloid disease (Nakayashiki et al. 2005). Either way, it is likely that interactions between the yeast prion proteins have specifically evolved, either to minimize the detrimental effects of amyloid formation or to regulate beneficial amyloid formation.For both Ure2p and Sup35p, the amino acid composition of the PFD is the predominant feature that drives prion formation. Scrambled versions of Ure2p and Sup35p (in which the order of the amino acids in the PFD was randomized while maintaining amino acid composition) are able to form prions when expressed in yeast as the sole copy Ure2p or Sup35p (Ross et al. 2004, 2005). To examine whether amino acid composition can similarly drive interactions between heterologous proteins, we tested whether the scrambled PFDs can interact with their wild-type counterparts to stimulate prion formation. When overexpressed, scrambled Ure2 PFDs promoted de novo prion formation by wild-type Ure2p, suggesting that the Ure2p PFD can promiscuously interact with compositionally similar PFDs during prion formation. When we searched the yeast proteome for proteins with regions of high compositional similarity to Ure2p, four of the top five proteins were able to efficiently stimulate [URE3] formation. However, there were limits to this promiscuity; overexpression of wild-type or scrambled Sup35 PFDs did not increase [URE3] levels. We propose that this ability to promiscuously interact may have evolved as a mechanism to regulate Ure2p activity and/or prion formation.     

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 October 2009-30 November 2009

This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus.     

A recombinant vaccine effectively induces c5a-specific neutralizing antibodies and prevents arthritis

Objectives

To develop and validate a recombinant vaccine to attenuate inflammation in arthritis by sustained neutralization of the anaphylatoxin C5a.

Methods

We constructed and expressed fusion protein of C5a and maltose binding protein. Efficacy of specific C5a neutralization was tested using the fusion protein as vaccine in three different arthritis mouse models: collagen induced arthritis (CIA), chronic relapsing CIA and collagen antibody induced arthritis (CAIA). Levels of anti-C5a antibodies and anti-collagen type II were measured by ELISA. C5a neutralization assay was done using a rat basophilic leukemia cell-line transfected with the human C5aR. Complement activity was determined using a hemolytic assay and joint morphology was assessed by histology.

Results

Vaccination of mice with MBP-C5a led to significant reduction of arthritis incidence and severity but not anti-collagen antibody synthesis. Histology of the MBP-C5a and control (MBP or PBS) vaccinated mice paws confirmed the vaccination effect. Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered.

Conclusions

Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases. Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity.     

Autophagy as a protective response to Bnip3-mediated apoptotic signaling in the heart

Bnip3 is a member of the 'BH3-only' Bcl-2 subfamily which has been implicated in apoptotic,(1) necrotic(2) and autophagic cell death.(3,4) We recently reported that Bnip3 is a key mediator of mitochondrial dysfunction and cell death in the ex vivo heart following ischemia/reperfusion (I/R).(5) Moreover, we found that Bnip3 was involved in upregulation of autophagy in I/R and that Bnip3-mediated mitochondrial dysfunction correlated with upregulation of autophagy. Using a model of simulated I/R and overexpression of Bnip3 in HL-1 cardiac myocytes, we determined that Bnip3-mediated upregulation of autophagic activity constituted a protective response against Bnip3 death signaling. Here we present additional evidence that enhanced autophagic activity functions as a cytoprotective pathway to oppose ischemia/reperfusion-related apoptosis.     

Development of the Minimum Information Specification for In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

We describe the creation process of the Minimum Information Specification for In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE). Modeled after the existing minimum information specification for microarray data, we created a new specification for gene expression localization experiments, initially to facilitate data sharing within a consortium. After successful use within the consortium, the specification was circulated to members of the wider biomedical research community for comment and refinement. After a period of acquiring many new suggested requirements, it was necessary to enter a final phase of excluding those requirements that were deemed inappropriate as a minimum requirement for all experiments. The full specification will soon be published as a version 1.0 proposal to the community, upon which a more full discussion must take place so that the final specification may be achieved with the involvement of the whole community.     

Lactic acid bacteria as probiotics

A number of Lactobacillus species, Bifidobacterium sp, Saccharomyces boulardii, and some other microbes have been proposed as and are used as probiotic strains, i.e. live microorganisms as food supplement in order to benefit health. The health claims range from rather vague as regulation of bowel activity and increasing of well-being to more specific, such as exerting antagonistic effect on the gastroenteric pathogens Clostridium difficile, Campylobacter jejuni, Helicobacter pylori and rotavirus, neutralising food mutagens produced in colon, shifting the immune response towards a Th2 response, and thereby alleviating allergic reactions, and lowering serum cholesterol (Tannock, 2002). Unfortunately, most publications are case reports, uncontrolled studies in humans, or reports of animal or in vitro studies. Whether or not the probiotic strains employed shall be of human origin is a matter of debate but this is not a matter of concern, as long as the strains can be shown to survive the transport in the human gastrointestinal (GI) tract and to colonise the human large intestine. This includes survival in the stressful environment of the stomach - acidic pH and bile - with induction of new genes encoding a number of stress proteins. Since the availability of antioxidants decreases rostrally in the GI tract production of antioxidants by colonic bacteria provides a beneficial effect in scavenging free radicals. LAB strains commonly produce antimicrobial substance(s) with activity against the homologous strain, but LAB strains also often produce microbicidal substances with effect against gastric and intestinal pathogens and other microbes, or compete for cell surface and mucin binding sites. This could be the mechanism behind reports that some probiotic strains inhibit or decrease translocation of bacteria from the gut to the liver. A protective effect against cancer development can be ascribed to binding of mutagens by intestinal bacteria, reduction of the enzymes beta-glucuronidase and beta-glucosidase, and deconjugation of bile acids, or merely by enhancing the immune system of the host. The latter has attracted considerable interest, and LAB have been tested in several clinical trials in allergic diseases. Characteristics ascribed to a probiotic strain are in general strain specific, and individual strains have to be tested for each property. Survival of strains during production, packing and storage of a viable cell mass has to be tested and declared.     

The influence of kainic acid on core temperature and cytokine levels in the brain

Excitotoxic brain injury is associated with hyperthermia, and there are data showing beneficial effects of hypothermia on neurodegeneration and that hyperthermia facilitates the neurodegeneration. Cytokines are inflammatory proteins that seem to be involved in the neuroinflammation associated with epilepsy. Core temperature changes caused by the epileptogenic glutamate analogue kainic acid (KA) were investigated in relation to changes in levels of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and interleukin-6 (IL-6), and the endogenous interleukin-1 receptor antagonist (IL-1ra). The temperature was measured every 10 min during the first hour, and at 90 and 120 min, and hourly until 8 h after KA-injection (10 mg/kg). The cytokines were measured in the hypothalamus, a site of temperature regulation, and in hippocampus, cerebellum, and frontal cortex. KA induced a brief hypothermia followed by hyperthermia. IL-1beta levels were increased after KA-administration in all brain regions examined and, excepting hippocampus, returned to baseline levels at 24 h. The hippocampal IL-1ra levels were significantly increased at 24 h, whereas no changes in IL-6 levels were observed. The changes in IL-1beta levels and in ratios between the levels of the three cytokines, may account for some of the temperature changes and the behavioural manifestations induced by KA.     

Effects of post-electrophoretic analysis on variance in gel-based proteomics

2D electrophoresis (2DE) is a prominent separation method for complex proteomes. Although recent advances have increased the utility of this method in quantitative proteomics studies, many sources of variance still exist. This review discusses the post-electrophoretic sources of variance in current 2DE analysis. The essential improvements in protein visualization and software algorithms that have made 2DE a leading quantitative proteomics method are briefly reviewed. A number of shortcomings in the post-electrophoretic analysis of 2DE data that require further attention are highlighted. Topics discussed include protein visualization and image acquisition, internal standards and normalization methods, background subtraction algorithms, normality of distribution, and the need for standardized tests for the evaluation of 2DE analysis software packages.     

LogoBar: bar graph visualization of protein logos with gaps

SUMMARY: LogoBar is a Java application to display protein sequence logos. In our software gaps are accounted for when calculating the information content present at each residue position in a multiple alignment. The resulting logo is displayed as a graph consisting of bars, although traditional letter representation is also possible. Amino acids are displayed from the bottom up with decreasing frequencies i.e. the most abundant residue is placed at the bottom of the logo. The bars can be color-coded according to user specifications. Gaps in the alignment are also displayed, either on top or at the bottom of the logo. Furthermore, residues can either be arranged according to their relative abundance or grouped according to user criteria to emphasize the conserved nature of particular positions. AVAILABILITY: LogoBar and further documentation is available at http://www.biosci.ki.se/groups/tbu/logobar/