The human ribosome-associated complex suppresses prion formation in yeast.

Many human diseases are associated with the misfolding of amyloidogenic proteins. Understanding the mechanisms cells employ to ensure the integrity of the proteome is therefore a crucial step in the development of potential therapeutic interventions. Yeast cells possess numerous prion-forming proteins capable of adopting amyloid conformations, possibly as an epigenetic mechanism to cope with changing environmental conditions. The ribosome-associated complex (RAC), which docks near the ribosomal polypeptide exit tunnel and recruits the Hsp70 Ssb to chaperone nascent chains, can moderate the acquisition of these amyloid conformations in yeast. Here we examine the ability of the human RAC chaperone proteins Mpp11 and Hsp70L1 to function in place of their yeast RAC orthologues Zuo1 and Ssz1 in yeast lacking endogenous RAC and investigate the extent to which the human orthologues can perform RAC chaperone activities in yeast. We found that the Mpp11/Hsp70L1 complex can partially correct the growth defect seen in RAC-deficient yeast cells, although yeast/human hetero species complexes were variable in this ability. The proportion of cells in which the Sup35 protein undergoes spontaneous conversion to a [PSI+ ] prion conformation, which is increased in the absence of RAC, was reduced by the presence of the human RAC complex. However, the toxicity in yeast from expression of a pathogenically expanded polyQ protein was unable to be countered by the human RAC chaperones. This yeast system can serve as a facile model for studying the extent to which the human RAC chaperones contribute to combating cotranslational misfolding of other mammalian disease-associated proteins.

Aggregation and Prion-Inducing Properties of the G-Protein Gamma Subunit Ste18 are Regulated by Membrane Association.

Yeast prions and mnemons are respectively transmissible and non-transmissible self-perpetuating protein assemblies, frequently based on cross-ß ordered detergent-resistant aggregates (amyloids). Prions cause devastating diseases in mammals and control heritable traits in yeast. It was shown that the de novo formation of the prion form [PSI+] of yeast release factor Sup35 is facilitated by aggregates of other proteins. Here we explore the mechanism of the promotion of [PSI+] formation by Ste18, an evolutionarily conserved gamma subunit of a G-protein coupled receptor, a key player in responses to extracellular stimuli. Ste18 forms detergent-resistant aggregates, some of which are colocalized with de novo generated Sup35 aggregates. Membrane association of Ste18 is required for both Ste18 aggregation and [PSI+] induction, while functional interactions involved in signal transduction are not essential for these processes. This emphasizes the significance of a specific location for the nucleation of protein aggregation. In contrast to typical prions, Ste18 aggregates do not show a pattern of heritability. Our finding that Ste18 levels are regulated by the ubiquitin-proteasome system, in conjunction with the previously reported increase in Ste18 levels upon the exposure to mating pheromone, suggests that the concentration-dependent Ste18 aggregation may mediate a mnemon-like response to physiological stimuli.

Self-Assembly of Antigen Proteins into Nanowires Greatly Enhances the Binding Affinity for High-Efficiency Target Capture.

High-efficiency target capture is an essential prerequisite for sensitive immunoassays. However, the current available immunoassay approaches are subject to deficient binding affinities between predator-prey molecules that greatly restrict the target capture efficiency and immunoassay sensitivity. Herein, we present a new strategy through the self-assembly of antigen proteins into nanowires to enhance the binding affinity between an antigen and antibody. Through the genetic fusion of antigen proteins (e.g., HIV p24) with the yeast amyloid protein Sup35 self-assembly domain, specific antigen nanowires (Ag nanowires) were constructed and demonstrated a remarkable enhancement in binding affinity compared with that of the monomeric antigen molecule. The Ag nanowires were further combined with magnetic beads to form a 3D magnetic probe based on a seed-induced self-assembly strategy. Taking advantage of both the strong binding affinity and the rapid magnetic separation and enrichment capacity, the specific 3D magnetic probe achieved a 100-fold improvement in detection sensitivity within a significantly shorter period of 20 min over that of the conventional enzyme-linked immunosorbent assay method.

Defining the protein complexome of translation termination factor eRF1: Identification of four novel eRF1-containing complexes that range from 20S to 57S in size.

The eukaryotic eRF1 translation termination factor plays an important role in recognizing stop codons and initiating the end to translation. However, which exact complexes contain eRF1 and at what abundance is not clear. We have used analytical ultracentrifugation with fluorescent detection system to identify the protein complexome of eRF1 in the yeast Saccharomyces cerevisiae. In addition to eRF1 presence in translating polysomes, we found that eRF1 associated with five other macromolecular complexes: 77S, 57S, 39S, 28S, and 20S in size. Generally equal abundances of each of these complexes were found. The 77S complex primarily contained the free 80S ribosome consistent with in vitro studies and did not appear to contain significant levels of the monosomal translating complex that co-migrates with the free 80S ribosome. The 57S and 39S complexes represented, respectively, free 60S and 40S ribosomal subunits bound to eRF1, associations not previously reported. The novel 28S and 20S complexes (containing minimal masses of 830 KDa and 500 KDa, respectively) lacked significant RNA components and appeared to be oligomeric, as eRF1 has a mass of 49 KDa. The majority of polysomal complexes containing eRF1 were both substantially deadenylated and lacking in closed-loop factors eIF4E and eIF4G. The thirteen percent of such translating polysomes that contained poly(A) tails had equivalent levels of eIF4E and eIF4G, suggesting these complexes were in a closed-loop structure. The identification of eRF1 in these unique and previously unrecognized complexes suggests a variety of new roles for eRF1 in the regulation of cellular processes.

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR.

The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as "rain" in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.

Characterization of aggregate load and pattern in living yeast cells by flow cytometry.

Protein aggregation is both a hallmark of and a driving force for a number of diseases. It is therefore important to identify the nature of these aggregates and the mechanism(s) by which the cell counteracts their detrimental properties. Currently, the study of aggregation in vivo is performed primarily using fluorescently tagged versions of proteins and analyzing the aggregates by fluorescence microscopy. While this strategy is considered the gold standard, it has several limitations, particularly with respect to its suitability for high-throughput screening (HTS). Here, using a GFP fusion of the well-characterized yeast prion amyloid protein [PSI+], we demonstrate that flow cytometry, which utilizes the same physical principles as fluorescence microscopy, can be used to determine the aggregate load and pattern in live and fixed yeast cells. Furthermore, our approach can easily be applied to high-throughput analyses such as screenings with a yeast deletion library.

Wild type huntingtin toxicity in yeast: Implications for the role of amyloid cross-seeding in polyQ diseases.

Proteins with expanded polyglutamine (polyQ) regions are prone to form amyloids, which can cause diseases in humans and toxicity in yeast. Recently, we showed that in yeast non-toxic amyloids of Q-rich proteins can induce aggregation and toxicity of wild type huntingtin (Htt) with a short non-pathogenic polyglutamine tract. Similarly to mutant Htt with an elongated N-terminal polyQ sequence, toxicity of its wild type counterpart was mediated by induced aggregation of the essential Sup35 protein, which contains a Q-rich region. Notably, polymerization of Sup35 was not caused by the initial benign amyloids and, therefore, aggregates of wild type Htt acted as intermediaries in seeding Sup35 polymerization. This exemplifies a protein polymerization cascade which can generate a network of interdependent polymers. Here we discuss cross-seeded protein polymerization as a possible mechanism underlying known interrelations between different polyQ diseases. We hypothesize that similar mechanisms may enable proteins, which possess expanded Q-rich tracts but are not associated with diseases, to promote the development of polyQ diseases.

Prions are affected by evolution at two levels.

Prions, infectious proteins, can transmit diseases or be the basis of heritable traits (or both), mostly based on amyloid forms of the prion protein. A single protein sequence can be the basis for many prion strains/variants, with different biological properties based on different amyloid conformations, each rather stably propagating. Prions are unique in that evolution and selection work at both the level of the chromosomal gene encoding the protein, and on the prion itself selecting prion variants. Here, we summarize what is known about the evolution of prion proteins, both the genes and the prions themselves. We contrast the one known functional prion, [Het-s] of Podospora anserina, with the known disease prions, the yeast prions [PSI+] and [URE3] and the transmissible spongiform encephalopathies of mammals.

Quantitative proteome analyses identify PrfA-responsive proteins and phosphoproteins in Listeria monocytogenes.

Protein phosphorylation is a major mechanism of signal transduction in bacteria. Here, we analyzed the proteome and phosphoproteome of a wild-type strain of the food-borne pathogen Listeria monocytogenes that was grown in either chemically defined medium or rich medium containing glucose. We then compared these results with those obtained from an isogenic prfA* mutant that produced a constitutively active form of PrfA, the main transcriptional activator of virulence genes. In the prfA* mutant grown in rich medium, we identified 256 peptides that were phosphorylated on serine (S), threonine (T), or tyrosine (Y) residues, with a S/T/Y ratio of 155:75:12. Strikingly, we detected five novel phosphosites on the virulence protein ActA. This protein was known to be phosphorylated by a cellular kinase in the infected host, but phosphorylation by a listerial kinase had not previously been reported. Unexpectedly, SILAC experiments with the prfA* mutant grown in chemically defined medium revealed that, in addition to previously described PrfA-regulated proteins, several other proteins were significantly overproduced, among them were several proteins involved in purine biosynthesis. This work provides new information for our understanding of the correlation among protein phosphorylation, virulence mechanisms, and carbon metabolism.

The processed isoform of the translation termination factor eRF3 localizes to the nucleus to interact with the ARF tumor suppressor.

The eukaryotic releasing factor eRF3 is a multifunctional protein that plays pivotal roles in translation termination as well as the initiation of mRNA decay. eRF3 also functions in the regulation of apoptosis; eRF3 is cleaved at Ala73 by an as yet unidentified protease into processed isoform of eRF3 (p-eRF3), which interacts with the inhibitors of apoptosis proteins (IAPs). The binding of p-eRF3 with IAPs leads to the release of active caspases from IAPs, which promotes apoptosis. Although full-length eRF3 is localized exclusively in the cytoplasm, p-eRF3 localizes in the nucleus as well as the cytoplasm. We here focused on the role of p-eRF3 in the nucleus. We identified leptomycin-sensitive nuclear export signal (NES) at amino acid residues 61-71 immediately upstream of the cleavage site Ala73. Thus, the proteolytic cleavage of eRF3 into p-eRF3 leads to release an amino-terminal fragment containing NES to allow the relocalization of eRF3 into the nucleus. Consistent with this, p-eRF3 more strongly interacted with the nuclear ARF tumor suppressor than full-length eRF3. These results suggest that while p-eRF3 interacts with IAPs to promote apoptosis in the cytoplasm, p-eRF3 also has some roles in regulating cell death in the nucleus.

Low activity of select Hsp104 mutants is sufficient to propagate unstable prion variants.

The molecular chaperone network plays a critical role in the formation and propagation of self-replicating yeast prions. Not only do individual prions differ in their requirements for certain chaperones, but structural variants of the same prion can also display distinct dependences on the chaperone machinery, specifically Hsp104. The AAA+ ATPase Hsp104 is a disaggregase required for the maintenance of most known yeast prions. As a key component in the propagation of prions, understanding how Hsp104 differs in its interaction with specific variants is crucial to understanding how prion variants may be selected or evolve. Here, we investigate two novel mutations in Hsp104, hsp104-G254D, and hsp104-G730D, which allow us to elucidate some mechanistic features of Hsp104 disaggregation and its requirement for activity in propagating specific prion variants. Both Hsp104 mutants propagate the [PSI+] prion to some extent, but show a high rate of prion loss. Both Hsp104-G254D and Hsp104-G730D display reduced biochemical activity, yet differ in their ability to efficiently resolubilize disordered, heat-aggregated substrates. Additionally, both mutants impair weak [PSI+] propagation, but are capable of propagating the less stable strong [PSI+] variant to some extent. One of the Hsp104 mutants also has the ability to propagate one variant of the [RNQ+] prion. Thus, our data suggest that changes in Hsp104 activity limit substrate disaggregation in a manner that depends more on the stability of the substrate than the nature of the aggregated species.

DNA aptamers detecting generic amyloid epitopes.

Amyloids are fibrillar protein aggregates resulting from non-covalent autocatalytic polymerization of various structurally and functionally unrelated proteins. Previously we have selected DNA aptamers, which bind specifically to the in vitro assembled amyloid fibrils of the yeast prionogenic protein Sup35. Here we show that such DNA aptamers can be used to detect SDS-insoluble amyloid aggregates of the Sup35 protein, and of some other amyloidogenic proteins, including mouse PrP, formed in yeast cells. The obtained data suggest that these aggregates and the Sup35 amyloid fibrils assembled in vitro possess common conformational epitopes recognizable by aptamers. The described DNA aptamers may be used for detection of various amyloid aggregates in yeast and, presumably, other organisms.

Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells.

Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

[Localization of polypeptides release factors and ribosome protein L11 in Euplotes octocarinatus].

Protozoan ciliates are a group of unicellular eukaryotes. The special characteristics of stop codons usage in termination of protein biosynthesis in ciliates cells makes them an ideal model to study the mechanism of stop codon recognition of polypeptides release factors. To localize the functional positions of biomolecules in ciliates cell, we constructed a macronuclear artificial chromosome containing a gene encoding red fluorescence protein (EoMAC_R) based on the structural characteristics of ciliates chromosome. Three factors, L11, eRF1a, and eRF3 that are involved in termination process of protein synthesis were colocalized in Euplotes octocarinatus cells by using novel EoMAC_R and the previously constructed EoMAC_G. The results indicated that protein synthesis mainly occurred inside the "C" shape macronucleus, suggesting that EoMAC could be a useful tool for localizing biomolecules in ciliates cell.

A positive regulatory role for LjERF1 in the nodulation process is revealed by systematic analysis of nodule-associated transcription factors of Lotus japonicus.

We have used reverse genetics to identify genes involved in legume-rhizobium symbiosis in Lotus japonicus. We obtained the sequences of 20 putative transcription factors from previously reported large-scale transcriptome data. The transcription factors were classified according to their DNA binding domains and patterns of expression during the nodulation process. We identified two homologues of Medicago truncatula MtHAP2-1, which encodes a CCAAT-binding protein and has been shown to play a role in nodulation. The functions of the remaining genes in the nodulation process have not been reported. Seven genes were found to encode proteins with AP2-EREBP domains, six of which were similar to proteins that have been implicated in ethylene and/or jasmonic acid signal transduction and defense gene regulation in Arabidopsis (Arabidopsis thaliana). We identified a gene, LjERF1, that is most similar to Arabidopsis ERF1, which is up-regulated by ethylene and jasmonic acid and activates downstream defense genes. LjERF1 showed the same pattern of up-regulation in roots as Arabidopsis ERF1. The nodulation phenotype of roots that overexpressed LjERF1 or inhibited LjERF1 expression using an RNA interference construct indicated that this gene functions as a positive regulator of nodulation. We propose that LjERF1 functions as a key regulator of successful infection of L. japonicus by Mesorhizobium loti.

Detection of Listeria monocytogenes in food using a combined enrichment/real-time PCR method targeting the prfA gene.

A combined enrichment/real-time PCR method for the detection of Listeria monocytogenes is presented. The method is based on a conventional PCR assay targeting the prfA gene, which has been validated and suggested as an international standard PCR method for identifying L. monocytogenes in food. This real-time PCR assay includes an internal amplification control. Inclusivity and exclusivity were 100% each when testing 100 L. monocytogenes isolates, 30 Listeria spp. isolates other than L. monocytogenes, and 29 non-Listeria isolates. The theoretical detection limit was one copy of the target gene per PCR reaction and the practical detection limit was about 5 copies per PCR. Using the combined enrichment/real-time PCR method, 7.5 CFU/25 ml of artificially contaminated raw milk, and 9, 1, and 1 CFU/15 g of artificially contaminated salmon, pâté, and green-veined cheese, respectively, were detected. When analyzing 76 naturally contaminated food samples of various types and comparing the results with the ISO 11290-1 standard method, the relative accuracy was 96%, the relative specificity 100%, and the relative sensitivity, 76.9%.

Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression.

Certain nascent peptide sequences, when within the ribosomal exit tunnel, can inhibit translation termination and/or peptide elongation. The 24 residue leader peptidyl-tRNA of the tna operon of E. coli, TnaC-tRNA(Pro), in the presence of excess tryptophan, resists cleavage at the tnaC stop codon. TnaC residue Trp12 is crucial for this inhibition. The approximate location of Trp12 in the exit tunnel was determined by crosslinking Lys11 of TnaC-tRNA(Pro) to nucleotide A750 of 23S rRNA. Methylation of nucleotide A788 of 23S rRNA was reduced by the presence of Trp12 in TnaC-tRNA(Pro), implying A788 displacement. Inserting an adenylate at position 751, or introducing the change U2609C in 23S rRNA or the change K90H or K90W in ribosomal protein L22, virtually eliminated tryptophan induction. These modified and mutated regions are mostly located near the putative site occupied by Trp12 of TnaC-tRNA(Pro). These findings identify features of the ribosomal exit tunnel essential for tna operon induction.

Expression of mitochondrial release factor in relation to respiratory competence in yeast.

Mitochondria have a single release factor that recognizes all stop codons in mRNAs. The yeast mitochondrial release factor, mRF1, is a protein of 43 kDa that emerges from its precursor by cleavage of a mitochondrial targeting sequence. mRF1 is localized exclusively in mitochondria, even when it is overproduced. A several-fold increase in mRF1 levels slightly inhibits the growth of wild-type cells on media containing a non-fermentable carbon source. A direct antisuppressor effect of overproduced mRF1 is observed, since the MRF1 gene on a multicopy plasmid causes Gly(-) phenotypes of the leaky mit(-) point mutations in mtDNA. We also examine steady-state mRF1 levels in a respiratory-deficient mrf1-780 mutant with inhibited mitochondrial translation. We show that both the mRF1 protein and the MRF1 transcript are elevated in mrf1-780 cells. A similar increase in mRF1 expression is observed in the rho(0) strain with no mitochondrial translation. This is indicative of retrograde signalling in the regulation of MRF1 expression. According to our hypothesis, inhibition of translation in the mrf1-780 strain is due to mitoribosome stalling at the stop codon and the observed elevated level of release factor is a secondary effect of respiratory deficiency.

[Characterization of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1]. / Kharakteristika missens-mutatsii v gene SUP45 drozhzhei Saccharomyces cerevisiae, kodiruiushchem faktor terminatsii translatsii eRF1.

Collection of missense mutations in the SUP45 gene of Saccharomyces cerevisiae encoding translation termination factor eRF1 has been obtained by different approaches. It has been shown that most of isolated mutations cause amino acid substitutions in the N-terminal part of eRF1 and do not decrease the eRF1 amount. Most of mutations studied do not abolish eRF1-eRF3 interaction. The role of the N-terminal part of eRF1 in stop codon recognition is discussed.

[A new method to measure the functional activity of class-1 translation termination factor eRF1]. / Novyi metod opredeleniia funktsional'noi aktivnosti factora terminatsii transliatsii pervogo klassa eRF1.

Termination of protein synthesis (hydrolysis of the last peptidyl-tRNA on the ribosome) takes place when the ribosomal A site is occupied simultaneously by one of the three stop codons and by a class-1 translation termination factor. The existing procedures to measure the functional activity of this factor both in vitro and in vivo have serious drawbacks, the main of which are artificial conditions for in vitro assays, far from those in the cell, and indirect evaluation of activity in in vivo systems. A simple reliable and sensitive system to measure the functional activity of class-1 translation termination factors could considerably expedite the study of the terminal steps of protein synthesis, at present remaining poorly known, especially in eukaryotes. We suggest a novel system to test the functional activity in vitro using native functionally active mRNA, rather than tri-, tetra-, or oligonucleotides as before. This mRNA is specially designed to contain one of the three terminating (stop) codons within the coding nucleotide sequence. Plasmids have been generated that carry the genes of suppressor tRNAs each of which is specific toward one of the three stop codons. They were shown to support normal synthesis of a reporter protein, luciferase, by reading through the stop codon within the coding mRNA sequence. We have demonstrated that human class-1 translation termination factor eRF1 is able to compete with suppressor tRNA for a stop codon and to completely prevent its suppressive effect at a sufficient concentration. Forms of eRF1 with point mutations in functionally essential regions have lower competitive ability, demonstrating the sensitivity of the method to the eRF1 structure. The enzymatic reaction catalyzed by the full-size reporter protein is accompanied by emission of light quanta. Therefore, competition between suppressor tRNA and eRF1 can be measured using a luminometer, and this allows precise kinetic measurements in a continuous automatic mode.