Christophe Cullin

Isolation of drugs active against mammalian prions using a yeast-based screening assay.

We have developed a rapid, yeast-based, two-step assay to screen for antiprion drugs. The method allowed us to identify several compounds effective against budding yeast prions responsible for the [PSI+] and [URE3] phenotypes. These inhibitors include the kastellpaolitines, a new class of compounds, and two previously known molecules, phenanthridine and 6-aminophenanthridine. Two potent promoters of mammalian prion clearance in vitro, quinacrine and chlorpromazine, which share structural similarities with the kastellpaolitines, were also active in the assay. The compounds isolated here were also active in promoting mammalian prion clearance. These results validate the present method as an efficient high-throughput screening approach to identify new prion inhibitors and furthermore suggest that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to humans.

Enhanced in vivo monooxygenase activities of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and human cytochrome b5

We have engineered yeast genomic DNA to construct a set of strains producing various relative amounts of yeast NADPH-P450 reductase (Yred) and human cytochrome b5 (Hb5). Expression of cDNAs encoding human P450 1A1, 1A2, 3A4, 19A and mouse P450 1A1 in the different oxido-reduction backgrounds thus constituted were achieved after strain transformation by plasmid-based P450-encoding expression cassettes. The results indicate that the level of Yred strongly affects all activities tested. In contrast, the amount of Hb5 affects activities in a manner that is dependent both on the P450 isoform considered and the Yred level. In a strain containing optimized amounts of Hb5 and Yred, human P450 3A4-specific testosterone-6 beta-hydroxylase activity can be enhanced as much as 73-fold in comparison with the activity observed in a wild-type strain. Bioconversion of sterols or xenobiotics was easily achieved in vivo using this new co-expression system.

Structural Characterization of Saccharomyces cerevisiae Prion-like Protein Ure2

Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.

Nitrogen catabolic repression controls the release of volatile thiols by Saccharomyces cerevisiae during wine fermentation

Volatile thiols such as 4-methyl-4-sulfanylpentan-2-one (4MSP) and 3-sulfanylhexan-1-ol (3SH) are aromatic molecules having an important organoleptic impact on white wines. These components are produced from inodorous nonvolatile cysteinylated precursors by Saccharomyces cerevisiae metabolic activity during alcoholic fermentation. Here we provide a new insight into the genetic determinism of the production of volatile thiols by yeast. Using a gene deletion approach, we investigated the role of three yeast beta-lyases and demonstrate that Irc7p, a putative cystathionine beta-lyase, is one of the main proteins catalyzing the 4MSP and 3SH release under enological conditions. Moreover, we demonstrate that Ure2p/Gln3p proteins mainly control the bioconversion of volatile thiols by the transcriptional regulation of the IRC7 gene through the general mechanism of nitrogen catabolic repression. Finally, our findings suggest that the enantiomer balance of 3SH may be modulated by activating specifically stereoselective enzymes such as Irc7p.

Internal initiation drives the synthesis of Ure2 protein lacking the prion domain and affects [URE3] propagation in yeast cells

The [URE3] phenotype in Saccharomyces cerevisiae is caused by the inactive, altered (prion) form of the Ure2 protein (Ure2p), a regulator of nitrogen catabolism. Ure2p has two functional domains: an N‐terminal domain necessary and sufficient for prion propagation and a C‐terminal domain responsible for nitrogen regulation. We show here that the mRNA encoding Ure2p possesses an IRES (internal ribosome entry site). Internal initiation leads to the synthesis of an N‐terminally truncated active form of the protein (amino acids 94–354) lacking the prion‐forming domain. Expression of the truncated Ure2p form (94–354) mediated by the IRES element cures yeast cells of the [URE3] phenotype. We assume that the balance between the full‐length and truncated (94–354) Ure2p forms plays an important role in yeast cell physiology and differentiation.

Cloning and characterization of a yeast cytochrome b5-encoding gene which suppresses ketoconazole hypersensitivity in a NADPH-P-450 reductase-deficient strain

Cytochrome P-450 (Cyp) 51 or lanosterol-C14-demethylase is the main target for antifungal compounds of the triazole family like ketoconazole (Kz). Disruption of the associated NADPH-P-450 reductase-encoding gene (YRED) is not lethal, but decreases by about 20-fold the Kz resistance (KzR) of wild-type (wt) Saccharomyces cerevisiae. Transformation of a YRED-disrupted strain by a yeast genomic library based on a multicopy vector allowed us to identify a suppressor of Kz hypersensitivity. Deletion analysis of the 5-kb cloned fragment indicated that yeast cytochrome b5-encoding gene (CYB5), which encodes a 120-amino-acid (aa) protein, is required and sufficient for the suppressor effect. The encoded polypeptide shares about 30% aa identity with mammalian cytochromes b5 (Cyb5). CYB5 disruption and tetrad analysis demonstrate that yeast Cyb5 is not required for growth in a Yred+ strain. Determination of the microsomal content of b-type cytochromes by differential spectra indicated the presence of a strongly decreased or null Cyb5 level in the disrupted strain. This confirms that we have cloned the gene encoding the major microsomal form of Cyb5 which appears not to be essential. Minor Cyb5 isoforms could also be present in yeast or other redox proteins could substitute for the pleiotropic roles of Cyb5 in the sterol and lipid biosynthesis pathways.

The yeast prion [URE3] can be greatly induced by a functional mutated URE2 allele

The non‐Mendelian element [URE3] of yeast is considered to be a prion form of the Ure2 protein. The [URE3] phenotype occurs at a frequency of 10−5 in haploid yeast strains, is reversible, and its frequency is increased by overexpressing the URE2 gene. We created a new mutant of the Ure2 protein, called H2p, which results in a 1000‐fold increase in the rate of [URE3] occurrence. To date, only the overexpression of various C‐terminal truncated mutants of Ure2p gives rise to a comparable level. The h2 allele is, thus, the first characterized URE2 allele that induces prion formation when expressed at a low level. By shuffling mutated and wild‐type domains of URE2, we also created the first mutant Ure2 protein that is functional and induces prion formation. We demonstrate that the domains of URE2 function synergistically in cis to induce [URE3] formation, which highlights the importance of intramolecular interactions in Ure2p folding. Additionally, we show using a green fluorescent protein (GFP) fusion protein that the h2 allele exhibits numerous filiform structures that are not generated by the wild‐type protein.

Rubber Elongation Factor (REF), a Major Allergen Component in Hevea brasiliensis Latex Has Amyloid Properties

REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom.

The polyphenol piceid destabilizes preformed amyloid fibrils and oligomers in vitro: hypothesis on possible molecular mechanisms.

Alzheimer’s disease (AD) is characterized by deposits of amyloid in various tissues. The neuronal cytotoxicity of Aβ peptides is attributed not only to various mechanisms but also to amyloid fibrils and soluble oligomeric intermediates. Consequently, finding molecules to prevent or reverse the oligomerization and fibrillization of Aβ could be of therapeutic value in the treatment of AD. We show that piceid, a polyphenol of the stilbene family, destabilized fibrils and oligomers to give back monomers that are not neurotoxic molecules. The mechanism of this destabilization could be a dynamic interaction between the polyphenol and the Aβ that could open the hydrophobic zipper and shift the reversible equilibrium “random coil⇔β-sheet” to the disordered structure.

A yeast model for amyloid-β aggregation exemplifies the role of membrane trafficking and PICALM in cytotoxicity

SUMMARY Alzheimer’s disease is the most common neurodegenerative disease, associated with aggregation of amyloid-β (Aβ) peptides. The exact mechanism of neuronal cell dysfunction in Alzheimer’s disease is poorly understood and numerous models have been used to decipher the mechanisms leading to cellular death. Yeast cells might be a good model to understand the intracellular toxicity triggered by Aβ peptides. Indeed, yeast has been used as a model to examine protein functions or cellular pathways that mediate the secretion, aggregation and subsequent toxicity of proteins associated with human neurodegenerative disorders. In the present study, we use the yeast Saccharomyces cerevisiae as a model system to study the effects of intracellular Aβ in fusion with green fluorescent protein. We sent this fusion protein into the secretory pathway and showed that intracellular traffic pathways are necessary for the generation of toxic species. Yeast PICALM orthologs are involved in cellular toxicity, indicating conservation of the mechanisms of toxicity from mammals to yeast. Finally, our model demonstrates the capacity for intracellular Aβ to cross intracellular membranes and target mitochondrial organelles.