Klaartje Pellens

Phosphorylation, lipid raft interaction and traffic of α-synuclein in a yeast model for Parkinson

Parkinsons disease is a neurodegenerative disorder characterized by the formation of Lewy bodies containing aggregated alpha-synuclein. We used a yeast model to screen for deletion mutants with mislocalization and enhanced inclusion formation of alpha-synuclein. Many of the mutants were affected in functions related to vesicular traffic but especially mutants in endocytosis and vacuolar degradation combined inclusion formation with enhanced alpha-synuclein-mediated toxicity. The screening also allowed for identification of casein kinases responsible for alpha-synuclein phosphorylation at the plasma membrane as well as transacetylases that modulate the alpha-synuclein membrane interaction. In addition, alpha-synuclein was found to associate with lipid rafts, a phenomenon dependent on the ergosterol content. Together, our data suggest that toxicity of alpha-synuclein in yeast is at least in part associated with endocytosis of the protein, vesicular recycling back to the plasma membrane and vacuolar fusion defects, each contributing to the obstruction of different vesicular trafficking routes.

Characterization of α-synuclein aggregation and synergistic toxicity with protein tau in yeast

A yeast model was generated to study the mechanisms and phenotypical repercussions of expression of α‐synuclein as well as the coexpression of protein tau. The data show that aggregation of α‐synuclein is a nucleation–elongation process initiated at the plasma membrane. Aggregation is consistently enhanced by dimethyl sulfoxide, which is known to increase the level of phospholipids and membranes in yeast cells. Aggregation of α‐synuclein was also triggered by treatment of the yeast cells with ferrous ions, which are known to increase oxidative stress. In addition, data are presented in support of the hypothesis that degradation of α‐synuclein occurs via autophagy and proteasomes and that aggregation of α‐synuclein disturbs endocytosis. Reminiscent of observations in double‐transgenic mice, coexpression of α‐synuclein and protein tau in yeast cells is synergistically toxic, as exemplified by inhibition of proliferation. Taken together, the data show that these yeast models recapitulate major aspects of α‐synuclein aggregation and cytotoxicity, and offer great potential for defining the underlying mechanisms of toxicity and synergistic actions of α‐synuclein and protein tau.

Protein folding diseases and neurodegeneration: Lessons learned from yeast

Budding yeast Saccharomyces cerevisiae has proven to be a valuable model organism for studying fundamental cellular processes across the eukaryotic kingdom including man. In this respect, complementation assays, in which the yeast protein is replaced by a homologous protein from another organism, have been very instructive. A newer trend is to use the yeast cell factory as a toolbox to understand cellular processes controlled by proteins for which the yeast lacks functional counterparts. An increasing number of studies have indicated that S. cerevisiae is a suitable model system to decipher molecular mechanisms involved in a variety of neurodegenerative disorders caused by aberrant protein folding. Here we review the current knowledge gained by the use of so-called humanized yeasts in the field of Huntingtons, Parkinsons and Alzheimers diseases.

Anti-Biofilm Strategies: How to Eradicate Candida Biofilms?

In nature, microorganisms prefer to reside in structured microbial communities, termed biofilms, rather than as free-floating planktonic cells. Advantageous for the microorganisms, but disadvantageous for human health, is the increased resistance/tolerance of the biofilm cells to antimicrobial treatment. In clinically relevant biofilms, Candida albicans is one of the most frequently isolated microorganisms in biofilms. This review primarily elaborates on the activity of the currently used antimycotics against Candida biofilms, the potential of antifungal lock therapy and sheds more light on new promising compounds resulting from the gradual shift of anti-biofilm research activities to natural products, plants and their extracts.

Antifungal activity in plants from Chinese traditional and folk medicine

ETHNOPHARMACOLOGICAL RELEVANCE From over 100 Chinese clinical trial publications, we retrieved 22 commercial preparations and 17 clinical prescriptions used as Traditional Chinese Medicine (TCM) for treating mycotic vaginitis, typically caused by Candida albicans. The 8 most frequently used plants as well as another 7 TCM and 18 folk medicinal plants used in the South of China for antifungal therapy were investigated for in vitro antifungal activity. MATERIALS AND METHODS For each plant we tested 4 extracts prepared with different solvents (water, ethanol, acetone, and n-hexane) for inhibition of Candida albicans and Saccharomyces cerevisiae growth in liquid culture. RESULTS Some plants have quite strong antifungal activity, such as Tujinpi (Pseudolarix kaempferi Gord.), of which each extract could significantly inhibit the growth of both tested fungi. In addition, the acetone extract of Kushen (Sophora flavescens Ait.), the ethanol, acetone, and hexane extracts of Guanghuoxiang (Pogostemon cablin (Blanco) Benth.) and Gaoliangjiang (Alpinia officinarum Hance), the hexane extract of Dingxiang (Eugenia caryophyllata Thunb.), and the ethanol and acetone extracts of Kulianpi (Melia toosendan Sieb. et Zucc.) and Laliao (Polygonum hydropiper L.), all inhibited Candida albicans growth by more than 50%. In some cases growth inhibition was even comparable to that by the clinically used antifungal miconazole, which we used as our positive control. CONCLUSIONS The majority of plants, whose clinical use for antifungal treatment is well supported within TCM or Chinese folk medicine, show in vitro antifungal activity against Candida albicans. Since Candida species represent the most common fungal pathogen of humans, these results provide more scientific evidence supporting the clinical application of these plants, and can serve as a starting point for new drug discovery from TCM and Chinese folk medicine.

A fungicidal piperazine-1-carboxamidine induces mitochondrial fission-dependent apoptosis in yeast

To unravel the working mechanism of the fungicidal piperazine-1-carboxamidine derivative BAR0329, we found that its intracellular accumulation in Saccharomyces cerevisiae is dependent on functional lipid rafts. Moreover, BAR0329 induced caspase-dependent apoptosis in yeast, in which the mitochondrial fission machinery consisting of Fis1 (Whi2), Dnm1 and Mdv1 is involved. Our data are consistent with a prosurvival function of Fis1 (Whi2) and a proapoptotic function of Dnm1 and Mdv1 during BAR0329-induced yeast cell death.

Design and Synthesis of a Series of Piperazine‐1‐carboxamidine Derivatives with Antifungal Activity Resulting from Accumulation of Endogenous Reactive Oxygen Species

In this study, we screened a library of 500 compounds for fungicidal activity via induction of endogenous reactive oxygen species (ROS) accumulation. Structure–activity relationship studies showed that piperazine‐1‐carboxamidine analogues with large atoms or large side chains substituted on the phenyl group at the R3 and R5 positions are characterized by a high ROS accumulation capacity in Candida albicans and a high fungicidal activity. Moreover, we could link the fungicidal mode of action of the piperazine‐1‐carboxamidine derivatives to the accumulation of endogenous ROS.

Novel fungicidal benzylsulfanyl-phenylguanidines

A series of substituted benzylsulfanyl-phenylamines was synthesized, of which four substituted benzylsulfanyl-phenylguanidines (665, 666, 667 and 684) showed potent fungicidal activity (minimal fungicidal concentration, MFC ≤ 10 μM for Candida albicans and Candida glabrata). A benzylsulfanyl-phenyl scaffold with an unsubstituted guanidine resulted in less active compounds (MFC=50-100 μM), whereas substitution with an unsubstituted amine group resulted in compounds without fungicidal activity. Compounds 665, 666, 667 and 684 also showed activity against single C. albicans biofilms and biofilms consisting of C. albicans and Staphylococcus epidermidis (minimal concentration resulting in 50% eradication of the biofilm, BEC50 ≤ 121 μM for both biofilm setups). Compounds 665 and 666 combined potent fungicidal (MFC=5 μM) and bactericidal activity (minimal bactericidal concentration, MBC for S. epidermidis ≤ 4 μM). In an in vivo Caenorhabditis elegans model, compounds 665 and 667 exhibited less toxicity than 666 and 684. Moreover, addition of those compounds to Candida-infected C. elegans cultures resulted in increased survival of Candida-infected worms, demonstrating their in vivo efficacy in a mini-host model.

P2-033: Molecular determinants of the phosphorylation and aggregation of human protein tau in a yeast model

aim of this study was to elucidate functional properties of IMPAS proteins. Methods: We applied mammalian cell culture proteolytic assays and soil nematode C.elegans model. Results and Conclusions: Three IMPAS genes in C.elegans (Ce-imp-1, Ce-imp-2, Ce-imp-3) and five paralogous human genes genes (IMP1-IMP5) were cloned and studied. Based on original alignment comparisons we predicted that human hIMP1/SPP and Ce-imp-2 are structural and functional orthologs. Inhibition of Ce-imp-2 gene by dsRNAi or in knockout mutant strains resulted in slow growth, uncoordinated movement, reduced brood size and larval death due to the incomplete shedding of cuticle (ICS) in the animals. The progeny of heterozygous deletion mutant Ce-imp-2 was analyzed by single worm PCR. The data demonstrated that homozygous mutants showed development pathology of late embryo death and ISC defects that is unrelated to Notch defects described for presenilin (sel-12, hop-1) mutants. This specific phenotype mimics features observed in worms with low function of megalin gene or on cholesterol diet. Inhibition of other IMPAS homologues in C.elegans (Ce-imp-1, Ce-imp-3) did not result in any obvious phenotype. We revealed that in co-expressing experiments with PS1 both human IMP1 and Ce-imp-2 were capable to cleave C-terminal part of PS1 in transmembrane domain. The data indicate that Ce-imp-2 is likely orthologous gene to hIMP1/SPP with similar proteolytic functions. The IMP1/Ce-imp-2induced and PS1-induced proteolytic cleavage assays were used to delineate essential domains and amino-acid signatures important for enzymatic or conformation properties critical for proteolytic functions of two types of intramembrane proteases (IMPAS and presenilin families).

P2-034: Deletion of genes responsible for vesicular transport in yeast causes mislocalization and enhanced toxicity of α-synuclein

-Synuclein is a small pre-synaptic protein involved in the pathology of Parkinson. The function of -synuclein is still unclear but was recently suggested to be important for vesicular transport. In order to define a role of -synuclein in vesicular transport and vesicular pathways, we expressed -synuclein as a native or as a GFP-tagged fusion protein in the yeast Saccharomyces cerevisiae. This is a suitable model since yeast expresses many functional orthologues of mammalian vesicular proteins. Similar to its localization in mammalian cells, both native -synuclein and -synuclein-GFP were bound to the cytoplasmic side of the plasma membrane in wild type yeast cells (Zabrocki et al., FEBS J., 272, 1386-1400, 2005). We screened mutant yeast strains lacking different proteins that are proven or suspected to be involved in vesicular transport. We observed distinct patterns of -synuclein mislocalization, that often coincided with enhanced toxicity of -synuclein. We provide evidence for connections of -synuclein to vesicular transport involving ESCRT I, II, III complexes, SNAREs, the HOPS complex and typical proteins of the Golgi complex. Many of these proteins are related to endosomal and ubiquitin-dependent transport, which links -synuclein to different vesicular transport systems.