The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines

Sequence variants or mutations in the GBA gene are numerically the most important risk factor for Parkinson disease (PD). The GBA gene encodes for the lysosomal hydrolase enzyme, glucocerebrosidase (GCase). GBA mutations often reduce GCase activity and lead to impairment of the autophagy-lysosomal pathway, which is important in the turnover of alpha-synuclein, accumulation of which is a key pathological hallmark of PD. Although the E326K variant is one of the most common GBA variants associated with PD, there is limited understanding of its biochemical effects. We have characterised homozygous and heterozygous E326K variants in human fibroblasts. We found that E326K variants did not cause significant loss of GCase protein or activity, endoplasmic reticulum (ER) retention or ER stress, in contrast to the L444P GBA mutation. This was confirmed in human dopaminergic SH-SY5Y neuroblastoma cell lines over-expressing GCase with either E326K or L444P protein. Despite no loss of GCase activity, a significant increase of insoluble alpha-synuclein aggregates in E326K and L444P mutants was observed. Notably, SH-SY5Y over-expressing E326K demonstrated a significant increase in lipid droplet number under basal conditions, which was exacerbated following treatment with the fatty acid oleic acid. Similarly, a significant increase in lipid droplet formation following lipid loading was observed in heterozygous and homozygous E326K fibroblasts. In conclusion, the work presented here demonstrates that the E326K mutation behaves differently to common loss of function GBA mutations, however lipid dyshomeostasis and alpha-synuclein pathology is still evident

Simultaneous detection and identification of pathogenic fungi in wheat using a DNA macroarray

The detection of economically important pathogens is a key element in sustainable wheat production and a prerequisite for crop protection. The objective of the project was to develop a DNA macroarray for fast and cost-effective detection of nine pathogenic fungi in wheat: Fusarium graminearum, Fusarium culmorum, Fusarium poae, Microdochium nivale var. majus, Microdochium nivale var. nivale, Puccinia recondita, Septoria tritici, Septoria nodorum and Pyrenophora tritici-repentis. Methodically, a macroarray is similar to a microarray but without the need for expensive equipment. PCR labelled samples of DNA are hybridized to pathogen-specific oligonucleotides (probes) anchored to a solid support. A positive reaction between an amplicon and a perfectly matched oligonucleotide generates a chemiluminescent signal which can be detected by a plate reader. The macroarray is sensitive enough to detect single nucleotide polymorphism (SNPs). Sample analysis is simple, fast, cost-effective, fully automated and suitable for high throughput screening. In this project, the nine wheat pathogens were detected within 6 hours simultaneously in a single sample using between one to four different species-specific probes for each pathogen. Species-specific detector oligonucleotides were designed based on the β-tubulin and/or succinate dehydrogenase region of fungal DNA. The detection limit of the DNA macroarray technique particularly depends on the pathogen-specific oligonucleotides deployed. The necessity for monitoring pathogenic fungi in wheat production and for prediction of crop yield has been recognized for a long time. The DNA macroarray responds very sensitively and has the potential to recognize pathogenic fungi earlier with reference to the cultivation period than a conventional PCR. This means that the DNA macroarray can detect genomic DNA from fungi in a lower potency than the conventional PCR. One benefit of the DNA macroarray for detection of fungal pathogens in wheat is its increased specificity and the other its application to a large number of microorganisms which can be detected in a single assay. This technology has been proven to be relatively cost-effective compared with real-time PCR or microarrays. This project was financially supported by the Commission of Technology and Innovation CTI in Berne, Switzerland

The Ctf18 RFC-like complex positions yeast telomeres but does not specify their replication time

Peer reviewedPreprin

The role of multiple marks in epigenetic silencing and the emergence of a stable bivalent chromatin state

We introduce and analyze a minimal model of epigenetic silencing in budding yeast, built upon known biomolecular interactions in the system. Doing so, we identify the epigenetic marks essential for the bistability of epigenetic states. The model explicitly incorporates two key chromatin marks, namely H4K16 acetylation and H3K79 methylation, and explores whether the presence of multiple marks lead to a qualitatively different systems behavior. We find that having both modifications is important for the robustness of epigenetic silencing. Besides the silenced and transcriptionally active fate of chromatin, our model leads to a novel state with bivalent (i.e., both active and silencing) marks under certain perturbations (knock-out mutations, inhibition or enhancement of enzymatic activity). The bivalent state appears under several perturbations and is shown to result in patchy silencing. We also show that the titration effect, owing to a limited supply of silencing proteins, can result in counter-intuitive responses. The design principles of the silencing system is systematically investigated and disparate experimental observations are assessed within a single theoretical framework. Specifically, we discuss the behavior of Sir protein recruitment, spreading and stability of silenced regions in commonly-studied mutants (e.g., sas2, dot1) illuminating the controversial role of Dot1 in the systems biology of yeast silencing.Comment: Supplementary Material, 14 page

Distinct Differences in Chromatin Structure at Subtelomeric X and Y' Elements in Budding Yeast

In Saccharomyces cerevisiae, all ends of telomeric DNA contain telomeric repeats of (TG1–3), but the number and position of subtelomeric X and Y' repeat elements vary. Using chromatin immunoprecipitation and genome-wide analyses, we here demonstrate that the subtelomeric X and Y' elements have distinct structural and functional properties. Y' elements are transcriptionally active and highly enriched in nucleosomes, whereas X elements are repressed and devoid of nucleosomes. In contrast to X elements, the Y' elements also lack the classical hallmarks of heterochromatin, such as high Sir3 and Rap1 occupancy as well as low levels of histone H4 lysine 16 acetylation. Our analyses suggest that the presence of X and Y' elements govern chromatin structure and transcription activity at individual chromosome ends

Effect of Differential N-linked and O-linked Mannosylation on Recognition of Fungal Antigens by Dendritic Cells

BACKGROUND. An experimental approach for improving vaccine efficacy involves targeting antigens to mannose receptors (MRs) on dendritic cells (DCs) and other professional antigen presenting cells. Previously, we demonstrated that mannosylated Pichia pastoris-derived recombinant proteins exhibited increased immunogenicity compared to proteins lacking mannosylation. In order to gain insight into the mechanisms responsible for this observation, the present study examined the cellular uptake of the mannosylated and deglycosylated recombinant proteins. METHODOLOGY/PRINCIPAL FINDINGS. Utilizing transfected cell lines, roles for the macrophage mannose receptor (MMR, CD206) and DC-SIGN (CD209) in the recognition of the mannosylated, but not deglycosylated, antigens were demonstrated. The uptake of mannosylated antigens into murine bone marrow-derived DCs (BMDCs) was inhibited by yeast mannans (YMs), suggesting a mannose-specific C-type lectin receptor-dependent process, while the uptake of deglycosylated antigens remained unaffected. In particular, antigens with both N-linked and extensive O-linked mannosylation showed the highest binding and uptake by BMDCs. Finally, confocal microscopy studies revealed that both mannosylated and deglycosylated P. pastoris-derived recombinant proteins localized in MHC class II+ compartments within BMDCs. CONCLUSIONS/SIGNIFICANCE. Taken together with our previous results, these data suggest that increased uptake by mannose-specific C-type lectin receptors is the major mechanism responsible for the enhanced antigenicity seen with mannosylated proteins. These findings have important implications for vaccine design and contribute to our understanding of how glycosylation affects the immune response to eukaryotic pathogens.National Institutes of Health (RO1 AI25780, RO1 AI37532

Leishmania actin binds and nicks kDNA as well as inhibits decatenation activity of type II topoisomerase

Leishmania actin (LdACT) is an unconventional form of eukaryotic actin in that it markedly differs from other actins in terms of its filament forming as well as toxin and DNase-1-binding properties. Besides being present in the cytoplasm, cortical regions, flagellum and nucleus, it is also present in the kinetoplast where it appears to associate with the kinetoplast DNA (kDNA). However, nothing is known about its role in this organelle. Here, we show that LdACT is indeed associated with the kDNA disc in Leishmania kinetoplast, and under in vitro conditions, it specifically binds DNA primarily through electrostatic interactions involving its unique DNase-1-binding region and the DNA major groove. We further reveal that this protein exhibits DNA-nicking activity which requires its polymeric state as well as ATP hydrolysis and through this activity it converts catenated kDNA minicircles into open form. In addition, we show that LdACT specifically binds bacterial type II topoisomerase and inhibits its decatenation activity. Together, these results strongly indicate that LdACT could play a critical role in kDNA remodeling

The Inheritance of Histone Modifications Depends upon the Location in the Chromosome in Saccharomyces cerevisiae

Histone modifications are important epigenetic features of chromatin that must be replicated faithfully. However, the molecular mechanisms required to duplicate and maintain histone modification patterns in chromatin remain to be determined. Here, we show that the introduction of histone modifications into newly deposited nucleosomes depends upon their location in the chromosome. In Saccharomyces cerevisiae, newly deposited nucleosomes consisting of newly synthesized histone H3-H4 tetramers are distributed throughout the entire chromosome. Methylation of lysine 4 on histone H3 (H3-K4), a hallmark of euchromatin, is introduced into these newly deposited nucleosomes, regardless of whether the neighboring preexisting nucleosomes harbor the K4 mutation in histone H3. Furthermore, if the heterochromatin-binding protein Sir3 is unavailable during DNA replication, histone H3-K4 methylation is introduced onto newly deposited nucleosomes in telomeric heterochromatin. Thus, a conservative distribution model most accurately explains the inheritance of histone modifications because the location of histones within euchromatin or heterochromatin determines which histone modifications are introduced