Inhibition of formation of α-synuclein inclusions by mannosylglycerate in a yeast model of Parkinson's disease

Background

Protein aggregation in the brain is a central hallmark in many neurodegenerative diseases. In Parkinson's disease, α-synuclein (α-Syn) is the major component of the intraneuronal inclusions found in the brains of patients. Current therapeutics is merely symptomatic, and there is a pressing need for developing novel therapies. Previously we showed that mannosylglycerate (MG), a compatible solute typical of marine microorganisms thriving in hot environments, is highly effective in protecting a variety of model proteins against thermal denaturation and aggregation in vitro.

Methods

Saccharomyces cerevisiae cells expressing eGFP-tagged α-Syn, were further engineered to synthesize MG. The number of cells with fluorescent foci was assessed by fluorescence microscopy. Fluorescence spectroscopy and transmission electron microscopy were used to monitor fibril formation in vitro.

Results

We observed a 3.3-fold reduction in the number of cells with α-Syn foci and mild attenuation of α-Syn-induced toxicity. Accordingly, sucrose gradient analysis confirmed a clear reduction in the size-range of α-Syn species in the cells. MG did not affect the expression levels of α-Syn or its degradation rate. Moreover, MG did not induce molecular chaperones (Hsp104, Hsp70 and Hsp40), suggesting the implication of other mechanisms for α-Syn stabilization. MG also inhibited α-Syn fibrillation in vitro.

Conclusions

MG acts as a chemical chaperone and the stabilization mechanism involves direct solute/protein interactions.

General significance

This is the first demonstration of the anti-aggregating ability of MG in the intracellular milieu. The work shows that MG is a good candidate to inspire the development of new drugs for protein-misfolding diseases.     

A cyto-biochemical study of the ‘canal’ formation in the yeast cell wall

Summary A distinction between the chemical composition of ultrastructurally modified regions and the rest of the cell wall (canals) of the yeast Schwanniomyces occidentalis was shown by cytochemical staining of cell wall polysaccharides. The formation of canals was induced by cultivation of yeasts on hydrocarbons and was parallelled by the enhancement of -glucosidase, -glucanase and -mannosidase activities which were all capable of degrading cell wall polysaccharides. The presence of cycloheximide prevented canal formation. We assume that these hydrolases modified definite cell wall regions transforming them into canals.     

Strategy for purification of aggregation prone β-glucosidases from the cell wall of yeast: a preparative scale approach

Purification of biotechnologically important proteins is of vital interest to the biotech industry. β-Glucosidases, belonging to Family 1 and Family 3 of the glycosylhydrolases, have varied applications as carbohydrate hydrolyzing and synthesizing enzymes. Obtaining high quantities of these enzymes is important for exploring their biosynthetic potential, structural information and catalytic activities. Classical methods for their preparation fail to deliver high yields because of adoption of several/hydroxyapatite chromatography steps. We report here a preparative method for purification of large quantities of two closely related cell bound β-glucosidases (BGL I and BGL II) from Pichia etchellsii that belong to Family 3 glycosylhydrolases. A combination of ion-exchange and gel filtration chromatography was used to process milligram quantities of protein with recoveries of up to 53%. A simple affinity based separation resulted in resolution of BGL I and BGL II with high recovery and high specific activities of 74IU/mg and 32IU/mg protein respectively. Peptide sequences of BGL II indicated it to be a novel member of Family 3. Methods reported here present a successful strategy for obtaining large quantities of these enzymes.     

Heteronuclear NMR studies of 13C-labeled yeast cell wall β-glucan oligosaccharides

Summary The structures of uniformly ¹³C-labeled -glucan octa- and undeca-oligosaccharides enzymatically prepared by the yeast cell wall glucanosyl transferase of Candida albicans were characterized by using a combination of HCCH-COSY, HCCH-TOCSY, and HMBC experiments. The oligosaccharide structures indicate that the cell wall glucanosyl transferase cleaves two glucosyl units from the reducing end of the initial linear (13) penta-oligosaccharide and subsequently transfers the remainder to another oligosaccharide at the nonreducing end via a (16) linkage. These results indicate that the combined action of cell wall glucanase and glucanosyl transferase activities could not only introduce intrachain (16) linkages within a single glucan strand, but also result in cross-linking of two initially separate glucan strands with concurrent introduction of intrachain (16) linkages. Since isolated fungal membranes only synthesize linear (13) glucan strands, wall-associated enzymes probably participate in the assembly of the final wall glucan structure during cell growth and division.     

Control of cell type in yeast by the mating type locus: The α1-α2 hypothesis

We have extended the genetic analysis of four mutants carrying defective MATα alleles in order to determine how the mating type locus controls yeast cell types: a, a, and $\text{a}\text{α}$. First, we have mapped the defect in the mutant VC73 to the mating type locus by diploid and tetraploid segregation analysis. Second, we have determined that the mutations in these strains define two complementation groups, MATα1 and MATα2. The MATα1 gene is proposed to be a positive regulator of α mating functions. The MATα2 gene product is proposed to have two roles, as a negative regulator of a-specific mating functions and as a regulator of $\text{a}\text{α}$ cell functions (required for sporulation, for inhibition of mating and other processes). This view of MATα leads to the prediction that matα1^?matα2^? mutants should have the mating ability of an a cell and that matα1^?matα2^?/MATα strains should mate as α and be unable to sporulate. Such double mutants have been constructed and behave as predicted. We therefore propose that a-specific mating functions in MATa cells are constitutively expressed due to the absence of the MATα2 gene product and that α-specific mating functions are not expressed due to the absence of the MATα1 gene product.     

Lactic fermentation of cellobiose by a yeast strain displaying β-glucosidase on the cell surface

The Aspergillus aculeatus beta-glucosidase 1 (bgl1) gene was expressed in a lactic-acid-producing Saccharomyces cerevisiae strain to enable lactic fermentation with cellobiose. The recombinant beta-glucosidase enzyme was expressed on the yeast cell surface by fusing the mature protein to the C-terminal half region of the alpha-agglutinin. The beta-glucosidase expression plasmids were integrated into the genome. Three strong promoters of S. cerevisiae, the TDH3, PGK1, and PDC1 promoters, were used for beta-glucosidase expression. The specific beta-glucosidase activity varied with the promoter used and the copy number of the bgl1 gene. The highest activity was obtained with strain PB2 that possessed two copies of the bgl1 gene driven by the PDC1 promoter. PB2 could grow on cellobiose and glucose minimal medium at the same rate. Fermentation experiments were conducted in non-selective-rich media containing 95 g l(-1) cellobiose or 100 g l(-1) glucose as a carbon source under microaerobic conditions. The maximum rate of L-lactate production by PB2 on cellobiose (2.8 g l(-1) h(-1)) was similar to that on glucose (3.0 g l(-1) h(-1)). This indicates that efficient fermentation of cellobiose to L-lactate can be accomplished using a yeast strain expressing beta-glucosidase from a mitotically stable genomic integration plasmid.     

Plant chaperonins: a role in microtubule-dependent wall formation?

Summary The cytosolic chaperonin containing t-complex peptide-1 (CCT) is involved in the correct folding of newly synthetized actin and tubulin molecules. To get insight into potential additional functions of plant CCT, the localization of the subunit CCT was followed throughout cell cycle, cell elongation, and cell differentiation in the tobacco cell culture VBI-O with relation to the microtubular cytoskeleton by double-immunofluorescence and confocal microscopy. The CCT subunit was found to colocalize with sites of microtubule nucleation such as nuclear envelope and preprophase band. In addition, CCT was associated with tubulin in sites of elevated wall synthesis such as phragmoplast or along secondary-wall thickenings. CCT and its substrate tubulin were found to be soluble during periods of cytoskeletal dynamics, whereas sedimentable, vesicle-bound forms of CCT and tubulin prevailed during cell differentiation. The sedimentability of CCT was increased by calcium, whereas it was detached from microsomes by ATP. CCT can bind to both polymerized microtubules and tubulin dimers. These data suggest an additional function of plant CCT in microtubule-driven transport of vesicles that contain cell-wall material.Abbreviations CCT cytosolic chaperonin containing t-complex polypeptide 1     

Growth inhibition of pathogenic yeast isolates by α-difluoromethylornithine: An inhibition of ornithine decarboxylase

A large body of evidence exists suggesting that polyamines can play essential roles in cellular growth and differentiation. We examined the ability of -difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, the major rate-limiting enzyme in polyamine biosynthesis, to inhibit the growth of Candida albicans, C. tropicalis, and C. parapsilosis. Substantial growth-inhibition was observed for all three species at DFMO concentrations ranging from 1 to 100 mM. C. tropicalis was significantly more susceptible to DFMO than C. albicans or C. parapsilosis. Depletion of cellular polyamine pools was seen in all 3 species following exposure to DFMO and polyamine depletion enhanced the susceptibility of the organisms to DFMO. The action of DFMO was specifically antagonized by exogenous polyamines. These data suggest that polyamines are important in the growth of Candida spp. and that inhibitors of polyamine biosynthesis may be useful as antifungal agents.     

Production of α-mannanase by B. subtilis from agro-industrial by-products: screening and optimization

We have demonstrated that a mixture of wheat bran (35 g l^-1), as a main substrate, and palm seed powder (10 g l^-1), as a co-substrate, is appropriate for -mannanase production by Bacillus subtilis. A 2ⁿ factorial experimental design was employed as a primary step for medium optimization. The enzyme activity titters obtained at the optimized growth condition were equivalent to about 319% of the -mannanse activity and 114% of the specific activity levels reached by a galactomannan-based culture.     

Control of yeast cell type by the mating type locus: Positive regulation of the α-specific STE3 gene by the MATα 1 product

The mating type locus (MAT) determines the three yeast cell types, a, α, and a/α. It has been proposed that alleles of this locus, MATa and MATα, encode regulators that control expression of unlinked genes necessary for mating and sporulation. Specifically, the α1 product of MATα is proposed to be a positive regulator of α-specific genes. To test this view, we have assayed RNA production from the α-specific STE3 gene in the three cell types and in mutants defective in MATα. The STE3 gene was cloned by screening a yeast genomic clone bank for plasmids that complement the mating defect of ste3 mutants. Using the cloned STE3 gene as a probe, we find that a cells produce STE3 RNA, whereas a and a/a cells do not. Furthermore, matα 1 mutants do not produce STE3 RNA, whereas matα 2 mutants do. These results show that the STE3 gene, required for mating only by α cells, is expressed only in α cells. They show also that production of RNA from the STE3 gene requires the α1 product of MATα. Thus α1 positively regulates at least one α-specific gene by increasing the level of that gene's RNA product.     

Efficient uptake of recombinant α-galactosidase A produced with a gene-manipulated yeast by Fabry mice kidneys

To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.     

Pectinous cell wall thickenings formation—A response of moss protonemata cells to lead

Lead poisoning constitutes one of most detrimental environmental hazards to all living organisms. Plants developed a variety of avoidance and tolerance mechanisms that are activated in response to lead exposure. Plant cell walls were suggested to play important role in these reactions by creating an efficient barrier to lead entry to the protoplasts, but the molecular mechanisms involved in such shielding reaction have not been elucidated. Tip growing protomemata of Funaria hygrometrica (Hedw.) were used as model for studying effects of lead exposure on plant cell walls (CWs). Forty-eight hour-treatment 4 μM PbCl₂ resulted in the appearance of cell wall thickenings (CWTs) at the tip of the apical cell, which is the lead entry site to the cell protoplast [Krzes?owska, M., Wo?ny, A., 1996. Lead uptake localization and changes in cell ultrastructure of Funaria hygrometrica protonemata. Biol. Plant. 38, 253–259]. The nature of these thickenings differed from the one of cell wall in unexposed plants as revealed by immunolabelling with monoclonal antibodies and histochemical analyses. The most striking difference was the appearance high amount of low-esterified (JIM5 epitope) and unesterified (PAM1 epitope) homogalacturonan, which were absent from the tip cell wall of control protonemata and are known as the compounds able to bind and immobilise Pb²⁺. Furthermore, the cell wall thickenings commonly contained callose and at least two kinds of lipid compounds known as the substances preventing metal ions entry to the protoplast.Observations in transmission electron microscope (TEM) showed that CWTs contained a few distinct, varied structurally regions. The dominant one was the region of a granular structure—never found in the control CW. This region contained both the highest amount of JIM5 pectins—and the most numerous lead deposits. In many cases gold particles, identifying JIM5 pectins, appeared to be bound to lead deposits. It indicated that JIM5 pectins which accumulated in CWTs were involved in immobilisation of high amounts of Pb²⁺. Because the region of lead accumulation occupied the largest volume of the CWTs, we concluded that CWTs appear to be a very important repository for Pb²⁺ in protonemata cells. Thus, we postulate that, CWTs localized at the tip of the apical cell—the main region of lead uptake [Krzes?owska, M., Wo?ny, A., 1996. Lead uptake localization and changes in cell ultrastructure of Funaria hygrometrica protonemata. Biol. Plant. 38, 253–259] rich in JIM5 pectins, callose and lipids function as the effective barrier against lead ions penetration into the protonema protoplast.The findings substantiate previous hypotheses that lead ions can be sequestered in cell walls and point to the possibility that capacity for lead binding might increase in cell response to lead.     

Methyl β-cyclodextrin reduces accumulation of reactive oxygen species and cell death in yeast

Stabilized F-actin structures have been shown to be detrimental to both mammalian and yeast cells. In yeast, stabilization of actin caused by addition of jasplakinolide, by point mutations in the act1 gene, or by deletion of certain genes that regulate F-actin leads to cell death with hallmarks of apoptosis. In particular, there is an elevation in the levels of reactive oxygen species, and we have shown the importance of the Ras/cAMP pathway for this effect. Here we show that in yeast cells deleted for end3, which functions to regulate actin organization during endocytosis, treatment of cells with methyl β-cyclodextrin reduces levels of reactive oxygen species and inhibits cell death progression. Methyl β-cyclodextrin is widely used to disrupt lipid rafts that contain cholesterol. The mechanism through which the rescue is achieved was investigated and we demonstrate that methyl β-cyclodextrin reduces accumulation of Ras2 at the plasma membrane in Δend3 cells. We use FRAP and live cell imaging to determine the possible mechanism through which methyl β-cyclodextrin functions to elicit this effect on Ras2 localization. Finally, we demonstrate that addition of methyl β-cyclodextrin to wild-type cells can act to protect cells from acute oxidative stress caused by addition of hydrogen peroxide.     

Stimulation of α-synuclein amyloid formation by phosphatidylglycerol micellar tubules

α-Synuclein (α-Syn) is a presynaptic protein that is accumulated in its amyloid form in the brains of Parkinson's patients. Although its biological function remains unclear, α-syn has been suggested to bind to synaptic vesicles and facilitate neurotransmitter release. Recently, studies have found that α-syn induces membrane tubulation, highlighting a potential mechanism for α-syn to stabilize highly curved membrane structures which could have both functional and dysfunctional consequences. To understand how membrane remodeling by α-syn affects amyloid formation, we have studied the α-syn aggregation process in the presence of phosphatidylglycerol (PG) micellar tubules, which were the first reported example of membrane tubulation by α-syn. Aggregation kinetics, β-sheet content, and macroscopic protein-lipid structures were observed by Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy, respectively. Collectively, the presence of PG micellar tubules formed at a stochiometric (L/P = 1) ratio was found to stimulate α-syn fibril formation. Moreover, transmission electron microscopy and solid-state nuclear magnetic resonance spectroscopy revealed the co-assembly of PG and α-syn into fibril structures. However, isolated micellar tubules do not form fibrils by themselves, suggesting an important role of free α-syn monomers during amyloid formation. In contrast, fibrils did not form in the presence of excess PG lipids (≥L/P = 50), where most of the α-syn molecules are in a membrane-bound α-helical form. Our results provide new mechanistic insights into how membrane tubules modulate α-syn amyloid formation and support a pivotal role of protein–lipid interaction in the dysfunction of α-syn.     

Continuous production of α-peptide using immobilized recombinant yeast cells: A model for continuous production of foreign peptide by recombinant yeast

α-Peptide, a portion of Escherichia coli β-galactosidase, was cloned downstream of the yeast α-factor promoter and the signal peptide by one of the authors. In this study, we utilized recombinant yeast cells, transformed the α-peptide secretion vector and attempted continuous production of α-peptide as a model of foreign peptide production. The continuous production of α-peptide was performed by using immobilized recombinant yeast cells on a column reactor, after characterizing the secretion, using minimal and complex medium. Utilizing minimal medium, with a productivity of 100 000 U h⁻¹ l⁻¹, α-peptide was continuously produced for more than 200 h. We then attempted to improve the productivity of α-peptide by alternating minimal and complex medium. Utilizing this medium changing method, 1.4 times higher α-peptide was produced during 150 h of operation compared with that achieved only by feeding minimal medium.     

Perturbation of endothelial junction proteins by Staphylococcus aureus α-toxin: inhibition of endothelial gap formation by adrenomedullin

Endothelial hyperpermeability is a hallmark of an inflammatory reaction and contributes to tissue damage in severe infections. Loss of endothelial cell–cell adhesion leads to intercellular gap formation allowing paracellular fluid flux. Although Staphylococcus aureus α-toxin significantly contributed to staphylococci disease, little is known about its mechanism of endothelial hyperpermeability induction. Here, we demonstrate that in a model of isolated perfused rat ileum discontinuation of capillary vascular-endothelial-cadherin (VE-cadherin) was observed after bolus application of S. aureus α-toxin being inhibited by the endogenous peptide adrenomedullin (ADM). In vitro, α-toxin exposure induced loss of immunoreactivity of VE-cadherin and occludin in human cultured umbilical vein endothelial cells. Likewise, ADM blocked α-toxin-related junctional protein disappearance from intercellular sites. Additionally, cyclic AMP elevation was shown to stabilize endothelial barrier function after α-toxin application. Although no RhoA activation was noted after endothelial α-toxin exposure, inhibition of Rho kinase and myosin light chain kinase blocked loss of immunoreactivity of VE-cadherin and occludin as well as intercellular gap formation. In summary, stabilization of endothelial junctional integrity as indicated by interendothelial immunostaining might be an interesting approach to stabilize endothelial barrier function in severe S. aureus infections.Andreas C. Hocke and Bettina Temmesfeld-Wollbrueck have contributed equally to this article.     

Aggresome formation and segregation of inclusions influence toxicity of α-synuclein and synphilin-1 in yeast

PD (Parkinson's disease) is a neurodegenerative disorder, caused by a selective loss of dopaminergic neurons in the substantia nigra, which affects an increasing number of the elderly population worldwide. One of the major hallmarks of PD is the occurrence of intracellular protein deposits in the dying neurons, termed Lewy bodies, which contain different proteins, including aggregated α-synuclein and its interacting protein synphilin-1. During the last decade, a number of groups developed yeast models that reproduced important features of PD and allowed the deciphering of pathways underlying the cytotoxicity triggered by α-synuclein. Here, we review the recent contributions obtained with yeast models designed to study the presumed pathobiology of synphilin-1. These models pointed towards a crucial role of the sirtuin Sir2 and the chaperonin complex TRiC (TCP-1 ring complex)/CCT (chaperonin containing TCP-1) in handling misfolded and aggregated proteins.