The anti-fungal ß-sitosterol targets the yeast oxysterol-binding protein Osh4.

BACKGROUND: ß-Sitosterol is a plant metabolite with a broad range of anti-fungal activity, however, this compound is not toxic against a few fungal species. The target of ß-sitosterol and the nature of its selective toxicity are not yet clear. Using a yeast model system and taking advantage of molecular biology and computational approaches, we identify the target and explain why ß-sitosterol is not toxic against some fungal pathogens. RESULTS: ß-Sitosterol (200 µg mL-1 ) is toxic against yeast cells expressing only Osh4 (an oxysterol-binding protein) and harbouring a upc2-1 mutation (which enables sterol uptake), but not against yeast strains expressing all seven Osh proteins and harbouring a upc2-1 mutation. Furthermore, ß-sitosterol is not toxic against yeast strains without the upc2-1 mutation irrespective of the number of Osh proteins being expressed. The deletion of COQ1 (a gene known to be highly induced upon deletion of OSH4) enhances the toxicity of ß-sitosterol in yeast cells expressing only Osh4 and harbouring the upc2-1 mutation. Molecular modelling suggests that ß-sitosterol binds to Osh4 and the binding mode is similar to the binding of cholesterol to Osh4. CONCLUSION: Our results indicate that the concentrations of ß-sitosterol, and Osh4, as well as its homologues within cells, are most likely the main determinants of ß-sitosterol toxicity. Furthermore, some fungal species do not take up sterols, e.g. Saccharomyces cerevisiae, under aerobic conditions. Therefore, sterol uptake may also contribute to the ß-sitosterol anti-fungal effect. These findings enable predicting the toxicity of ß-sitosterol against plant fungal pathogens. © 2019 Society of Chemical Industry.

Genetic, epigenetic and biochemical regulation of succinate dehydrogenase function.

Succinate dehydrogenase (SDH), complex II or succinate:quinone oxidoreductase (SQR) is a crucial enzyme involved in both the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), the two primary metabolic pathways for generating ATP. Impaired function of SDH results in deleterious disorders from cancer to neurodegeneration. SDH function is tailored to meet the energy demands in different cell types. Thus, understanding how SDH function is regulated and how it operates in distinct cell types can support the development of therapeutic approaches against the diseases. In this article we discuss the molecular pathways which regulate SDH function and describe extra roles played by SDH in specific cell types.

The anti-cancer compound Schweinfurthin A targets Osh2 and disrupts lipid metabolism in the yeast model.

Schweinfurthin A (Sch A) is a natural product with a selective and strong anti-cancer effect. Although it is known to target oxysterol binding proteins, the detailed mode of action is not well understood. Here, we provide strong evidence that yeast cells can be used as a eukaryotic model system to decipher the molecular modes of Sch A. We show that Sch A (100 µM) targets Osh2 (a yeast oxysterol binding protein homolog) genetically and taking advantage of computational chemistry indicate that the tetrahydro-2H-xanthene portion of Sch A forms H-bonds with residues Ser105, Val113, and Lys201, while its isoprenoid side chain is placed in a hydrophobic pocket lined by the side chains of Leu41, Leu45, Leu58, Met56, and Phe174 in Osh2. This model suggests that Sch A occupies the same binding pocket in Osh2 which is occupied by its natural substrate, ergosterol. Osh proteins transport sterol and PI(4)P in a cyclic manner between two membranes. Therefore, we suggest that Sch A interferes with this function of Osh2. In support of this hypothesis, we show that Sch A toxicity rate changes upon manipulating the enzymes that modify the levels of sterol and PI(4)P. This approach also informs how Sch A exerts its toxic effect in yeast cells. These enzymes include Coq1, Sac1, Plc1, Stt4, Pik1, and Mss4. We demonstrate that Coq1 an enzyme required for coenzyme Q synthesis (also involved in sterol metabolism indirectly), Sac1, and Stt4 the enzymes governing PI(4)P level modify Sch A toxicity and finally propose Sch A disrupts sterol/PI(4)P exchange between membranes by occupying the sterol/PI(4)P binding pocket in Osh2.

Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect.

Succinate dehydrogenase (SDH), also named as complex II or succinate:quinone oxidoreductases (SQR) is a critical enzyme in bioenergetics and metabolism. This is because the enzyme is located at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA); the two major pathways involved in generating energy within cells. SDH is composed of 4 subunits and is assembled through a multi-step process with the aid of assembly factors. Not surprisingly malfunction of this enzyme has marked repercussions in metabolism leading to devastating tumors such as paraganglioma and pheochromocytoma. It is already known that mutations in the genes encoding subunits lead to tumorigenesis, but recent discoveries have indicated that mutations in the genes encoding the assembly factors also contribute to tumorigenesis. The mechanisms of pathogenesis of tumorigenesis have not been fully understood. However, a multitude of signaling pathways including succinate signaling was determined. We, here discuss how defective SDH may lead to tumor development at the molecular level and describe how yeast, as a model system, has contributed to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH.

The assembly of succinate dehydrogenase: a key enzyme in bioenergetics.

Succinate dehydrogenase (SDH) also known as complex II or succinate:quinone oxidoreductase is an enzyme involved in both oxidative phosphorylation and tricarboxylic acid cycle; the processes that generate energy. SDH is a multi-subunit enzyme which requires a series of proteins for its proper assembly at several steps. This enzyme has medical significance as there is a broad range of human diseases from cancers to neurodegeneration related to SDH malfunction. Some of these disorders have recently been linked to defective assembly factors, reinvigorating further research in this area. Apart from that this enzyme has agricultural importance as many fungicides have been/will be designed targeting specifically this enzyme in plant fungal pathogens. In addition, we speculate it might be possible to design novel fungicides specifically targeting fungal assembly factors. Considering the medical and agricultural implications of SDH, the aim of this review is an overview of the SDH assembly factors and critical analysis of controversial issues around them.

CO2 Cycloaddition to Epoxides by using M-DABCO Metal-Organic Frameworks and the Influence of the Synthetic Method on Catalytic Reactivity.

A series of high-quality M2(BDC)2(DABCO) metal-organic frameworks (abbreviated as M-DABCO; M=Zn, Co, Ni, Cu; BDC=1,4-benzene dicarboxylate; DABCO=1,4-diazabicyclo[2.2.2]octane), were synthesized by using a solvothermal (SV) method, and their catalytic activity for the cycloaddition of CO2 to epoxides in the absence of a co-catalyst or solvent was demonstrated. Of these metal-organic frameworks (MOFs), Zn-DABCO exhibited very high activity and nearly complete selectivity under moderate reaction conditions. The other members of this MOF series (Co-DABCO, Ni-DABCO, and Cu-DABCO) displayed lower activity in the given sequence. Samples of Zn-DABCO, Co-DABCO, and Ni-DABCO were recycled at least three times without a noticeable loss in catalytic activity. The reaction mechanism can be attributed to structural defects along with the acid-base bifunctional characteristics of these MOFs. Moreover, we illustrate that the synthetic method of M-DABCO influences the yield of the reaction. In addition to the SV method, Zn-DABCO was synthesized by using spray drying due to its industrial attractiveness. It was found that the synthesis procedure clearly influenced the crystal growth and thus the physicochemical properties, such as surface area, pore volume, and gas adsorption, which in turn affected the catalytic performance. The results clarified that although different synthetic methods can produce isostructural MOFs, the application of MOFs, especially as catalysts, strongly depends on the crystal morphology and textural properties and, therefore, on the synthesis method.

4-Hydroxyphenylpyruvate Dioxygenase Inhibitors: From Chemical Biology to Agrochemicals.

The development of new herbicides is receiving considerable attention to control weed biotypes resistant to current herbicides. Consequently, new enzymes are always desired as targets for herbicide discovery. 4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is an enzyme engaged in photosynthetic activity and catalyzes the transformation of 4-hydroxyphenylpyruvic acid (HPPA) into homogentisic acid (HGA). HPPD inhibitors constitute a promising area of discovery and development of innovative herbicides with some advantages, including excellent crop selectivity, low application rates, and broad-spectrum weed control. HPPD inhibitors have been investigated for agrochemical interests, and some of them have already been commercialized as herbicides. In this review, we mainly focus on the chemical biology of HPPD, discovery of new potential inhibitors, and strategies for engineering transgenic crops resistant to current HPPD-inhibiting herbicides. The conclusion raises some relevant gaps for future research directions.

Yeast-based assays for detecting protein-protein/drug interactions and their inhibitors.

Understanding cellular processes at molecular levels in health and disease requires the knowledge of protein-protein interactions (PPIs). In line with this, identification of PPIs at genome-wide scale is highly valuable to understand how different cellular pathways are interconnected, and it eventually facilitates designing effective drugs against certain PPIs. Furthermore, investigating PPIs at a small laboratory scale for deciphering certain biochemical pathways has been demanded for years. In this regard, yeast two hybrid system (Y2HS) has proven an extremely useful tool to discover novel PPIs, while Y2HS derivatives and novel yeast-based assays are contributing significantly to identification of protein-drug/inhibitor interaction at both large- and small-scale set-ups. These methods have been evolving over time to provide more accurate, reproducible and quantitative results. Here we briefly describe different yeast-based assays for identification of various protein-protein/drug/inhibitor interactions and their specific applications, advantages, shortcomings, and improvements. The broad range of yeast-based assays facilitates application of the most suitable method(s) for each specific need.

Discovery of a butyrylcholinesterase-specific probe via a structure-based design strategy.

We report herein the structure-based design and application of a fluorogenic molecular probe (BChE-FP) specific to butyrylcholinesterase (BChE). This probe was rationally designed by mimicking the native substrate and optimized stepwise by manipulating the steric feature and the reactivity of the designed probe targeting the structural difference of the active pockets of BChE and AChE. The refined probe, BChE-FP, exhibits high specificity toward BChE compared to AChE, producing about 275-fold greater fluorescence enhancement upon the catalysis by BChE. Thus, BChE-FP is a specific BChE probe identified by the structure-based design and it can discriminate BChE from AChE. Furthermore, it has been successfully applied for imaging the endogenous BChE in living cells, as well as BChE inhibitor screening and characterization under physiological conditions.

Actin, Membrane Trafficking and the Control of Prion Induction, Propagation and Transmission in Yeast.

The model eukaryotic yeast Saccharomyces cerevisiae has proven a useful model system in which prion biogenesis and elimination are studied. Several yeast prions exist in budding yeast and a number of studies now suggest that these alternate protein conformations may play important roles in the cell. During the last few years cellular factors affecting prion induction, propagation and elimination have been identified. Amongst these, proteins involved in the regulation of the actin cytoskeleton and dynamic membrane processes such as endocytosis have been found to play a critical role not only in facilitating de novo prion formation but also in prion propagation. Here we briefly review prion formation and maintenance with special attention given to the cellular processes that require the functionality of the actin cytoskeleton.

Yeast Model of Amyloid-ß and Tau Aggregation in Alzheimer's Disease.

The amyloid-ß peptide (Aß) and the phosphorylated protein tau have been widely implicated in Alzheimer's disease and are the focus of most research. Both agents have been extensively studied in mammalian cell culture and in animal studies, but new research is focusing on yeast models. Yeast are eukaryotes, just like us, and are amenable to effects and expression of Aß and tau and appear able to 'report' with considerable relevance on the effects of these biomolecules. The use of yeast enables powerful new approaches to understanding how to overcome the effects of Aß and tau, and such advances could lead to new therapies to prevent the progression of Alzheimer's disease.

Hsp70/Hsp90 co-chaperones are required for efficient Hsp104-mediated elimination of the yeast [PSI(+)] prion but not for prion propagation.

The continued propagation of the yeast [PSI(+)] prion requires the molecular chaperone Hsp104 yet in cells engineered to overexpress Hsp104; prion propagation is impaired leading to the rapid appearance of prion-free [psi(-)] cells. The underlying mechanism of prion loss in such cells is unknown but is assumed to be due to the complete dissolution of the prion aggregates by the ATP-dependent disaggregase activity of this chaperone. To further explore the mechanism, we have sought to identify cellular factors required for prion loss in such cells. Sti1p and Cpr7p are co-chaperones that modulate the activity of Hsp70/Ssa and Hsp90 chaperones and bind to the C-terminus of Hsp104. Neither Sti1p nor Cpr7p is necessary for prion propagation but we show that deletion of the STI1 and CPR7 genes leads to a significant reduction in the generation of [psi(-)] cells by Hsp104 overexpression. Deletion of the STI1 and CPR7 genes does not modify the elimination of [PSI(+)] by guanidine hydrochloride, which inhibits the ATPase activity of Hsp104 but does block elimination of [PSI(+)] by overexpression of either an ATPase-defective mutant of Hsp104 (hsp104(K218T/K620T)) or a 'trap' mutant Hsp104 (hsp104(E285Q/E687Q)) that can bind its substrate but can not release it. These results provide support for the hypothesis that [PSI(+)] elimination by Hsp104 overexpression is not simply a consequence of complete dissolution of the prion aggregates but rather is through a mechanism distinct from the remodelling activity of Hsp104.