Yeast as a model for studying human neurodegenerative disorders

Protein misfolding and aggregation are central events in many disorders including several neurodegenerative diseases. This suggests that alterations in normal protein homeostasis may contribute to pathogenesis, but the exact molecular mechanisms involved are still poorly understood. The budding yeast Saccharomyces cerevisiae is one of the model systems of choice for studies in molecular medicine. Modeling human neurodegenerative diseases in this simple organism has already shown the incredible power of yeast to unravel the complex mechanisms and pathways underlying these pathologies. Indeed, this work has led to the identification of several potential therapeutic targets and drugs for many diseases, including the neurodegenerative diseases. Several features associated with these diseases, such as formation of protein aggregates, cellular toxicity mediated by misfolded proteins, oxidative stress and hallmarks of apoptosis have been faithfully recapitulated in yeast, enabling researchers to take advantage of this powerful model to rapidly perform genetic and compound screens with the aim of identifying novel candidate therapeutic targets and drugs. Here we review the work undertaken to model human brain disorders in yeast, and how these models provide insight into novel therapeutic approaches for these diseases.     

Synthesis of an alpha-aminophosphonate nucleoside as an inhibitor of S-adenosyl-L-homocysteine hydrolase

A phosphonic acid analogue of S-adenosyl-L-homocysteine was prepared by a novel method and the epimeric mixture separated. Preliminary studies indicate that each epimer causes time-dependent inactivation of S-adenosyl-L-homocysteine hydrolase, however each presented distinct kinetic characteristics.     

Unexpected inhibition of S-adenosyl-L-homocysteine hydrolase by a guanosine nucleoside

A series of shape-modified flexible nucleosides ('fleximers', 1, 2, and 3) was modeled, synthesized and subsequently assayed against S-adenosyl-L-homocysteine hydrolase (SAHase). No inhibitory activity was observed for the adenosine fleximer, which served as a substrate, but moderate inhibitory activity was exhibited by the guanosine fleximers. This is the first known report of a guanosine nucleoside analogue possessing activity against SAHase.     

S-adenosyl-L-homocysteine hydrolase, key enzyme of methylation metabolism, regulates phosphatidylcholine synthesis and triacylglycerol homeostasis in yeast: implications for homocysteine as a risk factor of atherosclerosis

In eukaryotes, S-adenosyl-L-homocysteine hydrolase (Sah1) offers a single way for degradation of S-adenosyl-L-homocysteine, a product and potent competitive inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases. De novo phosphatidylcholine (PC) synthesis requires three AdoMet-dependent methylation steps. Here we show that down-regulation of SAH1 expression in yeast leads to accumulation of S-adenosyl-L-homocysteine and decreased de novo PC synthesis in vivo. This decrease is accompanied by an increase in triacylglycerol (TG) levels, demonstrating that Sah1-regulated methylation has a major impact on cellular lipid homeostasis. TG accumulation is also observed in cho2 and opi3 mutants defective in methylation of phosphatidylethanolamine to PC, confirming that PC de novo synthesis and TG synthesis are metabolically coupled through the efficiency of the phospholipid methylation reaction. Indeed, because both types of lipids share phosphatidic acid as a precursor, we find in cells with down-regulated Sah1 activity major alterations in the expression of the INO1 gene as well as in the localization of Opi1, a negative regulatory factor of phospholipid synthesis, which binds and is retained in the endoplasmic reticulum membrane by phosphatidic acid in conjunction with VAMP/synaptobrevin-associated protein, Scs2. The addition of homocysteine, by the reversal of the Sah1-catalyzed reaction, also leads to TG accumulation in yeast, providing an attractive model for the role of homocysteine as a risk factor of atherosclerosis in humans.     

Yeast as a model system for studying endocytosis.

Endocytosis is the membrane trafficking process by which plasma membrane components and extracellular material are internalized into cytoplasmic vesicles and delivered to early and late endosomes, eventually either recycling back to the plasma membrane or arriving at the lysosome/vacuole. The budding yeast Saccharomyces cerevisiae has proven to be an invaluable system for identifying proteins involved in endocytosis and elucidating the mechanisms underlying internalization and postinternalization events. Through genetic studies in yeast and biochemical studies in mammalian cells, it has become apparent that multiple cellular processes are linked to endocytosis, including actin cytoskeletal dynamics, ubiquitylation, lipid modification, and signal transduction. In this review, we will highlight the most exciting recent findings in the field of yeast endocytosis. Specifically, we will address the involvement of the actin cytoskeleton in internalization, the role of ubiquitylation as a regulator of multiple steps of endocytosis in yeast, and the sorting of endocytosed proteins into the recycling and vacuolar pathways.     

Yeast as a model system for studying lipid homeostasis and function

Lipids are essential eukaryotic cellular constituents. Lipid metabolism has a strong impact on cell physiology, and despite good progress in this area, many important basic questions remain unanswered concerning the functional diversity of lipid species and on the mechanisms that cells employ to sense and adjust their lipid composition. Combining convenient experimental tractability, a large degree of conservation of metabolic pathways with other eukaryotes and the relative simplicity of its genome, proteome and lipidome, yeast represents the most advantageous model organism for studying lipid homeostasis and function. In this review we will focus on the importance of yeast as a model organism and some of the innovative advantages for the lipid research field.     

Purification and properties of recombinant Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

Recombinant S-adenosyl-L-homocysteine (SAH) hydrolase of the malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity and characterized. Comparison of the malaria parasite SAH hydrolase with that derived from the human gene indicated marked differences in kcat values. The values of both forward and reverse reactions of P. falciparum SAH hydrolase are more than 21-fold smaller than those of the human enzyme. Km values of the parasite and human SAH enzymes are 1.2 and 7.8 microM, respectively. On the other hand, IC50 values of neplanocin A, a strong inhibitor of SAH hydrolase and a growth inhibitor of P. falciparum, are 101 nM for the parasite enzyme and 47 nM for human enzyme. P. falciparum SAH hydrolase has been thought to be a target for a chemotherapeutic agent against malaria. This study may make it possible to develop a specific inhibitor for the parasite SAH hydrolase.     

Yeast as a model system to study RecQ helicase function

Mutations in the highly conserved RecQ helicase, BLM, cause the rare cancer predisposition disorder, Bloom's syndrome. The orthologues of BLM in Saccharomyces cerevisiae and Schizosaccharomyces pombe are SGS1 and rqh1⁺, respectively. Studies in these yeast species have revealed a plethora of roles for the Sgs1 and Rqh1 proteins in repair of double strand breaks, restart of stalled replication forks, processing of aberrant intermediates that arise during meiotic recombination, and maintenance of telomeres. In this review, we focus on the known roles of Sgs1 and Rqh1 and how studies in yeast species have improved our knowledge of how BLM suppresses neoplastic transformation.     

Synthesis and biological activity of novel S-adenosyl-L-homocysteine hydrolase inhibitors

Four potential S-adenosyl-L-homocysteine hydrolase inhibitors were prepared and tested against purified recombinant rat liver enzyme. Preliminary studies indicate that three of these compounds, 1, 2, and 4, caused time-dependent inactivation of S-adenosyl-L-homocysteine hydrolase but showed a biphasic nature. Compound 3 was found to be a rapid equilibrium inhibitor of this enzyme.     

4'-Fluorinated carbocyclic nucleosides: synthesis and inhibitory activity against S-adenosyl-L-homocysteine hydrolase

4'-Fluorinated analogue of 9-[(1'R,2'S,3'R)-2',3'-dihydroxy-cyclopentan-1'-yl]adenine (DHCaA) and their related analogues were systematically synthesized under the Mitsunobu and palladium(0) coupling conditions followed by fluorination with inversion of the configuration by using diethylaminosulfur trifluoride, respectively. 4'-beta-Fluoro DHCaA and 2-amino-4'-alpha-fluoro DHCaA demonstrated slight inhibitory activity against human and Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase, respectively.     

Synthesis of S-adenosyl-L-homocysteine hydrolase inhibitors and their biological activities.

Several nucleosides have been prepared as a possible inhibitor of human S-adenosyl-L-homocysteine (SAH) hydrolase for the development of anti-viral agents. Recently, SAH hydrolase has been considered as an attractive target for parasite chemotherapy for malaria. We report synthesis of several nucleosides including carbocyclic nucleosides and their inhibitory activities against recombinant malaria and human SAH hydrolases.     

Inhibitory effect of Epimedium extract on S-adenosyl-L-homocysteine hydrolase and biomethylation

In the present paper, the inhibitory effect of Epimedium extract on the activity of S-adenosyl-L-homocysteine (AdoHcy) Hydrolase was studied. The results showed that Epimedium extract inhibited the activity of recombinant human AdoHcy hydrolase in a dose-dependent manner. This inhibitory effect was also observed in hepatic cell line 7701 and hepatoma HepG2, however, the effect in 7701 cells was more potent than in HepG2 cells. The extract could significantly reduce AdoMet/AdoHcy ratio in 7701 cells in a dose-dependent manner, suggesting reduced biomethylation level in 7701 cells. In contrast, it resulted in elevated AdoMet/AdoHcy ratio in the HepG2 cells. The result of MALDI-MS assay indicated that epimedin A and ikarisoside F from the extract could bind to AdoHcy hydrolase. The present data suggested that Epimedium extract could inhibit the activity of AdoHcy hydrolase, thus regulating the cellular biomethylation as well as reducing cellular Hcy level. These results will provide new clues to the mechanisms of Epimedium in curing of cardiovascular disease and regulating tumor cell growth.     

Open-chain carbocyclic analogs of adenosine with dihalovinyl unit as potential inhibitors of S-adenosyl-L-homocysteine hydrolase

Vinylogously extended deoxyeritadenine derivatives were synthesized as acyclic/ carbocyclic analogues of the 6'-halo(homovinyl)adenosines, which are known to be potent inhibitors of S-adenosyl-L-homocysteine hydrolase. Swern oxidation of 9-[3-(t-butyldimethylsilyloxy)-4-hydroxybutyl]adenine (4) followed by Wittig olefination and desilylation gave access to ethyl 6-(adenin-9-yl)-4-hydroxy-2(E)-hexenoate (7) and 5-(adenin-9-yl)-1,1-dibromo-1-penten-3-ol (9). No inhibition of AdoHcy Hydrolase was observed with 7 and 9.     

Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum as deduced from the cDNA sequence

S-Adenosyl-L-homocysteine hydrolase has been cloned from a lambda gt11 cDNA library prepared from Dictyostelium discoideum that had been starved for 3 hours. The sequence of the cloned cDNA was determined and the deduced amino acid sequence was compared to the amino acid sequence of rat AdoHcy hydrolase. When the sequences from the two species were aligned, 74% of the amino acids were in identical positions. If conservative changes were taken into account the homology was 84%. Because differences have been reported in the binding characteristics of NAD+ to the D. discoideum and rat AdoHcy hydrolases, changes in the amino acids of the putative NAD+-binding site were of particular interest. Six changes were observed in this region but the changes appeared to be in regions that are not critical to the three dimensional folding of the NAD+-binding site.     

Synthesis of 2-modified aristeromycins and their analogs as potent inhibitors against Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

2-Modified aristeromycin derivatives and their related analogs were synthesized to investigate their inhibitory activity against human and Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH). 2-Fluoroaristeromycin showed a strong inhibitory activity against PfSAHH selectively and complete resistance to adenosine deaminase.     

Crystallization and preliminary X-ray crystallographic analysis of Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

S-adenosyl-l-homocysteine hydrolase from a malaria parasite Plasmodium falciparum (PfSAHH) has been crystallized by the vapor diffusion method. The crystals belong to an orthorhombic space group P212121 with the cell dimensions of a = 76.66 A, b = 86.31 A, and c = 335.6 A. There are four subunits (one tetramer) per asymmetric unit. X-ray diffraction data have been collected up to 2.8 A resolution. Self-rotation function studies suggest that the tetrameric PfSAHH molecule has the 222 point group symmetry.     

Evidence that a cAMP binding protein from Dictyostelium discoideum carries S-adenosyl-L-homocysteine hydrolase activity

A cAMP-adenosine binding protein partially purified from exponentially growing Dictyostelium discoideum cells carries S-adenosyl-L-homocysteine (SAH) hydrolase activity. This protein is present throughout the developmental cycle and has many properties in common with a cAMP binding activity previously reported from this laboratory (Gunzburg and Véron, 1981). Direct binding measurements with radioactive ligands indicate a dissociation constant of 0.2 microM for adenosine and 9 nM for cAMP, a value in good agreement with measurements of the rate constants for cAMP binding (k+1 = 2.4 X 10(4) M-1 sec-1) and dissociation (k-1 = 1.1 X 10(-4) sec-1). The binding of cAMP is completely abolished in the presence of 1 microM adenosine; a maximum 60 per cent inhibition of adenosine binding can be achieved with cAMP concentrations as high as 0.1 microM, suggesting that at least some of the cAMP and adenosine binding sites are not identical. The protein has a sedimentation coefficient of 9.2S and a native molecular weight of 190,000, as judged by gel filtration. Labeling with the photoaffinity ligand 8-azido-[3H]-cAMP followed by SDS polyacrylamide gel electrophoresis results in a single band of 47,000 MW, suggesting that the protein may be a tetramer. The physiological importance of the protein and its association with SAH hydrolase activity is discussed in relation to a possible role in the regulation of protein and phospholipid methylation that occurs during chemotaxis.