Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.
Synaptotagmin‐1 (Syt1) is a major Ca2+ sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t‐) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single‐molecule technique, alternating‐laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca2+, Syt1 mediates vesicle docking by directly binding to t‐SNARE/phosphatidylinositol 4,5‐biphosphate (PIP2) complex and increases the docking rate by 103 times. Second, synaptobrevin‐2 binding to t‐SNARE displaces Syt1 from SNAREpin. Third, with Ca2+, Syt1 rebinds to SNAREpin, which again requires PIP2. Thus without Ca2+, Syt1 may bring vesicles to the plasma membrane in proximity via binding to t‐SNARE/PIP2 to help SNAREpin formation and then, upon Ca2+ influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway.
CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7⌬ ura8⌬ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7⌬ ura8⌬ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32 P i -labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose-and time-dependent, and dependent on the concentrations of CTP synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation.CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) catalyzes the final step in the pyrimidine biosynthetic pathway (1). The end product CTP is required for the synthesis of nucleic acids and membrane phospholipids (2). Thus, CTP synthetase is an essential enzyme for the growth and metabolism of all organisms (2). In eukaryotes, CTP synthetase activity regulates the balance of nucleotide pools (3-9) and influences the pathways by which membrane phospholipids are synthesized (9 -11). The importance of understanding the mode of action and regulation of CTP synthetase is further highlighted by the fact that elevated CTP synthetase activity is a common property of several cancers in humans (12)(13)(14)(15)(16)(17)(18)(19)(20).CTP synthetase has been purified and characterized from bacteria (21-23), Saccharomyces cerevisiae (8, 24), and rat liver (25). In addition, crystal structures for the Escherichia coli (26) and Thermus thermophilus (27) enzymes have been solved. The enzymological properties of CTP synthetase enzymes from various sources are similar, although some differences have been identified (23). The enzyme catalyzes a complex set of reactions involving the ATP-dependent transfer of the amide nitrogen from glutamine (i.e. glutaminase reaction) to the C-4 position of UTP to generate CTP (Fig. 1) (21, 28). GTP activates the glutaminase reaction by accelerating the formation of a covalent glutaminyl enzyme intermediate (21,29). CTP synthetase exhibits positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP (8, 21, 23, 24, 29 -33). The positive c...
The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser 424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser 424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent K m values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser CTP synthetase is an essential enzyme in all organisms. The essential nature of this enzyme emanates from the fact that the product of its reaction CTP is required for the synthesis of nucleic acids and membrane phospholipids (1). The enzyme catalyzes the ATP-dependent transfer of the amide nitrogen of glutamine to the C-4 position of UTP to form CTP (2, 3). GTP stimulates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate (3-6). In eukaryotic cells, regulation of CTP synthetase activity plays an important role in the balance of nucleotide pools (6 -12) and in the synthesis of membrane phospholipids (12-14). The importance of understanding the regulation of CTP synthetase is further emphasized by the fact that unregulated levels of CTP synthetase activity is a common property of various human cancers (15-22).We utilize the yeast Saccharomyces cerevisiae as a model eukaryote to study the regulation of CTP synthetase and the impact of this regulation on phospholipid synthesis (Fig. 1). In yeast, CTP synthetase is encoded by the URA7 (10) and URA8 (11) genes. The yeast CTP synthetases (10, 11) contain a conserved glutamine amide transfer domain common to CTP synthetases from other organisms (24 -33). The URA7-encoded CTP synthetase is more abundant than the URA8-encoded enzyme (34) and is responsible for the majority of the CTP synthesized in vivo (11). Like CTP synthetase from mammalian cells (35), the yeast enzymes are allosterically regulated by their substrates and product CTP (6, 34).The S. cerevisiae URA7-encoded CTP synthetase is also regulated by phosphorylation. In vivo, CTP synthetase is phosphorylated on multiple serine residues (36). In vitro studies have shown that CTP synthetase is a substrate for protein kinase A (37) and for protein kinase C (36, 38). In S. cerevisiae, protein kinase A is the principal mediator of signals transmitted through the Ras-cAMP pathway (39, 40) whereas protein kinase C is required for the cell cycle (41-45) and plays a role maintaining cell wall integrity (46). Independently, the phosphorylation of CTP synthetase by protein kinase A (37) and by prote...
α-Synuclein (α-syn) is a major component of Lewy bodies found in synucleinopathies including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). Under the pathological conditions, α-syn tends to generate a diverse form of aggregates showing toxicity to neuronal cells and able to transmit across cells. However, mechanisms by which α-syn aggregates affect cytotoxicity in neurons have not been fully elucidated. Here we report that α-syn aggregates preferentially sequester specific synaptic proteins such as vesicle-associated membrane protein 2 (VAMP2) and synaptosomal-associated protein 25 (SNAP25) through direct binding which is resistant to SDS. The sequestration effect of α-syn aggregates was shown in a cell-free system, cultured primary neurons, and PD mouse model. Furthermore, we identified a specific blocking peptide derived from VAMP2 which partially inhibited the sequestration by α-syn aggregates and contributed to reduced neurotoxicity. These results provide a mechanism of neurotoxicity mediated by α-syn aggregates and suggest that the blocking peptide interfering with the pathological role of α-syn aggregates could be useful for designing a potential therapeutic drug for the treatment of PD.
Current single-molecule techniques do not permit the real-time observation of multiple proteins interacting closely with each other. We here report an approach enabling us to determine the single-molecule FRET kinetics of multiple protein-heterodimer interactions occurring far below the diffraction limit. We observed a strongly cooperative formation of multimeric SNARE complexes, which suggests that formation of the first SNARE complex triggers a cascade of SNARE complex formation.
The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose-and time-dependent and dependent on the concentrations of choline kinase (K m ؍ 27 g/ml) and ATP (K m ؍ 15 M). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V max /K m ؍ 17.5 mM ؊1 mol min
CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this study, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7⌬ ura8⌬ double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time-and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr 455 was a substrate for protein kinase A. A Thr 455 to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7⌬ ura8⌬ mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine.CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADPforming)) catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine (i.e. glutaminase reaction) to the C-4 position of UTP to generate CTP (1, 2). GTP activates the glutaminase reaction by accelerating the formation of a covalent glutaminyl enzyme intermediate (2, 3). The enzyme displays positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP (2-10). The kinetic behavior with respect to UTP and ATP is attributed to the nucleotide-dependent tetramerization of the enzyme (2,8,11,12). In fact, it is the tetrameric form of the enzyme that is active (2-10, 12).CTP synthetase is an essential enzyme for life because it catalyzes the formation of the CTP required for the synthesis of nucleic acids and membrane phospholipids (13). In eukaryotic cells, CTP synthetase activity controls the balance of nucleotide pools (8, 14 -19) and regulates the synthesis of membrane phospholipids (19 -21). The importance of understanding the regulation of CTP synthetase is highlighted by the fact that an unregulated level of its activity is a phenotype common to leukemia cells (22-24) and rapidly growing tumors of liver (25), colon (26), and lung (27). Indeed, CTP synthetase is a target of antiproliferative drug development for cancer therapy (28 -32, 34) and a target for parasite-based (35) and virally based (36) diseases.Two important modes of CTP synthetase regulati...
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