Three αSNAP and 10 ATP Molecules Are Used in SNARE Complex Disassembly by N-ethylmaleimide-sensitive Factor (NSF)

The fusion of intracellular membranes is driven by the formation of a highly stable four-helix bundle of SNARE proteins embedded in the vesicle and target membranes. N-Ethylmaleimide sensitive factor recycles SNAREs after fusion by binding to the SNARE complex through an adaptor protein, αSNAP, and using the energy of ATP hydrolysis to disassemble the complex. Although only a single molecule of αSNAP binds to a soluble form of the SNARE complex, we find that three molecules of αSNAP are used for SNARE complex disassembly. We describe an engineered αSNAP trimer that supports more efficient SNARE complex disassembly than monomeric αSNAP. Using the trimerized αSNAP, we find that N-ethylmaleimide-sensitive factor hydrolyzes 10 ATP molecules on average to disassemble a single SNARE complex.     

神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白

近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用.     

A Conserved Membrane Attachment Site in ��-SNAP Facilitates N-Ethylmaleimide-sensitive Factor (NSF)-driven SNARE Complex Disassembly

The ATPase NSF (N-ethylmaleimide-sensitive factor) and its SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) cofactor constitute the ubiquitous enzymatic machinery responsible for recycling of the SNARE (SNAP receptor) membrane fusion machinery. The enzyme uses the energy of ATP hydrolysis to dissociate the constituents of the SNARE complex, which is formed during the fusion of a transport vesicle with the acceptor membrane. However, it is still unclear how NSF and the SNAP adaptor work together to take the tight SNARE bundle apart. SNAPs have been reported to attach to membranes independently from SNARE complex binding. We have investigated how efficient the disassembly of soluble and membrane-bound substrates are, comparing the two. We found that SNAPs support disassembly of membrane-bound SNARE complexes much more efficiently. Moreover, we identified a putative, conserved membrane attachment site in an extended loop within the N-terminal domain of α-SNAP. Mutation of two highly conserved, exposed phenylalanine residues on the extended loop prevent SNAPs from facilitating disassembly of membrane-bound SNARE complexes. This implies that the disassembly machinery is adapted to attack membrane-bound SNARE complexes, probably in their relaxed cis-configuration.     

Luminescence detection of SNARE–SNARE interaction in Arabidopsis protoplasts

Membrane associated proteins SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) provide the minimal fusion machinery necessary for cellular vesicles to fuse to target organelle membranes in eukaryotic cells. Despite the conserved nature of the fusion machinery in all eukaryotes, it still remains challenging to identify functional SNARE pairs in higher plants. We developed a method based on a split-luciferase complementation assay for detecting changes in SNARE–SNARE interaction by luminescence within Arabidopsis protoplasts that express recombinant proteins at physiological levels in 96-well plates. The reliability of the assay was confirmed by three experiments. First, reduction of the SNARE–SNARE interaction caused by a single amino acid substitution adjacent to the SNARE motif in endosome-localized AtVAM3/SYP22 (syntaxin of plant 22) was detected by a reduction of luminescence. Second, reduction of the interaction between plasma-membrane localized SYP121 and VAMP722 in response to sodium azide was detected in real-time. Third, the results of 21 SNARE pairs investigated by this method largely agreed with the results from previously reported co-immunoprecipitation assays. Using the method, we newly identified the interaction between SYP121 and VAMP722 was significantly increased when the protoplasts were incubated in the light. Microscopic observation of transgenic Arabidopsis expressing GFP–SYP121 (green fluorescent protein tagged SYP121) from its own promoter suggested that the plasma-membrane localization of GFP–SYP121 is maintained by light. These suggested that the vesicle trafficking pathway mediated by SYP121 might be regulated by light in Arabidopsis. In general, this article demonstrated the method that can generate new biological insight of the SNARE protein interactions in plant cells.     

SNARE protein recycling by αSNAP and βSNAP supports synaptic vesicle priming

Neurotransmitter release proceeds by Ca(2+)-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and βSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and βSNAP expression on synaptic vesicle exocytosis, employing a new Ca(2+) uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca(2+) sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and βSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.     

Classification of α 1-antitrypsin (Pi) phenotypes by isoelectrofocusing

Summary Pi phenotypes have been determined by isoelectrofocusing in a sample of 538 healthy individuals from Southern Germany. Further subdivision of the common PiM phenotype is described. A procedure for the delineation of six common subtypes is presented. It is assumed that the six subtypes are determined by three alleles which are provisionally called Pi^Ma, Pi^Mb, and Pi^Mc. Their frequencies in this sample were 0.75, 0.06, and 0.15, respectively.Supported in part by INSERM, contract No. AT-FA-58     

民族生物学学科规范--美国国家科学基金(NSF)生物复杂性研讨会报告

SalickJan (MissouriBotanicalGarden) ;　　　AsaCheryl (St.LouisZoo) ;CamiloGerardo (St.LouisUniveristy) ;　　　FritzGayleJ .(WashingtonUniversity St.Louis) ;MillerJim (MissouriBotanicalGarden) ;　　　AlcornJanis (WorldResourcesInstitute) ;AndersonEugene (U     

Cardiac and neuroprotection regulated by α(1)-adrenergic receptor subtypes

Sympathetic nervous system regulation by the α(1)-adrenergic receptor (AR) subtypes (α(1A), α(1B), α(1D)) is complex, whereby chronic activity can be either detrimental or protective for both heart and brain function. This review will summarize the evidence that this dual regulation can be mediated through the different α(1)-AR subtypes in the context of cardiac hypertrophy, heart failure, apoptosis, ischemic preconditioning, neurogenesis, locomotion, neurodegeneration, cognition, neuroplasticity, depression, anxiety, epilepsy, and mental illness.     

Monocarboxylate transporter (MCT)-1 is up-regulated by PPARα

Peroxisome proliferator-activated receptor (PPAR)-α mediates an adaptive response to fasting by up-regulation of genes involved in fatty acid oxidation and ketone body synthesis. Ketone bodies are transferred in and out of cells by monocarboxylate transporter (MCT)-1. In this study we observed for the first time that activation of PPARα in rats by clofibrate treatment or fasting increased hepatic mRNA concentration of MCT1. In Fao rat hepatoma cells, incubation with the PPARα agonist WY 14,643 increased mRNA concentration of MCT1 whereas the PPARγ agonist troglitazone did not. To elucidate whether up-regulation of MCT1 is indeed mediated by PPARα we treated wild-type and PPARα-null mice with WY 14,643. In wild-type mice, treatment with WY 14,643 increased mRNA concentrations of MCT1 in liver, kidney and small intestine whereas no up-regulation was observed in PPARα-null mice.     

Alteration of Water Structure by Peptide Clusters Revealed by Neutron Scattering in the Small-Angle Region (below 1???1)

Solution scattering of neutrons and x-rays can provide direct information on local interactions of importance for biomolecular folding and structure. Here, neutron scattering experiments are combined with molecular-dynamics simulation to interpret the scattering signal of a series of dipeptides with varying degrees of hydrophobicity (GlyAla, GlyPro, and AlaPro) in concentrated aqueous solution (1:20 solute/water ratio) in which the peptides form large segregates (up to 50–60 amino acids). Two main results are found: 1), the shift to lower Q of the so-called water-ring peak (Q ≈ 2 Å⁻¹) arises mainly from an overlap of water-peptide and peptide-peptide correlations in the region of 1.3 < Q < 2 Å⁻¹, rather than from a shift of the water signal induced by the presence of the clusters; and 2), in the low-Q region (Q ≈ 0.6 Å⁻¹) a positive peak is observed originating from both the solute-solute correlations and changes in the water structure induced by the formation of the clusters. In particular, the water molecules are found to be more connected than in the bulk with hydrogen-bonding directions tangential to the exposed hydrophobic surfaces, and this effect increases with increasing peptide hydrophobicity. This work demonstrates that important information on the (hydrophobic) hydration of biomolecules can be obtained in the very-small-angle region.     

Highly selective biotransformation of (+)-(1S)- and (−)-(1R)-camphorquinone by Aspergillus wentii

Abstract

To clarify the structures of biotransformation products and metabolic pathways, the biotransformation of monoterpenoids, (+)- and (?)-camphorquinone (1a and b), has been investigated using Aspergillus wentii as a biocatalyst. Compound 1a was converted to (?)-(2S)-exo-hydroxycamphor (2a), (?)-(2S)-endo-hydroxycamphor (3a), (?)-(3S)-exo-hydroxycamphor (4a), (?)-(3S)-endo-hydroxycamphor (5a), and (+)-camphoric acid (6a). Compound 1b was converted to (+)-(2R)-exo-hydroxycamphor (2b), (+)-(2R)-endo-hydroxycamphor (3b), (+)-(3R)-exo-hydroxycamphor (4b), (+)-(3R)-endo-hydroxycamphor (5b), and (?)-camphoric acid (6b). Compound 1a mainly produced 2a (65.0%) with stereoselectivity, whereas 1b afforded 3b (84.3%) with high stereoselectivity. These structures were confirmed by gas chromatography–mass spectrometry, infrared, ¹H nuclear magnetic resonance (NMR), and ¹³C NMR spectral data. The products illustrate the marked ability of A. wentii for enzymatic oxidation and ketone reduction.     

Modulation of Dynamin-related Protein 1 (DRP1) Function by Increased O-linked-β-N-acetylglucosamine Modification (O-GlcNAc) in Cardiac Myocytes

O-linked-N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of the serine and threonine residues of cellular proteins is a dynamic process and affects phosphorylation. Prolonged O-GlcNAcylation has been linked to diabetes-related complications, including mitochondrial dysfunction. Mitochondria are dynamically remodeling organelles, that constantly fuse (fusion) and divide (fission). An imbalance of this process affects mitochondrial function. In this study, we found that dynamin-related protein 1 (DRP1) is O-GlcNAcylated in cardiomyocytes at threonine 585 and 586. O-GlcNAcylation was significantly enhanced by the chemical inhibition of N-acetyl-glucosaminidase. Increased O-GlcNAcylation decreases the phosphorylation of DRP1 at serine 637, which is known to regulate DRP1 function. In fact, increased O-GlcNAcylation augments the level of the GTP-bound active form of DRP1 and induces translocation of DRP1 from the cytoplasm to mitochondria. Mitochondrial fragmentation and decreased mitochondrial membrane potential also accompany the increased O-GlcNAcylation. In conclusion, this report shows, for the first time, that O-GlcNAcylation modulates DRP1 functionality in cardiac muscle cells.     

Linkage analysis of (1→6)-linked oligosaccharides by alkaline degradation

2-C-Methylglyceric acid was formed by sequential degradation of the chain in isomaltose oligosaccharides in aqueous barium hydroxide. The products of the stopping reaction, namely 6-O-substituted 3-deoxy-d-hexonic acids, were also obtained in considerable yield. The separation and determination of these products is of importance for the analysis of the linkage sequence in oligosaccharides.     

Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-β1 (TGF-β1) administration suppresses osteoblast differentiation

TGF-β1 can regulate osteoblast differentiation not only positively but also negatively. However, the mechanisms of negative regulation are not well understood. We previously established the reproducible model for studying the suppression of osteoblast differentiation by repeated or high dose treatment with TGF-β1, although single low dose TGF-β1 strongly induced osteoblast differentiation. The mRNA expression and protein level of insulin-like growth factor-1 (IGF-1) were remarkably decreased by repeated TGF-β1 administration in human periodontal ligament cells, human mesenchymal stem cells, and murine preosteoblast MC3T3-E1 cells. Repeated TGF-β1 administration subsequently decreased alkaline phosphatase (ALP) activity and mRNA expression of osteoblast differentiation marker genes, such as RUNX2, ALP, and bone sialoprotein (BSP). Additionally, repeated administration significantly reduced the downstream signaling pathway of IGF-1, such as Akt phosphorylation in these cells. Surprisingly, exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-β1 administration. These facts indicate that the key mechanism of inhibition of osteoblast differentiation induced by repeated TGF-β1 treatment is simply due to the down-regulation of IGF-1 expression. Inhibition of IGF-1 signaling using small interfering RNA (siRNA) against insulin receptor substrate-1 (IRS-1) suppressed mRNA expression of RUNX2, ALP, BSP, and IGF-1 even with single TGF-β1 administration. This study showed that persistence of TGF-β1 inhibited osteoblast differentiation via suppression of IGF-1 expression and subsequent down-regulation of the PI3K/Akt pathway. We think this fact could open the way to use IGF-1 as a treatment tool for bone regeneration in prolonged inflammatory disease.     

Tumor suppressor cell adhesion molecule 1 (CADM1) is cleaved by a disintegrin and metalloprotease 10 (ADAM10) and subsequently cleaved by γ-secretase complex

Cell adhesion molecule 1 (CADM1) is a type I transmembrane glycoprotein expressed in various tissues. CADM1 is a cell adhesion molecule with many functions, including roles in tumor suppression, apoptosis, mast cell survival, synapse formation, and spermatogenesis. CADM1 undergoes membrane-proximal cleavage called shedding, but the sheddase and mechanisms of CADM1 proteolysis have not been reported. We determined the cleavage site involved in CADM1 shedding by LC/MS/MS and showed that CADM1 shedding occurred in the membrane fraction and was inhibited by tumor necrosis factor-α protease inhibitor-1 (TAPI-1). An siRNA experiment revealed that ADAM10 mediates endogenous CADM1 shedding. In addition, the membrane-bound fragment generated by shedding was further cleaved by γ-secretase and generated CADM1-intracellular domain (ICD) in a mechanism called regulated intramembrane proteolysis (RIP). These results clarify the detailed mechanism of membrane-proximal cleavage of CADM1, suggesting the possibility of RIP-mediated CADM1 signaling.     

Backbone dynamics of (1–71)- and (1–36)bacterioopsin studied by two-dimensional 1H-15N NMR spectroscopy

Summary The backbone dynamics of uniformly ¹⁵N-labelled fragments (residues 1–71 and 1–36) of bacterioopsin, solubilized in two media (methanol-chloroform (1:1), 0.1 M ²HCO₂NH₄, or SDS micelles) have been investigated using 2D proton-detected heteronuclear ¹H-¹⁵N NMR spectroscopy at two spectrometer frequencies, 600 and 400 MHz. Contributions of the conformational exchange to the transverse relaxation rates of individual nitrogens were elucidated using a set of different rates of the CPMG spin-lock pulse train and were essentially suppressed by the high-frequency CPMG spin-lock. We found that most of the backbone amide groups of (1–71)bacterioopsin in SDS micelles are involved in the conformational exchange process over a rate range of 10³ to 10⁴ s^-1. This conformational exchange is supposed to be due to an interaction between two -helixes of (1–71)bacterioopsin, since the hydrolysis of the peptide bond in the loop region results in the disappearance of exchange line broadening. ¹⁵N relaxation rates and ¹H-¹⁵N NOE values were interpreted using the model-free approach of Lipari and Szabo [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559]. In addition to overall rotation of the molecule, the backbone N-H vectors of the peptides are involved in two types of internal motions: fast, on a time scale <20 ps, and intermediate, on a time scale close to 1 ns. The intermediate dynamics in the -helical stretches was mostly attributed to bending motions. A decrease in the order parameter of intermediate motions was also observed for residues next to Pro⁵⁰, indicating an anisotropy of the overall rotational diffusion of the molecule. Distinctly mobile regions are identified by a large decrease in the order parameter of intermediate motions and correspond to the N- and C-termini, and to a loop connecting the -helixes of (1–71)bacterioopsin. The internal dynamics of the -helixes on the millisecond and nanosecond time scales should be taken into account in the development of a model of the functioning bacteriorhodopsin.Abbreviations BO bacterioopsin - 2D two-dimensional - CPMG Carr-Purcell-Meiboom-Gill (Carr and Purcell, 1954) - SDS sodium dodecyl(²H₂₅) sulfate - R(S_x), R(S_z) ¹⁵N transverse and longitudinal relaxation rates, respectively     

Inhibition of Cytidine Deaminase by Derivatives of 1-(β-D-Ribofuranosyl)-Dihydropyrimidin-2-One (Zebularine)

Abstract

The 2′-deoxy and ara derivatives of 1-β-(D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) were synthesized by improved routes and tested for their inhibitory properties against cytidine deaminase. It was shown that the K_i′s of both compounds were comparable to that of the parent zebularine in inhibition studies with purified enzyme. In contrast to zebularine, 2′-deoxy and ara zebularine showed only nominal cytotoxicity against MOLT-4 and L1210 cells in vitro. A model compound for the inhibition of deoxycytidylate deaminase, 2′-deoxyzebularine 5′-monophosphate (6), was also prepared.     

Analysis of Aβ (1-40) and Aβ (1-42) monomer and fibrils by capillary electrophoresis

A method based on capillary electrophoresis (CE) with UV absorbance detection is presented to characterize synthetic amyloid beta (Aβ) peptide preparations at different aggregation states. Aggregation of Aβ (1-40) and Aβ (1-42) is closely linked to Alzheimer's disease (AD), and studying how Aβ peptides self-assemble to form aggregates is the focus of intense research. Developing methods capable of identifying, characterizing and quantifying a wide range of Aβ species from monomers to fully formed fibrils is critical for AD research and is a major analytical challenge. Monomer and fibril samples of Aβ (1-40) and Aβ (1-42) were prepared and characterized for this study. The monomer-equivalent concentration for each sample was determined by HPLC-UV, and aggregate formation was confirmed and characterized by transmission electron microscopy. The same samples were studied using CE with UV absorbance detection. Analysis by mass spectrometry of collected CE fractions was used to confirm the presence of Aβ for some CE-UV peaks. The CE-UV method reported here clearly indicates that monomeric and aggregated Aβ were electrophoretically separated, and substantial differences in the electrophoretic profiles between samples of Aβ (1-40) and Aβ (1-42) were observed. This CE-UV method can differentiate between Aβ monomer, oligomeric intermediates, and mature fibrils.