Single-Vesicle Fusion Assay Reveals Munc18-1 Binding to the SNARE Core Is Sufficient for Stimulating Membrane Fusion

1,2). According to their distribution in the cell, they are classified into v-SNARE, which includes synaptobrevin (VAMP), and t-SNAREs, which are composed of SNAP-25 and syntaxin for neuronal proteins. Sec1/Munc18 (SM) proteins are a family of cytoplasmic proteins with a conserved arch-shaped structure and play an essential role in intracellular membrane fusion. Along with SNARE proteins, which primarily mediate fusion of cellular transport vesicles with the target membrane, the SM proteins are thought to be central components of the exocytotic apparatus, which are required for membrane fusion (3−6). A gene knockout study has indicated that the SM proteins are crucial for synaptic membrane fusion, which is required for neurotransmitter release (7).Munc18 interacts with SNARE proteins at least in two different modes, namely, binding to Habc domain (amino acids 28−146) of syntanxin and binding to the SNARE core complex (8,9). In the first mode, Munc18 interacts with monomeric syntaxin through the N-terminal helical segment of syntaxin called the Habc domain to form a Munc18/syxntaxin binary complex (3−6). This Habc binding mode stabilizes the closed conformation of syntaxin during its transportation to the plasma membrane in vivo. Without this stabilization effect, the syntaxin level in sensory neurons was reduced by 70% in Munc18 knockout mice (10). In the second mode, Munc18 also binds to the SNARE core, a four helical bundle formed by syntaxin, VAMP, and SNAP25 (8,11,12). Ensemble proteoliposome fusion experiments showed that Munc18 binds preassembled SNARE complexes, and effectively promotes SNARE-mediated fusion with full-length syntaxin (11). An interaction called N-peptide binding between Munc18 and N-terminal peptides (amino acids 1−24) of syntaxin besides this SNARE core/Munc18 interaction is believed to be critical for this fusion promotion effect (8,11). However, how Munc18 activates fusion remains unclear in part due to the inability of the ensemble in vitro fusion assay to dissect different steps of the fusion reaction.For an unambiguous dissection of the protein-mediated membrane fusion mechanism, in vitro characterizations of reconstituted fusion machinery and regulators are required. Traditionally, in vitro studies rely on ensemble lipid mixing of proteoliposomes reconstituted with SNARE proteins, which cannot distinguish different stages of fusion such as docking, hemifusion and full fusion (13). Recently, new techniques have been developed for observing membrane fusion processes at the single-vesicle level (13−17). The single-vesicle fusion assay we developed could distinguish between different stages of docking, hemifusion, and full fusion via fluorescence resonance energy transfer (FRET) between the donor and acceptor fluorophores incorporated into the separate proteoliposomes reconstituted with t- and v-SNARE proteins, respectively. In addition, the single-vesicle fusion assay also allows us to describe the kinetics of transitions between different stages of fusion and postfusion pathways such as the kiss-and-run event (13) and to discover the dual functions of fusion regulator protein complexin I that inhibits SNARE complex formation and docking but enhances the fusion of docked vesicles together with calcium ions (18).Figure ​Figure1a1a illustrates our single-vesicle lipid-mixing assay. The v-SNARE vesicles carrying vesicle-associated membrane protein (VAMP) and the acceptor fluorophores were immobilized on a polymer-coated quartz surface via biotinylated lipids. The t-SNARE vesicles containing syntaxin and SNAP-25 and doped with the donor fluorophores were added together with Munc18, and the sample was incubated at 37 °C. After a 12-min incubation, the sample was transferred to a dual-color total internal reflection (TIR) fluorescence microscope (19), and FRET measurements of individual vesicles at 37 °C were performed 20 min after the reaction began. Passivation of quartz slides via coating with poly(ethylene glycol) (20) was essential in minimizing nonspecific binding of the vesicles to the surface and in keeping the proteins functional (Figure ​(Figure2)2) (13). The multiple intermediate states of fusion are classified according to their different FRET efficiency values as characterized previously (13). A finite but low-efficiency distribution ≤0.25 suggests close contact or docking between the donor and the acceptor vesicles without a high degree of lipid mixing. The final efficiency distribution around 0.35 indicates a hemifusion state. FRET efficiency distribution ≥0.5 is assigned as full fusion where both inner and outer leaflets have been mixed (13). The lipid composition of vesicles used in this study, 15 mol % PS (phospho-l-serine), 45 mol % PC (phosphocholine), and 40 mol % cholesterol, and the 200:1 lipid/protein ratio were chosen to emulate the composition of the native synaptic vesicles (18,21).Open in a separate windowFigure 1Single-vesicle FRET assay for Munc18-1 in neuronal SNARE-mediated fusion. (a), Schematics of the single-vesicle assay. (left) Acceptor-labeled v-SNARE vesicles are immobilized on a bottom quartz surface of a flow chamber. Donor-labeled t-SNARE vesicles, mixed with preset amount of Munc18-1, are introduced to the chamber space using a flow system. (right) Some t-SNARE vesicles dock to single v-SNARE vesicles through formation of trans-SNARE complexes, and Munc18-1 binds to the trans-SNARE complexes. Membrane fusion between t- and v-SNARE vesicles and resultant lipid mixing will cause an increase in the FRET efficiency. (b, c) FRET efficiency, E, distributions of single-vesicle complexes for various concentrations of Munc18-1 (0, 0.2, and 1 μM) in neuronal SNARE-mediated fusion with (b) syntaxin-full and (c) syntaxin-HT. Docking (or early fusion steps) shows low E values that are smaller than 0.25, and the full fusion state gives E ≈ 0.8 (13,18). To make the comparison clearer, we normalized histograms by the total number of liposomes per experiment, which is more than one thousand for all experiments (13,18).Open in a separate windowFigure 2Laser-excited (532 nm) images of single-vesicle fusion experiments with Munc18-1. Acceptor-labeled v-vesicles are directly tethered to the surface via biotin−neutravidin linker and the donor-labeled t-vesicles are added. Because the laser excites the acceptor only very weakly, bright fluorescent spots are seen only when the t-vesicles are present: (a) t-vesicles containing syntaxin-full and SNAP-25, (b) t-vesicles containing syntaxin-HT and SNAP-25, and (c) protein-free t-vesicles. Green and red rectangles denote the donor and acceptor emission detection channels, respectively. Panels a and b show docked t-SNARE vesicles in the donor channel and bright v-SNARE vesicles through FRET in the acceptor channel. Strong FRET signal demonstrates that binding of t-SNARE vesicles to the surface is specially achieved via interaction with the surface-immobilized v-SNARE vesicles. Panel c only shows dim v-SNARE vesicles in the acceptor channel without docking of t-vesicles, demonstrating that the nonspecific adhesion of the t-SNARE vesicles to the surface is minimal. In all experiments, 1 μM Munc18-1 was used.The resulting single-vesicle FRET efficiency histograms of the reaction product showed that Munc18-1 promotes full fusion represented at the FRET efficiency ≥0.5 in a concentration-dependent manner whether the full-length syntaxin 1A (syntaxin-full, amino acids 1−288) or the truncated syntaxin 1A lacking the N-peptide and Habc domain (syntaxin-HT (Habc-truncated), amino acids 168−288) was used (Figure ​(Figure1b,c).1b,c). After 20 min reaction, we observed more than 50% full fusion populations (Figure ​(Figure3),3), which is much faster than the previous report of several hours (11). The fusion promotion activity of Munc18-1 is dependent on SNAP-25 for both syntaxin-full and syntaxin-HT cases because omitting SNAP-25 led to a significant reduction in full fusion population (Figure ​(Figure3a,b).3a,b). Because SNAP-25 is required for the formation of the complete SNARE complex, it is likely that the interaction between Munc18-1 and the SNARE core complex promotes fusion. Furthermore, this fusion promotion activity of Munc18-1 does not seem to require additional interactions with the N-peptide of syntaxin 1A.Open in a separate windowFigure 3Fraction of vesicle complexes showing full fusion for various reaction conditions. One micromolar Munc18-1 was used. (a) Results obtained with the full-length syntaxin. (b) Results obtained with a syntanxin-HT. Controls without SNAP-25 led to a significant reduction in full fusion, likely due to the formation of incomplete SNARE complexes, which cannot mediate efficient fusion (18). The full fusion population is calculated by summing all normalized populations with E values >0.5 (13,18). Error bars denote the SD of three to five independent experiments with different batches of SNARE and Munc18 proteins.Spin labeling electron paramagnetic resonance (EPR) has proven effective in detecting the intermolecular interaction such the one between syntaxin 1A and Munc18-1. The seven native cysteines in wild-type Munc18 hamper the site-specific attachment of the nitroxide spin labels. However, we could at least nonselectively spin-label native cysteines to primitively probe the putative intermolecular interaction. For this purpose, wild-type Munc18-1 was labeled with the methanethiosulfonate spin label. The EPR spectra for spin labeled Munc18-1 in solution, with syntaxin 1A (full-length and HT), and with the SNARE complex in membrane were measured at room temperature (Figure ​(Figure4).4). The broadened line shape of the EPR spectrum indicates that the Munc18-1 binds to full-length syntaxin directly (Figure ​(Figure4b,4b, left panel). The EPR spectrum did not show any change when incubated with syntaxin-HT only (Figure ​(Figure4c,4c, left panel). These data are consistent with the previous conclusion that the Habc domain of syntaxin plays an important role in the interaction between monomeric syntaxin and Munc18 in solution.Open in a separate windowFigure 4Electron paramagnetic resonance (EPR) spectra of Munc18-1. (a) Munc18-1 only (left panel) and Munc18-1with protein-free membrane in the fusion buffer (right panel). The same EPR sharp spectra indicate that there is no measurable interaction between Munc18-1 and membrane. (b) Munc18-1 with syntaxin-full protein (left panel) and Munc18-1with membrane-associated SNARE complex containing syxtaxin-full (right panel). Both EPR spectra are broadened in comparison to the spectrum of Munc18-1 only (a, left panel) indicating interactions. (c) Munc18-1 with syntaxin-HT protein (left panel) and Munc18-1 with membrane-associated SNARE complex containing syntaxin-HT (right panel). The sharp EPR spectrum on the left indicates that there is no interaction between Munc18-1 and syntaxin-HT, while the broadened EPR spectrum on the right suggests interactions. Arrows indicate the quaternary interaction between Munc18 and SNAREs, and circles show the spin−spin interaction due to the clustering of spin-labeled Munc18.We found a different result when the EPR spectrum from Munc18 was measured with the SNARE complex in the context of membrane. After full-length syntaxin or syntaxin-HT was reconstituted into membrane and formed a ternary complex with SNAP-25 and soluble VAMP 2 (amino acids 1−89), spin-labeled Munc18-1 was added to the complex. The EPR spectra of Munc18-1 were broadened for SNARE complexes containing both full-length syntaxin and syntaxin-HT, indicating that the N-peptide of syntaxin 1A is not necessary for Munc18-1 interaction with the membrane-associated SNARE complexes (Figure ​(Figure4b,c,4b,c, right panels). Interestingly, Rothman’s group (11) also found that Munc18-1 still binds to the membrane-associated SNARE complex even with the Habc domain removed (syntaxin-HT) or the N-peptide mutated (L8A). Overall, our work shows that Munc18-1 could bind to the SNARE core complex reconstituted into the lipid membrane even in the absence of the N-peptide interaction, and this SNARE core/Munc18 binding mode is likely to be responsible for fusion acceleration by Munc18-1. Meanwhile, the EPR analysis showed that there is no direct interaction between the membrane and Munc18-1 (Figure ​(Figure4a,4a, right panel), which rules out the possibility that the spectral broadening observed in the presence of SNARE complex (Figure ​(Figure4c,4c, right panel) was caused by lipid molecules. We note here that we not only have the EPR line broadening (Figure ​(Figure4,4, indicated by arrows) due to the quaternary interaction between Munc18 and SNAREs, but we also see some extra line broadening (Figure ​(Figure4,4, indicated by circles) due to the spin−spin interaction. The spin−spin interaction is most likely due to the clustering of spin-labeled Munc18, perhaps reflecting the binding of several Munc18 molecules to the oligomeric supramolecular SNARE complex (22).It has been debated whether the main function of Munc18 is for vesicle docking on the plasma membrane or whether it also assists membrane fusion (5,23). The role in vesicle docking is supported by impaired dense-core granule docking in adrenal chromaffin cells of Munc18-1 knockout mice (24). Other observations, however, suggest that SM proteins function also at a late, postdocking stage of membrane fusion (11,25). Our data on single-vesicle lipid mixing and EPR spectroscopy highlight the importance of Munc18 binding to the SNARE core complex in stimulating membrane fusion. For both roles, the syntaxin N-terminal domain containing N-peptide and Habc domain is regarded as an essential component for Munc18’s dual interactions with syntaxin for vesicle docking as well as with the SNARE complex for fusion stimulation (3−6,8,11,12).Our EPR data support the requirement of syntaxin N-terminal domain in the interaction between monomeric syntaxin and Munc18. However, the interaction between SNARE complex and Munc18 to promote fusion, in the context of membranes, is not dependent upon the N-terminal domain of syntaxin. Recently, Fasshauer’s group (26) found that the interaction between Munc18 and the syntaxin N-terminal domain blocks the SNARE complex formation. However, a truncated syntaxin 1A (amino acids 25−262) could bind to Munc18-1 with high affinity and a SDS-resistant SNARE complex together with SNAP-25 and VAMP could be formed. Based on our data and other groups’ findings (9,26,27), we propose that for the stimulation of membrane fusion, Munc18-1 interacts with the SNARE complex in a mode where the Habc-domain is not required.In conclusion, Munc18-1 promotes neuronal SNARE-mediated fusion not only with the full-length syntaxin 1A but also with Habc-truncated syntaxin 1A. The SNARE complex/Munc18 interaction is mainly responsible for this effect. Furthermore, Munc18-1 accelerates vesicle fusion significantly more rapidly than previously observed. With the advent of new single-molecule imaging technologies, these protein−protein interactions critical for fusion may also become observable.     

Increasing Resistance of Tubular Epithelial Cells to Apoptosis by shRNA Therapy Ameliorates Renal Ischemia-Reperfusion Injury

Renal tubular epithelial cells (TEC) die by apoptosis or necrosis in renal ischemia-reperfusion injury (IRI). Fas/Fas ligand-dependent fratricide is critical in TEC apoptosis, and Fas promotes renal IRI. Therefore, targeting Fas or caspase-8 may have therapeutic potential for renal injury in kidney transplant or failure. RNA silencing by short hairpin RNA (shRNA) is a novel strategy to down-regulate protein expression. Using this approach, silencing of Fas or caspase-8 by shRNA to prevent TEC apoptosis and IRI was evaluated. IRI was induced by renal artery clamping for 45 or 60 min at 32 degrees C in uninephrectomized C57BL/6 mice. Here, we showed that Fas or pro-caspase-8 expression was significantly knocked down in TEC by stable expression of shRNA, resulting in resistance to apoptosis induced by superoxide, IFN-gamma/TNF-alpha and anti-Fas antibody. Inferior vena cava delivery of pHEX-small interfering RNA targeting Fas or pro-caspase-8 resulted in protection of kidney from IRI, indicated by reduction of renal tubular injury (necrosis and apoptosis) and serum creatinine or blood urea nitrogen. Our data suggest that shRNA-based therapy targeting Fas and caspase-8 in renal cells can lead to protection of kidney from IRI. Attenuation of pro-apoptotic proteins using genetic manipulation strategies such as shRNA might represent a novel strategy to promote kidney allograft survival from rejection or failure.     

自制中药包加压理疗裤在预防和控制腹腔镜疝修补后并发症的临床研究

目的分析自制中药包加压理疗裤在腹腔镜腹股沟疝修补术后并发症的防治效果。 方法收集2019年8月至2021年8月山西省中医药研究院普外科收治的腹股沟疝患者128例,按照随机数字表法,随机分为对照组和试验组,每组患者64例。2组患者均接受腹腔镜腹股沟疝修补术。对照组术后于腹股沟区沙袋加压并穿戴传统疝气带。试验组术后穿戴我科自制中药包加压理疗裤。对比2组临床指标及并发症发生情况。 结果试验组术后疼痛视觉模拟评分较对照组低,住院时间较对照组短,PZB服务质量量表评分较对照组高（P<0.05）。术后24 h及7 d,试验组血肿及血清肿发生率、阴囊肿胀及阴囊血肿发生率低于对照组（P<0.05）。术后30 d,2组血肿及血清肿发生率、阴囊肿胀及阴囊血肿发生率差异无统计学意义（P>0.05）。 结论腹腔镜腹股沟修补术后穿戴中药包加压理疗裤可减轻疼痛程度,降低血肿、血清肿及阴囊肿胀等术后并发症发生率,缩短住院时间,提高干预满意度。     

符合米兰标准和杭州标准的肝癌肝移植受者预后回顾性分析

目的比较符合米兰标准和符合杭州标准的肝细胞肝癌(HCC)患者行肝移植术后生存和肿瘤复发情况,验证杭州标准的临床应用价值。方法回顾性分析2006年1月至2011年12月中国肝移植注册登记的肝移植手术,并在浙江大学医学院附属第一医院、重庆医科大学附属第一医院两家肝移植中心接受随访调查受者的临床资料,196例肝癌肝移植受者纳入研究。将符合米兰标准的HCC患者作为米兰标准组,共90例(45.9%);超出米兰标准但符合杭州标准者作为杭州标准组,共40例(20.4%);肿瘤结节直径之和8 cm,术前AFP400 ng/m L且组织学分级为低度分化者作为超出杭州标准组,共66例(33.7%)。比较3组受者术后生存率、无瘤生存率,并评判预后。结果米兰标准组受者术后1、3、5年生存率和1、3、5年无瘤生存率分别为88.9%、73.3%、60.0%和84.4%、66.7%、51.1%,杭州标准组受者术后1、3、5年生存率和1、3、5年无瘤生存率分别为80.0%、65.0%、50.0%和75.0%、55.0%、45.0%,超出杭州标准组受者术后1、3、5年生存率和1、3、5年无瘤生存率分别为57.6%、30.3%、18.1%和45.5%、27.3%、18.1%。对3组受者术后累积生存率和无瘤生存率进行比较,米兰标准组和杭州标准组受者术后1、3、5年生存率和1、3、5年无瘤生存率差异均无统计学意义(P均0.05),米兰标准组受者术后1、3、5年生存率和无瘤生存率均显著高于超出杭州标准组受者(P均0.05),杭州标准组受者术后1、3、5年生存率和1、3、5年无瘤生存率均显著高于超出杭州标准组受者(P均0.05)。结论同米兰标准一样,杭州标准也具有很强的科学性,能够显著拓展受益人群,让更多未符合米兰标准的患者能够施行肝移植术。同时杭州标准能有效预测肝移植受者预后,其创新性提出的生物学标准AFP水平和肿瘤分化程度是影响肝癌肝移植受者术后预后的关键性因素。     

山羊颈椎次全切除减压非融合后生物力学特性

目的 探讨山羊颈椎次全切除减压非融合后生物力学特性.方法 选取成年雄性山羊6只造模(模型组),术前行颈椎X线检查,排除骨性结构异常,并测量节段性Cobb角(SCA)和颈椎Cobb角(CCA)于C4行颈椎次全切除术,并植入可动人工颈椎(ACV).术后行颈椎正侧位X线检查,再次测量SCA和CCA.术后6个月处死山羊,并以6...     

Metabolism of the new synthetic cannabinoid EG-018 in human hepatocytes by high-resolution mass spectrometry

Purpose

The present study aims to recommend appropriate urinary marker metabolites for documenting EG-018 consumption by investigating its metabolism in human hepatocytes.

Methods

For metabolite profiling, 10 µM EG-018 was incubated in human hepatocytes for 3 h. Metabolite identification in hepatocyte samples was accomplished with high-resolution mass spectrometry via information-dependent data acquisition.

Results

EG-018 was highly metabolized in human hepatocytes. A total of eight metabolites were characterized, mainly generated from hydroxylation and carbonylation on the pentyl chain. Dihydrodiol formation, N-dealkylation, and glucuronidation of hydroxylated metabolites were the other major pathways.

Conclusions

The primary metabolites of EG-018 in human hepatocyte incubation were pentyl hydroxylated EG-018 (M6) and pentyl carbonylated EG-018 (M8). These two metabolites are proposed as the best urinary markers for confirming EG-018 intake.

     

机械通气辅助及血压调控对重症颅脑损伤患者脑氧代谢的影响

目的观察机械通气辅助及血压调控对重症颅脑损伤患者脑氧代谢的影响。方法56例重症颅脑外伤患者(3相似文献   

Postnatal development of nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the retina of the golden hamster

The histochemical method was used to investigate the postnatal development of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) -positive neurons in retinas of the golden hamster. NADPH-d-positive neurons were discernible in the retina at postnatal day (P)1. From P4 onward to adulthood, when the retina acquired its laminated characteristics, NADPH-d- positive neurons were observed in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Results showed that NADPH-d-positive neurons in INL and GCL followed different time courses and patterns in their development. NADPH-d-positive neurons in INL underwent a sharp increase from P4 to P8 (3.6-fold), followed by a decrease to 46% of the maximum at P12. This value was maintained relatively constant to the adult level. The mean diameters of NADPH-d-positive neurons in INL, which were smaller than those in the GCL for all ages, increased from P8 to P12 and from P20 to adulthood. As for neurons in the GCL, the increase in cell number was not so apparent for the earlier postnatal days until P20; thereafter, an obvious increase to the adult level was observed. The mean diameters of the NADPH-d-positive cell bodies in the GCL increased with age, except for P16-P20, during which time there was a slight and insignificant decrease. The tendency of changes in cell density was basically similar to that of the total number for both the INL and the GCL. Between P12 and P20, the density distribution map of the NADPH-d-positive neurons underwent dramatic changes: The highest density shifted from the upper central retina at the earlier postnatal days to the lower central retina in the adult. The two waves of increase in NADPH-d-positive neurons coincide with the process of axonal elongation and synaptogenesis and the acquisition of visual function and experience. It is suggested that these NADPH-d-positive neurons are related to these two developmental events.     

高效液相色谱法测定血浆中洛美沙星浓度及其在人体内药代动力学

建立了人血浆洛美沙星的反相离子对高效液相色谱测定方法。该法简便易行,精密度好,方法回收率95～102%,日内、日间RSD为2.1～7.8%,血药浓度在0.125～5.012 μg/ml范围内呈线性关系,相关系数0.9998,当S/N=2时,最小检测浓度为25 ng/ml。健康志愿者口服400 mg洛美沙星,药代动力学过程符合一室模型,消除相半衰期为5.5 h。     

Cadmium Pollution in Paddy Soil as Affected by Different Rice (Oryza sativa L.) Cultivars

No abstract available.