A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion

Yeast vacuoles undergo priming, docking, and homotypic fusion, although little has been known of the connections between these reactions. Vacuole-associated Vam2p and Vam6p (Vam2/6p) are components of a 65S complex containing SNARE proteins. Upon priming by Sec18p/NSF and ATP, Vam2/6p is released as a 38S subcomplex that binds Ypt7p to initiate docking. We now report that the 38S complex consists of both Vam2/6p and the class C Vps proteins [Reider, S. E. and Emr, S. D. (1997) Mol. Biol. Cell 8, 2307-2327]. This complex includes Vps33p, a member of the Sec1 family of proteins that bind t-SNAREs. We term this 38S complex HOPS, for homotypic fusion and vacuole protein sorting. This unexpected finding explains how Vam2/6p associates with SNAREs and provides a mechanistic link of the class C Vps proteins to Ypt/Rab action. HOPS initially associates with vacuole SNAREs in "cis" and, after release by priming, hops to Ypt7p, activating this Ypt/Rab switch to initiate docking.     

Trans-SNARE complex assembly and yeast vacuole membrane fusion

cis-SNARE complexes (anchored in one membrane) are disassembled by Sec17p (alpha-SNAP) and Sec18p (NSF), permitting the unpaired SNAREs to assemble in trans. We now report a direct assay of trans-SNARE complex formation during yeast vacuole docking. SNARE complex assembly and fusion is promoted by high concentrations of the SNARE Vam7p or Nyv1p or by addition of HOPS (homotypic fusion and vacuole protein sorting), a Ypt7p (Rab)-effector complex with a Sec1/Munc18-family subunit. Inhibitors that target Ypt7p, HOPS, or key regulatory lipids prevent trans-SNARE complex assembly and ensuing fusion. Strikingly, the lipid ligand MED (myristoylated alanine-rich C kinase substrate effector domain) or elevated concentrations of Sec17p, which can displace HOPS from SNARE complexes, permit full trans-SNARE pairing but block fusion. These findings suggest that efficient fusion requires trans-SNARE complex associations with factors such as HOPS and subsequent regulated lipid rearrangements.     

The CORVET complex promotes tethering and fusion of Rab5/Vps21-positive membranes

Membrane fusion along the endocytic pathway occurs in a sequence of tethering, docking, and fusion. At endosomes and vacuoles, the CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) tethering complexes require their organelle-specific Rabs for localization and function. Until now, despite the absence of experimental evidence, it has been assumed that CORVET is a membrane-tethering factor. To test this theory and understand the mechanistic analogies with the HOPS complex, we set up an in vitro system, and establish CORVET as a bona-fide tether for Vps21-positive endosome/vacuole membranes. Purified CORVET binds to SNAREs and Rab5/Vps21-GTP. We then demonstrate that purified CORVET can specifically tether Vps21-positive membranes. Tethering via CORVET is dose-dependent, stimulated by the GEF Vps9, and inhibited by Msb3, the Vps21-GAP. Moreover, CORVET supports fusion of isolated membranes containing Vps21. In agreement with its role as a tether, overexpressed CORVET drives Vps21, but not the HOPS-specific Ypt7 into contact sites between vacuoles, which likely represent vacuole-associated endosomes. We therefore conclude that CORVET is a tethering complex that promotes fusion of Rab5-positive membranes and thus facilitates receptor down-regulation and recycling at the late endosome.     

Structure of a C-terminal fragment of its Vps53 subunit suggests similarity of Golgi-associated retrograde protein (GARP) complex to a family of tethering complexes

The Golgi-associated retrograde protein (GARP) complex is a membrane-tethering complex that functions in traffic from endosomes to the trans-Golgi network. Here we present the structure of a C-terminal fragment of the Vps53 subunit, important for binding endosome-derived vesicles, at a resolution of 2.9 Å. We show that the C terminus consists of two α-helical bundles arranged in tandem, and we identify a highly conserved surface patch, which may play a role in vesicle recognition. Mutations of the surface result in defects in membrane traffic. The fold of the Vps53 C terminus is strongly reminiscent of proteins that belong to three other tethering complexes—Dsl1, conserved oligomeric Golgi, and the exocyst—thought to share a common evolutionary origin. Thus, the structure of the Vps53 C terminus suggests that GARP belongs to this family of complexes.     

Antigenicity of a recombinant Ro (SS-A) fusion protein

The antigenicity of the 60-kd human Ro (SS-A) synthesized in vitro from its complementary DNA as a β-galactosidase fusion protein (β-gal—Ro) was evaluated by Western blotting. In this analysis, almost all the anti-Ro (SS-A)-positive sera that bound β-gal-Ro also bound affinity-purified 60-kd human Ro (SS-A) (P > 0.005). Three of the 27 anti-Ro (SS-A) precipitinpositive sera, however, did not show reactivity on Western blot analysis, which suggests that in some sera, antigenicity to Ro (SS-A) is destroyed by denaturation. Of the 22 sera that were reactive with β-gal-Ro, 2 were not reactive with affinity-purified human Ro (SS-A). Two serum samples that did not react with β-gal-Ro were also reactive with affinity-purified human Ro (SS-A). Nevertheless, except for a small percentage of Ro (SS-A) precipitin-positive sera, the frequency of antibody binding to the fusion protein was similar to the frequency of binding to the purified antigen in Western blots. Recombinant Ro (SS-A) antigen may therefore be valuable in the serologic evaluation of anti-Ro (SS-A) autoantibodies.     

Structures of the atlastin GTPase provide insight into homotypic fusion of endoplasmic reticulum membranes

The generation of the tubular network of the endoplasmic reticulum (ER) requires homotypic membrane fusion that is mediated by the dynamin-like, membrane-bound GTPase atlastin (ATL). Here, we have determined crystal structures of the cytosolic segment of human ATL1, which give insight into the mechanism of membrane fusion. The structures reveal a GTPase domain and athree-helix bundle, connected by a linker region. One structure corresponds to a prefusion state, in which ATL molecules in apposing membranes interact through their GTPase domains to form a dimer with the nucleotides bound at the interface. The other structure corresponds to a postfusion state generated after GTP hydrolysis and phosphate release. Compared with the prefusion structure, the three-helix bundles of the two ATL molecules undergo a major conformational change relative to the GTPase domains, which could pull the membranes together. The proposed fusion mechanism is supported by biochemical experiments and fusion assays with wild-type and mutant full-length Drosophila ATL. These experiments also show that membrane fusion is facilitated by the C-terminal cytosolic tails following the two transmembrane segments. Finally, our results show that mutations in ATL1 causing hereditary spastic paraplegia compromise homotypic ER fusion.     

The protein SdhA maintains the integrity of the Legionella-containing vacuole

Legionella pneumophila directs the formation of a specialized vacuole within host cells, dependent on protein substrates of the Icm/Dot translocation system. Survival of the host cell is essential for intracellular replication of L. pneumophila. Strains lacking the translocated substrate SdhA are defective for intracellular replication and activate host cell death pathways in primary macrophages. To understand how SdhA promotes evasion of death pathways, we performed a mutant hunt to identify bacterial suppressors of the ΔsdhA growth defect. We identified the secreted phospholipase PlaA as key to activation of death pathways by the ΔsdhA strain. Based on homology between PlaA and SseJ, a Salmonella protein associated with vacuole degradation, we determined the roles of SdhA and PlaA in controlling vacuole integrity. In the absence of sdhA, the Legionella-containing vacuole was unstable, resulting in access to the host cytosol. Both vacuole disruption and host cell death were largely dependent on PlaA. Consistent with these observations, the ΔsdhA strain colocalized with galectin-3, a marker of vacuole rupture, in a PlaA-dependent process. Access of ΔsdhA strains to the macrophage cytosol triggered multiple responses in the host cell, including degradation of bacteria, induction of the type I IFN response, and activation of inflammasomes. Therefore, we have demonstrated that the Legionella-containing vacuole is actively stabilized by the SdhA protein during intracellular replication. This vacuolar niche affords the bacterium protection from cytosolic host factors that degrade bacteria and initiate immune responses.     

Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein

Class I viral fusion proteins share common mechanistic and structural features but little sequence similarity. Structural insights into the protein conformational changes associated with membrane fusion are based largely on studies of the influenza virus hemagglutinin in pre- and postfusion conformations. Here, we present the crystal structure of the secreted, uncleaved ectodomain of the paramyxovirus, human parainfluenza virus 3 fusion (F) protein, a member of the class I viral fusion protein group. The secreted human parainfluenza virus 3 F forms a trimer with distinct head, neck, and stalk regions. Unexpectedly, the structure reveals a six-helix bundle associated with the postfusion form of F, suggesting that the anchor-minus ectodomain adopts a conformation largely similar to the postfusion state. The transmembrane anchor domains of F may therefore profoundly influence the folding energetics that establish and maintain a metastable, prefusion state.     

Antigenicity of a recombinant Ro (SS-A) fusion protein.

The antigenicity of the 60-kd human Ro (SS-A) synthesized in vitro from its complementary DNA as a beta-galactosidase fusion protein (beta-gal-Ro) was evaluated by Western blotting. In this analysis, almost all the anti-Ro (SS-A)-positive sera that bound beta-gal-Ro also bound affinity-purified 60-kd human Ro (SS-A) (P less than 0.005). Three of the 27 anti-Ro (SS-A) precipitin-positive sera, however, did not show reactivity on Western blot analysis, which suggests that in some sera, antigenicity to Ro (SS-A) is destroyed by denaturation. Of the 22 sera that were reactive with beta-gal-Ro, 2 were not reactive with affinity-purified human Ro (SS-A). Two serum samples that did not react with beta-gal-Ro were also reactive with affinity-purified human Ro (SS-A). Nevertheless, except for a small percentage of Ro (SS-A) precipitin-positive sera, the frequency of antibody binding to the fusion protein was similar to the frequency of binding to the purified antigen in Western blots. Recombinant Ro (SS-A) antigen may therefore be valuable in the serologic evaluation of anti-Ro (SS-A) autoantibodies.     

Purification and architecture of the ubiquitous Wave complex

The Wave proteins are major activators of the Arp2/3 complex. The ubiquitous Wave-2 is required for actin polymerization at the leading edge of migrating cells. Here we purify Wave-2 from HeLa cells. Five proteins, Sra, Nap, Wave-2, Abi, and Hspc, are copurified, indicating that they form a tight complex. These proteins are only present in the complexed form, with the exception of Hspc, which displays a free pool. We reconstitute the Wave-2 complex by cotranslating in vitro the five subunits and use this system together with specific immunoprecipitations to study the molecular architecture of the complex. The complex is organized around a core of Nap and Abi. Sra is a peripheral subunit recruited on the Nap side, whereas the Wave and Hspc subunits are recruited on the Abi side of the core.     

重组结核杆菌融合蛋白(EC)的稳定性与有效性研究

目的考察重组结核杆菌融合蛋白(EC)[该制品名称是国家药典委员会确定的药品中文通用名称,"EC"为重组融合蛋白"结核分枝杆菌早期分泌性抗原靶6(ESAT-6)和培养滤液蛋白10(CFP-10)"](以下简称"EC")原液的储存稳定性、成品储存及使用的稳定性与有效性。方法 (1)原液储存稳定性研究:将3批次EC原液储存于-70℃,观察0～36个月,保存0、3、6、9、12、18、24、36个月时取样进行致敏效应试验、鉴别试验、效价检测,以及等电点、分子量、蛋白质含量、纯度(电泳法)等检测,其中致敏效应试验用豚鼠分为试验组(注射EC原液)与对照组(注射稳定剂),观察注射后动物反应,两组动物反应应无差异;鉴别试验为卡介苗致敏后豚鼠分别皮内注射EC原液和结核菌素纯蛋白衍生物(TB-PPD),观察测量豚鼠皮肤试验检测结果,EC原液皮肤试验应呈阴性反应(硬结或红晕平均直径<5mm),而TB-PPD皮肤试验应呈阳性反应(硬结平均直径≥5mm);效价检测为结核分枝杆菌活菌致敏后6只豚鼠分别皮内注射3个稀释度的EC原液及参考品,观察测量豚鼠皮肤试验检测结果,不同稀释度的EC原液与参考品皮肤试验局部...     

Lipid interaction of the C terminus and association of the transmembrane segments facilitate atlastin-mediated homotypic endoplasmic reticulum fusion

The homotypic fusion of endoplasmic reticulum (ER) membranes is mediated by atlastin (ATL), which consists of an N-terminal cytosolic domain containing a GTPase module and a three-helix bundle followed by two transmembrane (TM) segments and a C-terminal tail (CT). Fusion depends on a GTP hydrolysis-induced conformational change in the cytosolic domain. Here, we show that the CT and TM segments also are required for efficient fusion and provide insight into their mechanistic roles. The essential feature of the CT is a conserved amphipathic helix. A synthetic peptide corresponding to the helix, but not to unrelated amphipathic helices, can act in trans to restore the fusion activity of tailless ATL. The CT promotes vesicle fusion by interacting directly with and perturbing the lipid bilayer without causing significant lysis. The TM segments do not serve as mere membrane anchors for the cytosolic domain but rather mediate the formation of ATL oligomers. Point mutations in either the C-terminal helix or the TMs impair ATL's ability to generate and maintain ER morphology in vivo. Our results suggest that protein-lipid and protein-protein interactions within the membrane cooperate with the conformational change of the cytosolic domain to achieve homotypic ER membrane fusion.     

Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing

Membrane fusion entails organelle docking and subsequent mixing of membrane bilayers and luminal compartments. We now present an in vitro assay of fusion, using yeast vacuoles bearing domains of either Fos or Jun fused to complementary halves of beta-lactamase. Upon fusion, these proteins associate to yield beta-lactamase activity. This assay complements the standard fusion assay (activation of pro-Pho8p in protease-deficient vacuoles by proteases from pho8Delta vacuoles). Both the beta-lactamase and pro-Pho8p activation assays of fusion show the same long kinetic delay between SNARE pairing and luminal compartment mixing. Lipid-mixing occurs rapidly after SNARE pairing but well before aqueous compartment mixing. These results support a model in which SNARE pairing leads to rapid hemifusion, followed by slow further lipid rearrangement and aqueous compartment mixing.     

Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly

The endosomal sorting complexes required for transport (ESCRT) machinery functions in HIV-1 budding, cytokinesis, multivesicular body biogenesis, and other pathways, in the course of which it interacts with concave membrane necks and bud rims. To test the role of membrane shape in regulating ESCRT assembly, we nanofabricated templates for invaginated supported lipid bilayers. The assembly of the core ESCRT-III subunit CHMP4B/Snf7 is preferentially nucleated in the resulting 100-nm-deep membrane concavities. ESCRT-II and CHMP6 accelerate CHMP4B assembly by increasing the concentration of nucleation seeds. Superresolution imaging was used to visualize CHMP4B/Snf7 concentration in a negatively curved annulus at the rim of the invagination. Although Snf7 assemblies nucleate slowly on flat membranes, outward growth onto the flat membrane is efficiently nucleated at invaginations. The nucleation behavior provides a biophysical explanation for the timing of ESCRT-III recruitment and membrane scission in HIV-1 budding.The endosomal sorting complexes required for transport (ESCRTs) are an ancient and conserved system for membrane scission (1, 2). ESCRT membrane remodeling activities are important in the budding of HIV-1 and other viruses from host cell membranes (3); cytokinesis (4); lysosomal transport (5); and more recently discovered functions that include membrane repair, exosome biogenesis, and nuclear envelope reformation (6). The ESCRTs are unique in that they promote membrane budding and sever membrane necks by working from the inner face of the bud (1, 2).The ESCRTs consist of the upstream complexes ESCRT-I, ESCRT-II, and ALIX, which recognize cargo, the ESCRT-III complex responsible for membrane scission, and the AAA+ ATPase VPS4, which releases and recycles ESCRT-III (1, 2). In this study, we focus on the human ESCRT-III subunit CHMP4B, which is considered a core component of the membrane scission machinery and is essential for HIV-1 budding (7). Its yeast counterpart is Snf7. CHMP4 can be recruited and activated through two different pathways in human cells. The first proceeds through ESCRT-I, ESCRT-II, and CHMP6, and the second through ALIX (3). The ESCRT-II– and CHMP6-dependent pathway functions downstream of ESCRT-I, which is in turn the essential link between HIV-1 Gag and the ESCRTs (3). Although there is uncertainty over whether ESCRT-II itself is essential in HIV-1 budding, the most recent virological data suggest that ESCRT-II is important for the efficient release of HIV-1 (8). Moreover, ESCRT-II and CHMP6 were required to bridge Gag and ESCRT-I to the rest of ESCRT-III in a reconstituted system (9).Most concepts of ESCRT recruitment to HIV-1 budding sites have focused on protein–protein interactions between the PTAP and YPXL late domain motifs of the Gag p6 domain and ESCRT-I and ALIX, respectively (3). ESCRT-I is recruited to HIV-1 budding sites simultaneously with Gag (10). However, ESCRT-III is recruited after a time lag and only to Gag that has already assembled on the plasma membrane (10, 11). In principle, either the oligomerization of Gag or its membrane association might trigger ESCRT-III recruitment to Gag-ESCRT-I assemblies. In one recent report, ESCRT-III assembly was visualized by superresolution light microscopy within the center of the Gag shell (12). This observation led to a model for virus scaffolding of ESCRT-III assembly, which downplayed the direct role of membrane shape. Another group, also using superresolution imaging, noted a displacement of the ESCRT-III localization closer to the plasma membrane than the mean position of Gag (13), consistent with ESCRT-III localization predominantly to the bud neck (3). The latter model implies that ESCRT-III could be a coincidence detector, responsive both to the presence of upstream interacting proteins and to membrane shape. Whereas an abundant literature describes the role of viral late domains and other protein interactions in ESCRT recruitment, almost no data are available on the role of membrane curvature in initiating ESCRT-III assembly.In this study, we set out to characterize the recruitment and assembly of purified ESCRT complexes on membranes of a defined geometry approximating that of an early stage HIV-1 budding site. At early stages, budding profiles with broad necks have been visualized in thin-section EM and in cryo-EM tomograms (14–17). During the process of their formation, HIV-1 budding intermediates are 50–100 nm deep and slightly over 100 nm wide. The well-developed methods for studying protein interactions with positively curvature membranes (18) cannot be applied to this type of geometry. Here, we used a focused ion beam to fabricate a 100-nm-deep invaginated template for negative curvature, which approximates the shape of a nascent HIV-1 bud. When coated with a supported lipid bilayer, we term this structure an invaginated supported lipid bilayer (invSLB). We went on to measure CHMP4B/Snf7 assembly in real time, which allowed us to dissect in vitro, and in real time, the role of membrane shape in the nucleation and growth of ESCRT polymers.     

融合蛋白胸腺素α1-干扰素α抗乙型肝炎病毒活性的实验研究

目的观察融合蛋白胸腺素α1-干扰素α(TA1-IFN)体外抗乙型肝炎病毒(HBV)作用,并与胸腺素α1、干扰素α两者联合(TA1+IFN)应用的体外抗HBV作用进行比较。方法HepG22.2.15细胞接种后24h,换用含5种不同浓度(8000、4000、2000、1000、500U／ml)药物的培养基,37℃、体积分数5％CO2条件下培养,每3d换用原浓度含药培养液1次,并于第6天收集培养液,用Abbott诊断试剂盒,分别检测不同组药物作用后上清液中乙型肝炎表面抗原(HBsAg)、乙型肝炎e抗原(HBeAg)的含量,并计算其抑制率;同时用四甲基偶氮唑盐比色分析法检测不同组药物对HepG22.2.15细胞的细胞毒性作用。结果TA1-IFN体外对HBsAg、HBeAg的抑制率与药物浓度呈剂量依赖关系,并且在药物浓度达8000U／ml后趋于稳定,此时TA1-IFN对HBsAg、HBeAg抑制率分别为72.2％±0.8％、60.4％±1.1％,细胞存活率为85.2％±2.0％;而相应浓度的TA1-IFN对HBsAg、HBeAg抑制率为40.0％±0.7％、34.5％±3.2％,细胞存活率为70.0％±1.9％,两者HBsAg、HBeAg抑制率及细胞存活率比较差异均有统计学意义(P值均<0.05)。结论融合蛋白TA1-IFN体外具有良好的抗HBV作用,其体外抗HBV活性比TA1-IFN联合应用强,且细胞毒性比TA1+IFN联合应用时小,本实验为融合蛋白TA1-IFN的临床研究提供了重要的理论     

Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission

The final stage of cytokinesis is abscission, the cutting of the narrow membrane bridge connecting two daughter cells. The endosomal sorting complex required for transport (ESCRT) machinery is required for cytokinesis, and ESCRT-III has membrane scission activity in vitro, but the role of ESCRTs in abscission has been undefined. Here, we use structured illumination microscopy and time-lapse imaging to dissect the behavior of ESCRTs during abscission. Our data reveal that the ESCRT-I subunit tumor-susceptibility gene 101 (TSG101) and the ESCRT-III subunit charged multivesicular body protein 4b (CHMP4B) are sequentially recruited to the center of the intercellular bridge, forming a series of cortical rings. Late in cytokinesis, however, CHMP4B is acutely recruited to the narrow constriction site where abscission occurs. The ESCRT disassembly factor vacuolar protein sorting 4 (VPS4) follows CHMP4B to this site, and cell separation occurs immediately. That arrival of ESCRT-III and VPS4 correlates both spatially and temporally with the abscission event suggests a direct role for these proteins in cytokinetic membrane abscission.     

Structural basis of Vps33A recruitment to the human HOPS complex by Vps16

The multisubunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for late endosome-lysosome and autophagosome-lysosome fusion in mammals. We have determined the crystal structure of the human HOPS subunit Vps33A, confirming its identity as a Sec1/Munc18 family member. We show that HOPS subunit Vps16 recruits Vps33A to the human HOPS complex and that residues 642–736 are necessary and sufficient for this interaction, and we present the crystal structure of Vps33A in complex with Vps16(642–736). Mutations at the binding interface disrupt the Vps33A–Vps16 interaction both in vitro and in cells, preventing recruitment of Vps33A to the HOPS complex. The Vps33A–Vps16 complex provides a structural framework for studying the association between Sec1/Munc18 proteins and tethering complexes.Eukaryotic cells tightly regulate the movement of macromolecules between their membrane-bound compartments. Multiple proteins and protein complexes interact to identify vesicles or organelles destined to fuse, bring them into close proximity, and then fuse their membranes, thereby allowing their contents to mix (1). Multisubunit tethering complexes modulate key steps in these fusion events by recognizing specific Rab-family small GTPases on the membrane surfaces, physically docking the membranes and then recruiting the machinery that effects the membrane fusion (2, 3).In metazoans, the multisubunit tethering complex homologous to the yeast homotypic fusion and vacuole protein sorting (HOPS) complex (4–7) is required for the maturation of endosomes (8); the delivery of cargo to lysosomes (9) and lysosome-related organelles, such as pigment granules in Drosophila melanogaster (10); and the fusion of autophagosomes with late endosomes/lysosomes (11). The mammalian HOPS complex comprises six subunits (Vps11, Vps16, Vps18, Vps33A, Vps39, and Vps41) (4–6). Homologs of HOPS components can be identified in almost all eukaryotic genomes (12) and are thought to be essential; for example, removal of the Vps33A homolog carnation (car) in Drosophila is lethal during larval development (13).HOPS components have been identified in animal models of human disease. A missense point mutation in the murine Vps33a gene gives rise to the buff mouse phenotype, characterized by pigmentation, platelet activity, and motor deficiencies (14). This phenotype closely resembles the clinical presentation of Hermansky–Pudlak syndrome (HPS) (15), and a mutation in the human VPS33A gene has been observed in a patient with HPS who lacked mutations at other known HPS loci (14). In metazoans, there is a second homolog of yeast Vps33 called Vps33B, but disruption of the VPS33B gene in humans gives rise to a clinical phenotype distinct from HPS (16).Human Vps33A is predicted to be a member of the Sec1/Munc18 (SM) family of proteins (7, 17) that, together with SNAREs, comprise the core machinery essential for membrane fusion in eukaryotes (18). Three SNAREs with glutamine residues at the center of their SNARE domain (Q_a-, Q_b-, and Q_c-SNAREs) and one with a central arginine residue (R-SNARE) associate to form a four-helical bundle, the trans-SNARE complex. Formation of this trans-SNARE complex by SNAREs on adjacent membranes drives the fusion of these membranes (18). SM proteins are essential regulators of this process, promoting membrane fusion by correctly formed (cognate) SNARE complexes (18). Although a comprehensive understanding of how SM proteins achieve this still remains elusive, it is clear that SM proteins bind directly both to individual SNAREs and to SNARE complexes (18, 19). Most SM proteins bind strongly and specifically to an N-terminal segment of their cognate Q_a-SNARE, the N-peptide, and this interaction is thought to recruit the SM protein to the site of SNARE-mediated fusion (20, 21).When considered as a whole, the HOPS complex has the functional characteristics of an SM protein: It binds SNAREs and SNARE complexes (5, 22–24), and yeast HOPS has been shown to promote SNARE-mediated membrane fusion (25, 26). Recent biochemical analysis of Vps33, the yeast Vps33A homolog, shows it to be capable of binding isolated SNARE domains and SNARE complexes but not the N-terminal domain or full cytosolic portion of the Q_a-SNARE Vam3 (23, 24). Data from the yeast HOPS complex are consistent with a model whereby Vps33 provides the SM functionality of HOPS, accelerating SNARE-mediated fusion, whereas the rest of the HOPS complex recruits Vps33 (and thus SM function) to the site of SNARE-mediated fusion (24).Although a recent EM study has defined the overall topology of the yeast HOPS complex (27), atomic resolution insights into the assembly of the HOPS complex have thus far been unavailable. Here, we present the 2.4-Å resolution structure of human Vps33A, confirming its structural identity as an SM protein. We have mapped the HOPS epitope that binds Vps33A to a helical fragment comprising residues 642–736 of Vps16, solved the structure of this complex to 2.6-Å resolution, and identified mutations at the binding interface that disrupt the Vps33A–Vps16 complex both in vitro and in cultured cells.     

Molecular architecture of human prion protein amyloid: a parallel, in-register beta-structure

Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and alpha-helical prion protein, PrP(C), to the beta-sheet-rich PrP(Sc). This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrP(C) monomer, structures of the pathogenic PrP(Sc) or synthetic PrP(Sc)-like aggregates remain elusive. Here we have used site-directed spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrP(C). Our data show that, in contrast to earlier, largely theoretical models, the con formational conversion of PrP(C) involves major refolding of the C-terminal alpha-helical region. The core of the amyloid maps to C-terminal residues from approximately 160-220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of beta-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to beta-structure.     

重组结核杆菌融合蛋白(EC)临床应用专家共识

中国是结核病高负担国家之一,结核病发病例数与潜伏性结核感染(latent tuberculosis infection,LTBI)人数庞大,给我国结核病防控工作带来了巨大的挑战。有效识别结核病和LTBI对控制结核病疫情有重要意义。菌阴肺结核和LTBI的诊断依赖于结核感染的免疫学诊断方法;现行结核感染免疫学检测方法主要是结核菌素皮肤试验(tuberculin skin test,TST)、γ干扰素释放试验(interferon gamma release assays,IGRA)和抗原抗体检测。在现行的三类方法基础上,研发出了敏感度高、特异度强、试验操作简单、可用于LTBI和结核病诊断的新产品和新技术——重组结核杆菌融合蛋白(EC)[该制品名称是国家药典委员会确定的药品中文通用名称,“EC”为重组融合蛋白“结核分枝杆菌早期分泌性抗原靶6(ESAT-6)和培养滤液蛋白10(CFP-10)”](简称“EC”)。目前,已完成EC的Ⅰ、Ⅱ和Ⅲ期临床试验。其中Ⅲ期临床试验中对1559名健康人群的筛查中发现,EC与IGRA的检测结果具有较高的特异度,且两者之间具有较高的一致性(88.77%);对791例临床诊断为结核病患者的临床研究发现,EC、结核感染T淋巴细胞斑点试验(T-SPOT.TB)、结核菌素纯蛋白衍生物(TB-PPD)检测均具有良好的敏感度,且三者之间具有较高的一致性;对479名未感染结核分枝杆菌人员的研究发现,EC与T-SPOT.TB的两次检测阴性一致率较高(88.20%和93.17%);在卡介苗接种对检测结果影响的研究中发现,EC和T-SPOT.TB基本不受卡介苗的影响;对394例临床诊断非结核性疾病患者的临床研究发现,EC与T-SPOT.TB阴性符合率较高,且一致性较好(87.21%)。基于EC在用于诊断结核感染安全且有效的基础上,EC通过了国家药品监督管理局药品审批而准予上市。经广泛征求有关结核病防控、临床和研究等领域的专家意见,在系统总结相关技术和方法的应用特点的基础上,结合EC的临床试验结果,形成了EC临床应用的专家共识。本共识介绍了EC的临床应用建议,包括使用对象、使用方法、结果判读,以及临床意义和使用范围。