生物膜融合研究进展

生物膜的融合是一个基本的生命过程,在生物的生长发育中有着重要作用.通过融合,两套独立的双层脂分子合二为一,完成一定的生物功能.膜融合分子机制的关键在于其主要成分融合蛋白.I、II类病毒融合蛋白形成"发夹",胞内囊泡与目标膜各提供的融合蛋白形成"类亮氨酸拉链".这些结构将独立的膜拉近,继而促使他们合为一体.细胞与细胞间融合蛋白的作用机制目前还未明确.在各种膜融合中,脂双层的变化可能是类似的,但介导融合的分子机制应该是不同的.目前,对于膜融合很多方面的理解还停留在假说阶段.理解了膜融合的过程和分子机制不仅极大地促进生物学的发展,而且为相关的疾病治疗打下坚实的基础.     

囊膜病毒膜融合分子机制研究进展

囊膜病毒通过病毒与宿主细胞膜融合的方式感染宿主,病毒囊膜蛋白介导了膜融合过程。根据这些囊膜蛋白在病毒囊膜表面的排列、蛋白结构及其在融合肽中的位置不同,可将囊膜病毒分为三类,其利用这些囊膜特殊的蛋白分子与受体相互作用完成膜融合。在分子水平上研究这一过程有助于认识病毒侵染的本质和发现关键环节,达到预防与治疗病毒病的目的。     

融合蛋白与病毒入膜机制研究进展

包膜病毒感染细胞的第一步即病毒与靶细胞膜的融合,它由病毒包膜上的融合蛋白诱发,融合蛋白与受体分子相互作用后暴露出融合肽,它伸向靶膜使两膜紧密接近后,多肽周围的脂质分子进一步重排,通过中间态最后发生融合,本文将介绍近年来病毒融合蛋白及入膜机制研究进展。     

神经递质释放的胞吐过程膜融合机制的新观点

胞吐作用(excocytosis)是与胞吞作用(endo-Cytosis)方向相反的细胞活动过程,前者是细胞将某种要释放或分泌的物质排到细胞外面,后者是物质进入细胞的过程。胞吐作用是真核细胞一种极复杂的机能活动,是某些物质从细胞中释放或分泌的共同途径。例如:胰腺消化酶原的分泌,胰岛素从β-细胞的分泌、多肽激素从垂体细胞的分泌以及神经递质从神经末梢的释     

Ⅱ类囊膜病毒膜融合的分子机制

病毒囊膜与宿主细胞膜的膜融合是囊膜病毒入侵的重要过程,病毒囊膜融合糖蛋白的一系列结构变化引发此过程.综述了Ⅱ类囊膜病毒、弹状病毒及疱疹病毒融合蛋白结构与功能研究的最新进展,介绍了软件分析并定位融合蛋白功能区域的方法.Ⅱ类病毒与Ⅰ类病毒融合蛋白的融合前结构不同,但融合后结构(发夹三聚体结构)相似.弹状病毒与疱疹病毒的融合蛋白集合了Ⅰ/Ⅱ类融合蛋白的某些特征,但其结构变化及融合过程各不相同,被归为新型融合蛋白.上述研究为基础设计的以病毒融合过程为靶标的抑制子,可为抗病毒新药的研制提供新思路.     

囊膜病毒膜融合的分子机制

囊膜病毒可能采用相似的病毒-宿主细胞膜融合机制,即病毒表面糖蛋白结合到宿主细胞受体后,启动了病毒融合蛋白的一系列构象变化,根据囊膜蛋白构象变化特征,囊膜病毒可采用两种以上的方式发生膜融合,并据此分为两类：Ⅰ型病毒膜融合和Ⅱ型病毒膜融合.Ⅱ型病毒膜融合以黄病毒为代表,其分子机制与Ⅰ型病毒膜融合不同,但不很清楚.而Ⅰ型病毒膜融合中,如艾滋病毒,流感病毒等,在囊膜蛋白变构形成稳定折叠的发夹三聚体结构时,拉近了两膜之间的距离,此过程释放出来的能量进一步促使两膜融合.膜融合使病毒蛋白及病毒RNA基因组释放到宿主细胞内而感染宿主.以上述研究为基础设计的C肽/N肽小分子抑制子, 可以在病毒糖蛋白中间体构象形成的短时间内,高效、特异地竞争结合其配体,从而阻止糖蛋白的进一步折叠,达到抑制病毒入侵的目的,为病毒疾病的防治提供了新思路和策略.针对艾滋病毒设计的C肽,即T20或Enfuvirtide在临床应用效果很好.以艾滋病毒和流感病毒为例,主要对Ⅰ型病毒膜融合的研究进展进行了讨论.     

杆状病毒出芽病毒粒子膜融合蛋白的研究进展

杆状病毒是一类感染节肢动物的病原微生物,其基因组为双链环状DNA,大小为80～180kb。杆状病毒科包括核多角体病毒属(Nucleopolyhedrovirus,NPV)和颗粒体病毒属(Granulovirus,GV),其中NPV根据病毒粒子在包膜中的粒子数目不同而划分为多核衣壳核多角体病毒(multiple nucleocapsid     

杆状病毒出芽病毒粒子膜融合蛋白的研究进展

杆状病毒是一类感染节肢动物的病原微生物,其基因组为双链环状DNA,大小为80～180kb.     

细菌生物被膜分散及分子调控机制研究进展

生物被膜分散(Biofilm Dispersal)是生物被膜发展后期细菌响应营养物、低浓度的一氧化氮、D-氨基酸、自诱导肽(Autoinducing Peptide,AIP)、酰基高丝氨酸内酯(Acyl Homoserine Lactones,AHL)、腺苷三磷酸(Adenosine Triphosphate,ATP)等信号变化而做出的一种程序性反应,有利于细菌从恶劣的生物被膜内部环境中脱离出来寻找新的定殖位点。此外,由生物被膜引起的细菌短暂的抗生素耐受性在分散过程中会恢复正常水平,这有助于治疗由致病菌引起的难治愈的生物被膜相关疾病。目前生物被膜分散的相关研究正处于起步阶段,本文希望通过综述生物被膜分散现象、信号分子及调控机制,可以更好地了解细菌生物被膜分散对于防控病原微生物和应用有益微生物的重要意义。     

Molecular mechanisms of membrane fusion: Steps during phospholipid and exocytotic membrane fusion

Exocytosis is considered as four separate steps: adhesion, fusion/pore formation, pore widening, and content discharge. Experiments on both synthetic and natural membranes are presented to show each of these steps. Major differences are seen in the two fusing systems. These differences are discussed in terms of molecular mechanisms of fusion.     

A dual-functional paramyxovirus F protein regulatory switch segment: activation and membrane fusion

Many viral fusion-mediating glycoproteins couple alpha-helical bundle formation to membrane merger, but have different methods for fusion activation. To study paramyxovirus-mediated fusion, we mutated the SV5 fusion (F) protein at conserved residues L447 and I449, which are adjacent to heptad repeat (HR) B and bind to a prominent cavity in the HRA trimeric coiled coil in the fusogenic six-helix bundle (6HB) structure. These analyses on residues L447 and I449, both in intact F protein and in 6HB, suggest a metamorphic region around these residues with dual structural roles. Mutation of L447 and I449 to aliphatic residues destabilizes the 6HB structure and attenuates fusion activity. Mutation of L447 and I449 to aromatic residues also destabilizes the 6HB structure despite promoting hyperactive fusion, indicating that 6HB stability alone does not dictate fusogenicity. Thus, residues L447 and I449 adjacent to HRB in paramyxovirus F have distinct roles in fusion activation and 6HB formation, suggesting this region is involved in a conformational switch.     

Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from −31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.     

The protein coat in membrane fusion: lessons from fission

Multiple cell biological processes involve two opposite rearrangements of membrane configuration, referred to as fusion and fission. While membrane intermediates in protein-mediated fusion have been studied in some detail, the global force which drives sequential stages of the fusion reaction from early local intermediates to an expanding fusion pore remains unknown. Fusion proceeds via stages, which are analogous but in the opposite direction to that of membrane budding-off and fission driven by protein coats. On the basis of this analogy, we propose that an interconnected coat formed by membrane-bound activated fusion proteins surrounding the membrane contact zone generates the driving force for fusion. This fusion protein coat has a strongly curved intrinsic shape opposite to that of the protein coat driving fission. To relieve internal stresses, the fusion protein coat spontaneously bends out of the initial shape of the membrane surface. This bending produces elastic stresses in the underlying lipid bilayer and drives its fusion with the apposing membrane. The hypothesis that 'bystander' proteins (i.e. fusion proteins outside the contact zone) generate the driving force for fusion offers a new interpretation for a number of known features of the fusion reaction mediated by the prototype fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other fusion reactions.     

SNARE protein analog‐mediated membrane fusion

Fusion of lipid membranes to form a single bilayer is an essential process for life and provides important biological functions including neurotransmitter release. Membrane fusion proteins facilitate approximation of interacting membranes to overcome the energy barrier. In case of synaptic transmission, proteins involved are known as soluble N‐ethylmaleimide‐sensitive‐factor attachment receptor (SNARE) proteins. The SNAREs from synaptic vesicles interact with the SNAREs from the target membrane to form a coiled‐coil bundle of four helices, thus pulling the membranes tightly together and initiating fusion. However, it remains unclear how these proteins function at molecular level. Natural systems are often too complex to obtain unambiguous results. Simple model systems mimicking natural proteins in synthetic lipid bilayers are powerful tools for obtaining insights into this essential biological process. An important advantage of such systems is their well‐defined composition, which can be systematically varied in order to fully understand events at molecular level. In this review, selected model systems are presented based upon specific interactions between recognition units embedded in separate lipid bilayers mimicking native SNARE protein‐mediated membrane fusion. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Difference in capacities for virion-to-virion fusion of young and aged HVJ (Sendai virus): A model of membrane fusion

Summary Young and aged HVJ virions differ structurally and morphologically due to changes that occur during aging in vitro or in ovo. Young virions soon after their budding off are rodshaped, rigid and relatively uniform in size, whereas virions that have aged in vitro after their formation are round, nonrigid and variable in size. These changes during aging seem to be due to the variation of M protein, a skeletal protein that is associated with both the envelope membrane proteins and nucleocapsid strands in the virions. The capacities for virion-to-virion fusion of young and aged virions were compared to clarify the relation between the membrane fusion and membrane-associating skeletal proteins. On treatment with polyethylene glycol (PEG), aged virions readily fused, forming large virion vesicles, but young virions were resistant to fusion. Further, aged virions fused even on incubation at 37°C without the fusogen. Thus the capacity for virion-to-virion fusion evidently increases during aging of virions. This result suggests that skeletal proteins associating with the biological membrane are important for preventing membrane fusion, and that virion-to-virion fusion is a good model system for use in studies on the mechanism of membrane fusion.     

Membrane fusion mechanisms: the influenza hemagglutinin paradigm and its implications for intracellular fusion

The mechanism of membrane fusion induced by the influenza virus hemagglutinin (HA) has been extensively characterized. Fusion is triggered by low pH, which induces conformational changes in the protein, leading to insertion of a hydrophobic 'fusion peptide' into the viral membrane and the target membrane for fusion. Insertion perturbs the target membrane, and hour glass-shaped lipidic fusion intermediates, called stalks, fusing the outer monolayers of the two membranes, are formed. Stalk formation is followed by complete fusion of the two membranes. Structures similar to those formed by HA at the pH of fusion are found not only in many other viral fusion proteins, but are also formed by SNAREs, proteins involved in intracellular fusion. Substances that inhibit or promote HA-induced fusion because they affect stalk formation, also inhibit or promote intracellular fusion, cell–cell fusion and even intracellular fission similarly. Therefore, the mechanism of influenza HA-induced fusion may be a paradigm for many intracellular fusion events.     

Virus membrane fusion

Membrane fusion of enveloped viruses with cellular membranes is mediated by viral glycoproteins (GP). Interaction of GP with cellular receptors alone or coupled to exposure to the acidic environment of endosomes induces extensive conformational changes in the fusion protein which pull two membranes into close enough proximity to trigger bilayer fusion. The refolding process provides the energy for fusion and repositions both membrane anchors, the transmembrane and the fusion peptide regions, at the same end of an elongated hairpin structure in all fusion protein structures known to date. The fusion process follows several lipidic intermediate states, which are generated by the refolding process. Although the major principles of viral fusion are understood, the structures of fusion protein intermediates and their mode of lipid bilayer interaction, the structures and functions of the membrane anchors and the number of fusion proteins required for fusion, necessitate further investigations.     

Palaeontological evidence for membrane fusion between a unit membrane and a half-unit membrane

Membrane fusion is of fundamental importance for many biological processes and has been a topic of intensive research in past decades with several models being proposed for it. Fossils had previously not been considered relevant to studies on membrane fusion. But here two different membrane fusion patterns are reported in the same well-preserved fossil plant from the Miocene (15–20 million years old) at Clarkia, Idaho, US. Scanning electron microscope, transmission electron microscope, and traditional studies reveal the vesicles in various states (even transient semi-fusion) of membrane fusion, and thus shed new light on their membrane structure and fusion during exocytoses. The new evidence suggests that vesicles in plant cells may have not only a unit membrane but also a half-unit membrane, and that a previously overlooked membrane fusion pattern exists in plant cells. This unexpected result from an unexpected material not only marks the first evidence of on-going physiological activities in fossil plants, but also raises questions on membrane fusion in recent plants.     

Membrane fusion in cells: molecular machinery and mechanisms

Membrane fusion is a sine qua non process for cell physiology. It is critical for membrane biogenesis, intracellular traffic, and cell secretion. Although investigated for over a century, only in the last 15 years, the molecular machinery and mechanism of membrane fusion has been deciphered. The membrane fusion event elicits essentially three actors on stage: anionic phospholipids - phosphatidylinositols, phosphatidyl serines, specific membrane proteins, and the calcium ions, all participating in a well orchestrated symphony. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion. Target membrane proteins, SNAP-25 and syntaxin (t- SNARE) and secretory vesicle-associated membrane protein (v-SNARE) or VAMPwere discovered in the 1990's and suggested to be the minimal fusion machinery. Subsequently, the molecular mechanism of SNARE-induced membrane fusion was discovered. It was demonstrated that when t-SNARE-associated lipid membrane is exposed to v-SNARE-associated vesicles in the presence of Ca(2+), the SNARE proteins interact in a circular array to form conducting channels, thus establishing continuity between the opposing bilayers. Further it was proved that SNAREs bring opposing bilayers close to within a distance of 2-3 Angstroms, allowing Ca(2+) to bridge them. The bridging of bilayers by Ca(2+) then leads to the expulsion of water between the bilayers at the contact site, allowing lipid mixing and membrane fusion. Calcium bridging of opposing bilayers leads to the release of water, both from the water shell of hydrated Ca(2+) ions, as well as the displacement of loosely coordinated water at the phosphate head groups in the lipid membrane. These discoveries provided for the first time, the molecular mechanism of SNARE-induced membrane fusion in cells. Some of the seminal discoveries are briefly discussed in this minireview.