哺乳动物精卵黏附和融合的分子基础

哺乳动物受精是一个多分子参与的多级过程,在精卵融合形成一个受精卵时达到顶点。新的研究已表明:精卵黏附后,精子膜从赤道区开始与卵膜融合;有精子膜上的去整合蛋白金属蛋白酶蛋白家族(ADAM)、Izumo、附睾蛋白DE、卵膜上的整合蛋白、四次跨膜蛋白CD9、CD81、糖基磷脂酰肌醇锚定蛋白等多种分子参与精卵黏附和融合过程。     

糖基化磷脂酰肌醇锚定蛋白转化法—一门全新的细胞 …

糖基化磷脂酰肌醇（ＧＰＩ）锚定蛋白是一种新型细胞表面蛋白,它只通过ＧＰＩ结构锚定于细胞膜表面而不跨越其磷脂膜双层结构;当它与细胞共同孵育时,可自动整合至细胞膜表面。ＧＰＩ锚定蛋白转化法利用ＧＰＩ锚定信号将目的蛋白直接整合至靶细胞表面,超越了目的蛋白靶细胞自身合成的机制,具有对靶细胞选择性不高,转化过程迅速等优点,在抗原提呈细胞工程,肿瘤疫苗及移植物抗排斥反应等研究领域得到了广泛应用。     

受精素和整合素在哺乳动物精卵质膜融合过程中是必需的吗?

精子膜蛋白受精素(fertilin)和卵子膜蛋白整合素(integrin)是精卵质膜融合侯选蛋白中最受关注之一,认为它们之间的相互作用介导了精卵质膜的融合。最近的基因剔除研究结果显示,受精素被剔除的精子和整合素α6β1被剔除的卵子仍具有一定的受精能力,因此,受精素和卵子整合素在精卵质膜融合中的重要作用值得进一步研究。     

腺苷及其类似物对猪红细胞膜上磷脂酰肌醇磷酸化的抑制作用

本文研究了腺苷及其类似物对猪红细胞膜上磷脂酰肌醇磷酸化的影响。研究结果表明:1、腺苷对磷脂酰肌醇磷酸化有明显的抑制作用,IC_(50)=15μmol/L;动力学分析表明,这种抑制作用机理是与ATP竞争性的;2、腺嘌呤、AMP、ADP、5＇-氯-5＇-脱氧腺苷、阿糖腺苷、2＇-脱氧腺苷对磷脂酰肌醇磷酸化有不同程度的抑制作用;3、cAMP对磷脂酰肌醇磷酸化也有抑制作用,这提示了cAMP与肌醇脂质信使系统有联系;5、6-氯-嘌呤核苷(100μmol/L)对该磷酸化无显著抑制作用。     

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.     

疱疹病毒膜融合的分子机制

囊膜病毒与宿主细胞的膜融合是病毒入侵宿主细胞的重要过程,这一过程涉及到病毒囊膜表面糖蛋白与宿主细胞表面受体之间的相互作用和构象变化．疱疹病毒有多个糖蛋白及不同类型的细胞作用受体,相应的受体-糖蛋白复合体构成方式也有多种,其引致的膜融合机制被认为是目前病毒融合机制研究中最复杂的,近年来被广泛研究并取得突破性进展．从病毒糖蛋白与相应受体的结构与功能、受体-糖蛋白复合体的形成与入侵途径,以及膜融合模式几个方面,全面综述疱疹病毒膜融合的分子机制,并展望了未来研究趋势．     

人骨髓基质细胞中糖基化磷脂酰肌醇特异性磷脂酶DcDNA的克隆

为探讨人糖基化磷脂酰肌醇特异性磷脂酶D(GPI PLD)cDNA的结构及功能 ,应用RT PCR从人骨髓基质细胞中克隆了长约 2 6kb的GPI PLDcDNA ,包含完整阅读框架 ,编码 2 3个氨基酸的信号肽及 817个氨基酸的成熟肽 .该cDNA与人胰腺GPI PLDcDNA几乎百分之百同源 ,与人肝脏GPI PLDcDNA同源性为 95 %,氨基酸同源性为 94 %,3者对应的结构基因只有 1个 ,位于人类第 6号染色体上 ,基因组序列长约 80kb ,包括 2 5个外显子 .构建克隆的GPI PLDcDNA的真核表达载体 ,通过脂质体转染能表达GPI锚定的胎盘型碱性磷酸酶 (PLAP)而无GPI PLD活性的G9细胞 ,同时设立对照组检测GPI PLDcDNA的功能 .结果显示 ,对照组细胞几乎检测不到GPI PLD活性 ,其表达的PLAP主要位于细胞膜上 ;而转染GPI PLDcDNA的G9细胞能检测到较高水平的GPI PLD活性 ,而且大部分酶活性存在于培养液中 ,其表达的PLAP也主要被释放入培养液 .结果证实 ,从人骨髓基质细胞中克隆的GPI PLDcDNA有生物学功能 ,它能释放细胞膜上GPI锚定蛋白质 .     

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.     

胆固醇对金属离子磷脂酰甘油单分子膜成膜的影响

目的：本文主要研究不同条件胆固醇（Cholesterol,简称Chol）和金属离子（钾、镁离子）对磷脂酰甘油（Phosphatidylglycerols,简称PG）相互作用后形成的单分子膜的影响。方法：首先以金属离子作为亚相,研究胆固醇的量对磷脂酰甘油单分子膜的影响;其次加入同等量的胆固醇量,亚相为不同金属离子时,对磷脂酰甘油单分子膜的影响,最后分析磷脂酰甘油LB膜的π-A曲线,即曲线外扩、相变点、膜压等等变化特征。结果：随着胆固醇的增多,金属离子磷脂酰甘油单分子膜形成π-A曲线变化逐渐明显;当加入同等量的胆固醇时,随着金属离子价态的逐渐增高,磷脂酰甘油单分子膜形成的π-A曲线的变化也逐渐明显。结论：其一：胆固醇对金属离子磷脂酰甘油单分子膜成膜质量是有影响的。其二,金属离子对胆固醇与磷脂酰甘油混合形成的单分子膜同样也是有的影响。     

Membrane Fusion

The fusion of biological membranes results in two bilayer-based membranes merging into a single membrane. In this process the lipids have to undergo considerable rearrangement. The nature of the intermediates that are formed during this rearrangement has been investigated. Certain fusion proteins facilitate this process. In many cases short segments of these fusion proteins have a particularly important role in accelerating the fusion process. Studies of the interaction of model peptides with membranes have allowed for increased understanding at the molecular level of the mechanism of the promotion of membrane fusion by fusion proteins. There is an increased appreciation of the roles of several independent segments of fusion proteins in promoting the fusion process.Many of the studies of the fusion of biological membranes have been done with the fusion of enveloped viruses with other membranes. One reason for this is that the number of proteins involved in viral fusion is relatively simple, often requiring only a single protein. For many enveloped viruses, the structure of their fusion proteins has certain common elements, suggesting that they all promote fusion by an analogous mechanism. Some aspects of this mechanism also appears to be common to intracellular fusion, although several proteins are involved in that process which is more complex and regulated than is fusion.     

融合标签技术在膜蛋白结构研究中的应用

膜蛋白高级结构的研究包括不同的层次,即膜蛋白拓扑学结构的研究、利用核磁共振技术和蛋白质晶体衍射技术对三维结构的研究,以及膜蛋白复合体的研究。在研究过程中,如果能够基于膜蛋白的拓扑学结构预测,选择合适的蛋白质或多肽融合标签,利用基因融合技术在基因水平上对膜蛋白进行改造,可以产生含有融合标签的重组膜蛋自,不仅具有原有膜蛋白的功能活性,还具有融合标签所特有的生理生化特性,将会极大地促进膜蛋白结构和功能的研究。我们就目前膜蛋白结构研究中所涉及的融合标签技术及其应用策略和所取得的进展做一简述。     

Structures and Mechanisms of Viral Membrane Fusion Proteins: Multiple Variations on a Common Theme

Recent work has identified three distinct classes of viral membrane fusion proteins based on structural criteria. In addition, there are at least four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. Viral fusion proteins also contain different types of fusion peptides and vary in their reliance on accessory proteins. These differing features combine to yield a rich diversity of fusion proteins. Yet despite this staggering diversity, all characterized viral fusion proteins convert from a fusion-competent state (dimers or trimers, depending on the class) to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide, attached to the target membrane, and the transmembrane domain, attached to the viral membrane, into close proximity thereby facilitating the union of viral and target membranes. During these conformational conversions, the fusion proteins induce membranes to progress through stages of close apposition, hemifusion, and then the formation of small, and finally large, fusion pores. Clearly, highly divergent proteins have converged on the same overall strategy to mediate fusion, an essential step in the life cycle of every enveloped virus.     

On the Mechanism of Intracellular Membrane Fusion: In Search of the Genuine Fusion Factor

Intracellular membrane fusion events require a general protein machinery that functions in vesicular traffic and in assembly and maintenance of organelles. An array of cytosolic and integral membrane proteins are currently identified, and in conjunction with ongoing detailed structural studies, rapid progress is made in understanding basic features of the overall mechanism of the fusion machinery, but above all a proper appreciation of its enormous complexity. Thus a highly sophisticated level of regulation of the different steps involved in tethering, docking and merging itself is apparent. Apart from the relevance of protein–protein interactions, also a role of distinct lipids is gradually emerging, particularly in fusion. However, although various suggestions have been made recently, largely based upon in vitro studies, the identity of the actual fusion factor(s) remains to be determined.     

Modification of the Membrane Components of Synaptosomes and the Fusion Process

A model system consisting of synaptic vesicles and synaptic membrane fragments isolated from brain synaptosomes was used for studying the role of the state of membrane components in membrane fusion. It is concluded that the state of proteins and lipids of biological membranes influences considerably the membrane's ability to fuse. This state can be changed by the regulation of cell enzyme systems (proteolytic enzymes, phospholipases), and regulation by nitric oxide is an important aspect of this control.     

Insights into a Structure-Based Mechanism of Viral Membrane Fusion

A number of different viral spike proteins, responsible for membrane fusion, show striking similarities in their core structures. The prospect of developing a general structure-based mechanism seems plausible in light of these newly determined structures. Influenza hemagglutinin (HA) is the best-studied fusion machine, whose action has previously been described by a hypothetical spring-loaded model. This model has recently been extended to explain the mechanism of other systems, such as HIV gp120–gp41. However, evidence supporting this idea is insufficient, requiring re-examination of the mechanism of HA-induced membrane fusion. Recent experiments with a shortened construct of HA, which is able to induce lipid mixing, have provided evidence for an alternative scenario for HA-induced membrane fusion and perhaps that of other viral systems.     

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.     

Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing

Membrane fusion is induced by SNARE complexes that are anchored in both fusion partners. SNAREs zipper up from the N to C terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3, and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial leucine-valine sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a prerequisite for fusion. Lipid-anchored Vti1 was fully active, and lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion, and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion.     

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution

Microvilli are found on the surface of many cell types, including the mammalian oocyte, where they are thought to act in initial contact of sperm and oocyte plasma membranes. CD9 is currently the only oocyte protein known to be required for sperm-oocyte fusion. We found CD9 is localized to the oocyte microvillar membrane using transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that CD9 null oocytes, which are unable to fuse with sperm, have an altered length, thickness and density of their microvilli. One aspect of this change in morphology was quantified using TEM by measuring the radius of curvature at the microvillar tips. A small radius of curvature is thought to promote fusibility and the radius of curvature of microvillar tips on CD9 wild-type oocytes was found to be half that of the CD9 null oocytes. We found that oocyte CD9 co-immunoprecipitates with two Ig superfamily cis partners, EWI-2 and EWI-F, which could have a role in linking CD9 to the oocyte microvillar actin core. We also examined latrunculin B-treated oocytes, which are known to have reduced fusion ability, and found altered microvillar morphology by SEM and TEM. Our data suggest that microvilli may participate in sperm-oocyte fusion. Microvilli could act as a platform to concentrate adhesion/fusion proteins and/or provide a membrane protrusion with a low radius of curvature. They may also have a dynamic interaction with the sperm that serves to capture the sperm cell and bring it into close contact with the oocyte plasma membrane.