Dynamics and pathways of assembly in membrane pore formation

膜孔形成中的组装动力学和途径

基本信息

批准号：
BB/J006254/1
负责人：
Bart Hoogenboom
金额：
$ 33.64万
依托单位：
University College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2012
资助国家：
英国
起止时间：
2012 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FJ006254%2F1
关键词：
Dynamics pathways assembly membrane pore

项目摘要

Pore-forming proteins are crucial armaments in the continuous battle between living organisms and the pathogens that threaten their fitness and survival. These proteins act on cells, which are the micrometre-scaled, basic units of all forms of life. Cells are separated and protected from their environment by a thin membrane. Pathogens such as bacteria can release pore-forming proteins ("toxins") that drill holes in the membranes of healthy cells in the host organism, to release nutrients for the bacteria, to invade these cells and/or kill them. Patients affected by bacterial pneumonia, for example, suffer from the devastating effects of such a toxin, pneumolysin, on lung tissue. The immune system, however, uses a similar mechanism to kill germs and infected or cancerous cells, thus preventing them from doing further damage to the organism. It secretes related, but somewhat different pore-forming proteins to perforate the membranes of such unwanted invaders.To perform these tasks, pore-forming proteins have developed sophisticated drilling mechanism. These proteins can convert from a soluble form in the aqueous, cellular environment into a very different form, in which 20-50 protein molecules assemble into a ring-shaped pore bound to the membrane. We can look at these forms with X-rays or electrons to deduce their three-dimensional structures. Thanks to such experiments, we now have a reasonably clear picture of the soluble proteins and their pore structure in the membrane. For some pore-forming proteins, scientists have even identified the changes inside the proteins which make this transition possible.However, if we wish to design drugs that prevent such pores from being formed, as in the example of bacterial pneumonia indicated above, it would be useful to know more about the steps in their formation. It is exactly this pore assembly that is still largely enigmatic. In this project, we will try to answer some specific questions about membrane pore formation. We would like to know how the proteins assemble on the membrane. Do they assemble one by one, or do they first form larger units that subsequently assemble in a pore? Do the proteins first need to assemble on the membrane, or can they dock in the membrane and assemble in pores afterwards? And at what point in this process will the membrane that is surrounded by the assembled protein be extruded to create a hole?To investigate the dynamics of this process, we rely on a technique called atomic force microscopy. Atomic force microscopy is the small-scale equivalent of reading Braille: With a tiny artificial finger, we feel the pore-forming proteins while they assemble on the membrane. Whereas X-ray crystallography and electron microscopy are limited to static samples, atomic force microscopy can probe active proteins while they are at work. We will thus apply atomic force microscopy to the membranes that are being exposed to attack by pore-forming proteins. Meanwhile, we will benefit from the more detailed views provided by electron microscopy to identify intermediate assemblies of pore-forming proteins, that are trapped by chemical bonds or by lowering the temperature. Electron microscopy will thus provide highly detailed pictures of pore forming proteins in different states of assembly and atomic force microscopy will enable us to see how the proteins transit between these different states.

成孔蛋白是生物体与威胁其健康和生存的病原体之间持续战斗的关键武器。这些蛋白质作用于细胞，细胞是所有生命形式的微米级基本单位。细胞通过薄膜与环境隔离并受到保护。细菌等病原体可以释放成孔蛋白（“毒素”），在宿主有机体的健康细胞膜上钻孔，为细菌释放营养物质，侵入这些细胞和/或杀死它们。例如，患有细菌性肺炎的患者会遭受肺炎球菌溶血素这种毒素对肺组织的破坏性影响。然而，免疫系统使用类似的机制来杀死细菌和受感染的细胞或癌细胞，从而防止它们对有机体造成进一步的损害。它分泌相关但有些不同的成孔蛋白来刺穿这些不需要的入侵者的细胞膜。为了执行这些任务，成孔蛋白已经开发出复杂的钻孔机制。这些蛋白质可以从水性细胞环境中的可溶形式转化为一种非常不同的形式，其中 20-50 个蛋白质分子组装成与膜结合的环形孔。我们可以用 X 射线或电子观察这些形式，从而推断出它们的三维结构。通过这些实验，我们现在对可溶性蛋白质及其在膜中的孔结构有了相当清晰的了解。对于一些成孔蛋白，科学家甚至已经确定了蛋白质内部的变化，使得这种转变成为可能。然而，如果我们希望设计出药物来阻止这种孔的形成，就像上面提到的细菌性肺炎的例子一样，那就需要了解更多关于它们形成的步骤是有用的。正是这种孔隙组合在很大程度上仍然是个谜。在这个项目中，我们将尝试回答一些有关膜孔形成的具体问题。我们想知道蛋白质如何在膜上组装。它们是一个接一个地组装，还是首先形成更大的单元，然后在孔隙中组装？蛋白质是否首先需要在膜上组装，或者它们可以停靠在膜中然后在孔中组装？在这个过程中的什么时候，被组装的蛋白质包围的膜会被挤压以形成一个洞？为了研究这个过程的动力学，我们依靠一种称为原子力显微镜的技术。原子力显微镜相当于阅读盲文的小规模技术：用一根微小的人造手指，我们可以感觉到成孔蛋白质在膜上组装时的情况。 X 射线晶体学和电子显微镜仅限于静态样品，而原子力显微镜可以在活性蛋白质工作时对其进行探测。因此，我们将原子力显微镜应用于受到成孔蛋白攻击的膜。同时，我们将受益于电子显微镜提供的更详细的视图，以识别通过化学键或降低温度捕获的成孔蛋白质的中间组装体。因此，电子显微镜将提供不同组装状态下的成孔蛋白的高度详细的图像，而原子力显微镜将使我们能够看到蛋白质如何在这些不同状态之间转变。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Antimicrobial peptide capsids of de novo design.

DOI：
10.1038/s41467-017-02475-3
发表时间：
2017-12-22
期刊：
Nature communications
影响因子：
16.6
作者：
De Santis E;Alkassem H;Lamarre B;Faruqui N;Bella A;Noble JE;Micale N;Ray S;Burns JR;Yon AR;Hoogenboom BW;Ryadnov MG
通讯作者：
Ryadnov MG

Lipid specificity of the immune effector perforin.