Amyloids and Oligomers. Curvilinear and annular structures and their interaction with exosomes and whole cells

背景：有许多蛋白质聚集在一起形成有序的纤维组装，称为淀粉样蛋白。淀粉样蛋白可以在酵母和细菌中发挥作用。它们也代表了潜在的新型纳米材料。此外，蛋白质的错误折叠和自我组装，如Pron蛋白，与疯牛病(BSE)和绵羊瘙痒病等疾病有关。而淀粉样β蛋白(Ab)与人类阿尔茨海默氏症有关。除了长的未分枝的纤维外，通常还会形成特定的前纤维集合体。这些结构包括环形(甜甜圈环)状结构以及更多的曲线状原纤维。这些前纤维结构通常对脂双层有很高的亲和力，它们与脂膜的相互作用会破坏细胞的过程和平衡，这会导致一连串的事件，最终导致细胞死亡。AB-聚集体对细胞有毒性，因为它通过细胞膜的‘地毯’破坏细胞的完整性，或通过形成跨越细胞壁的小通道，从而导致钙离子不受控制地流入细胞。了解这些过程是揭示抗体引起细胞毒性的机制的关键。目前，在类天然条件下对这些相互作用的高分辨率分子成像一直是有限的，而离子通道测量主要集中在重组的人工脂双层上，这与细胞膜中抗体通道的形成行为很不接近。目的：-我们想要研究抗体与脂膜相互作用的基本过程。在低温条件下，使用强大的电子显微镜，我们将成像环状(甜甜圈)结构，这些结构形成了跨越类脂膜的通道。利用低温电子断层扫描，我们将获得在接近自然条件下，抗体覆盖在细胞膜表面的三维分子细节。此外，我们的目标是用电生理学方法探索细胞膜上AB42离子通道形成的机制细节。我们将确定不同的抗体制备方法在神经元上形成的抗体通道的大小和数量。通过这种方式，我们将揭示AB42离子通道在生理相关条件下对细胞过程的可能影响。我们将探索在神经元的胞外膜和胞内膜表面形成抗体通道。意义：-前纤维集合体的结构将通知更广泛的淀粉样蛋白区域。我们希望这项研究将使我们更深入地了解不同的抗体组装如何影响细胞活力。这些研究将为更广泛的淀粉样蛋白领域提供信息。我们希望我们的研究能够强调抗体在痴呆症早期破坏细胞膜完整性中的关键作用。我们的专家团队能够很好地研究这些基本的相互作用。

Background:- There are a number of proteins that clump together to form ordered fibrillary assembles known as amyloids. Amyloids can have functional roles in yeast and bacteria. They also represent potential novel nanomaterials. Furthermore, misfolding and self-assembly of proteins, such as the prion protein, are link with diseases including, mad-cow disease (BSE) and scrapie in sheep. While amyloid beta protein (Ab) is linked to Alzheimer's in humans.In addition to long unbranched fibrils, specific pre-fibrillary assemblies are often formed. These include annular (donut-ring) shaped structures as well as more curvilinear protofibrils. These prefibrillar structures often have a high affinity for lipid bilayers, their interaction with the lipid membrane can disrupt cellular processes and balance; this causes a cascade of events culminating in cell death. Ab-aggregates are toxic to cells by disrupting cell integrity through the 'carpeting' of the cell membrane, or via the formation of small channels that span the cell-wall, thereby causing an uncontrolled flow of calcium ions into the cell. Understanding these processes is key to uncovering the mechanism by which Ab causes cell toxicity. At present, high resolution molecular imaging of these interactions, in native-like conditions, has been limited, while ion channel measurements have focused on reconstituted artificial lipid bilayers, which poorly approximate the behaviour of Ab channel formation in cellular membranes. Aims:- We want to investigate the fundamental process of Ab interactions with lipid membranes. Using powerful electron microscopes under cryogenic conditions we will image the annular (donut-ring) structures that form channels across lipid membranes. Using cryo-electron tomography, we will obtain three-dimensional molecular details of Ab carpeting the cell derived membrane surface under near native conditions. Furthermore, we aim to probe the mechanistic details of Ab42 ion-channel formation in cellular membranes using electrophysiology methods. We will determine the size and number of Ab channels that typically form on a neuron with different Ab preparations. In this way we will uncover the likely effects that Ab42 ion channels have on cellular processes under physiologically relevant conditions. We will explore Ab channel formation on both the extra- and intra-cellular membrane face of neurons. Significance:- The structures of prefibrillar assemblies will inform the wider amyloid field. We hope this study will give us a deeper understanding of how different Ab assemblies impact cell viability. These studies will inform the wider amyloid field. We expect our studies to highlight the key role of Ab in disrupting membrane integrity in the early stages of dementia. Our team of experts is well placed to investigate these fundamental interactions.