Advancing our knowledge of viral membrane fusion and of IDP-membrane interactions by ESR

通过 ESR 增进我们对病毒膜融合和 IDP-膜相互作用的了解

• 批准号: 10552109
• 负责人: Jack H Freed
• 金额: $ 41万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552109
• 关键词: 2019-nCoV; Binding; Biological Process; C-terminal; COVID-19 pandemic; Calorimetry; Cell Wall; Cells; Circular Dichroism; Development; Disease; Ebola virus; Electron Spin Resonance Spectroscopy; Emerging Communicable Diseases; Exocytosis; Goals; HIV; Human; Influenza; Influenza Hemagglutinin; Knowledge; Learning; Membrane; Membrane Fusion; Membrane Proteins; Methodology; Methods; Middle East Respiratory Syndrome; Middle East Respiratory Syndrome Coronavirus; Modeling; Nature; Nerve Degeneration; Organism; Peptides; Property; Proteins; Role; SARS coronavirus; Severe Acute Respiratory Syndrome; Structure; Techniques; Tertiary Protein Structure; Testing; Titrations; Transmembrane Domain; Viral; Virus; Virus Diseases; biophysical techniques; combat; fighting; flu; nervous system disorder; new therapeutic target; pathogenic virus

For many years Freed has pioneered the development of electron-spin resonance (ESR) methods for the study of proteins and their dynamical effects on membranes. The goal of this proposal is to use those ESR techniques to advance our knowledge of 1) viral membrane fusion and 2) Intrinsic Disordered Protein (IDP)-membrane interactions in conjunction with other biophysical methods. The ongoing COVID19 pandemic has unveiled how limited are our knowledge and methods to combat emerging infectious diseases caused by viral pathogens. A key step in SARS-CoV-2 (“SARS-2”) viral infection is membrane fusion initiated by its fusion peptide (FP) domain in its Spike protein. In fact, membrane fusion is key for all enveloped viruses such as SARS-CoV-1 (“SARS-1”), MERS-CoV, EBOV, influenza and HIV. However, the mechanism of viral membrane fusion is still unclear. We have extensively demonstrated that FP-induced membrane ordering is a prerequisite for viral membrane fusion in all these viruses. We have shown that membrane ordering for some viruses including SARS-1, MERS and EBOV is strongly Ca2+-dependent. Most recently we have shown that SARS-2 FP interacts with membranes more strongly than SARS-1, and it depends very specifically on Ca2+. However, the exact role of Ca2+, as well as the mechanism of membrane ordering are still unclear. The FP only initiates membrane fusion. We have proposed that the transmembrane domain (TMD) of influenza hemagglutinin is important for finalizing membrane fusion. However, this remains to be tested to see if it is applicable to SARS-2. Thus, we plan to continue studies of the mechanism of membrane fusion of SARS-2 as well as other Ca2+-dependent viruses. IDPs lack a stable tertiary structure in solution. After membrane binding, they can either undergo a disorder- to-order transition or still remain in the absence of a stable structure. The viral FPs are such examples. This IDP- membrane interaction localizes IDPs to their target membranes, facilitating interactions with other membrane proteins, and helping to remodel membrane properties. Understanding the structure/function relationships underlying IDP-membrane interactions is a significant challenge because of their highly variable and dynamic nature. We have been using complexin, a key exocytosis regulator related to neurodegeneration, as a model to delineate the IDP-membrane binding mode. We have found that complexins from different organisms have very different modes, which is likely to reflect their different biological functions. We plan to continue to study the membrane-binding C-terminal domain of complexins of several species to determine the mechanisms that govern complexin-membrane binding modes. These findings will be useful for human therapies. ESR is a powerful methodology to study the dynamic and structural properties of SARS FPs and other IDPs, as we have shown. We will employ our well-developed ESR methods to achieve these goals. This will be supplemented by other biophysical methods, including Isothermal Titration Calorimetry and Circular Dichroism.

多年来，Freed 一直致力于开发用于研究的电子自旋共振 (ESR) 方法 该提案的目标是使用这些 ESR 技术。 增进我们对 1) 病毒膜融合和 2) 内在无序蛋白 (IDP) 膜的了解 与其他生物物理方法相结合的相互作用。 持续不断的新冠病毒大流行揭示了我们的抗击知识和方法是多么有限 由病毒病原体引起的新发传染病。SARS-CoV-2（“SARS-2”）病毒感染的一个关键步骤是。 膜融合是由其 Spike 蛋白中的融合肽 (FP) 结构域引发的。 对于所有有包膜病毒（例如 SARS-CoV-1（“SARS-1”）、MERS-CoV、EBOV、流感和 HIV）来说都是关键。 病毒膜融合的机制目前还不清楚。我们已经大体证明了FP诱导的。 我们已经证明，膜排序是所有这些病毒中病毒膜融合的先决条件。 包括 SARS-1、MERS 和 EBOV 在内的一些病毒的膜排序强烈依赖于 Ca2+。 最近我们发现 SARS-2 FP 与膜的相互作用比 SARS-1 更强，这取决于 然而，Ca2+ 的确切作用以及膜排序机制是非常具体的。 仍不清楚 FP 仅启动膜融合。 流感血凝素的检测对于完成膜融合很重要，但这仍有待测试。 看看它是否适用于SARS-2 因此，我们计划继续研究膜融合的机制。 SARS-2 以及其他 Ca2+ 依赖性病毒。 IDP 在溶液中缺乏稳定的三级结构，在膜结合后，它们可能会发生紊乱。 病毒 FP 就是这样的例子。 膜相互作用将 IDP 定位到其目标膜，促进与其他膜的相互作用 蛋白质，并帮助重塑膜特性。 潜在的 IDP-膜相互作用是一个重大挑战，因为它们具有高度可变性和动态性 我们一直在使用复合蛋白（一种与神经退行性变相关的关键胞吐调节因子）作为模型。 描述 IDP 膜结合模式 我们发现来自不同生物体的络合蛋白具有很大的差异。 不同的模式，这很可能反映了它们不同的生物学功能，我们计划继续研究。 几个物种的复合蛋白的膜结合 C 端结构域，以确定其机制 这些发现将有助于人类治疗。 ESR 是研究 SARS FP 和其他 IDP 动态和结构特性的强大方法，如 我们已经表明，我们将采用我们成熟的 ESR 方法来实现这些目标。 辅以其他生物物理方法，包括等温滴定量热法和圆二色性。