Advanced fluorescence spectroscopy to unravel functional oligomerization of a newly discovered Ca2+ channel and its role in heart failure.

内质网（Endoplasmic reticulum， ER）的内稳态正成为心力衰竭的一个越来越有趣的靶点，因为错误折叠或未折叠的蛋白质的积累很容易导致内质网应激，而内质网应激反过来又在心血管疾病的发展中发挥重要作用。内质网跨膜蛋白可以发挥重要的信号作用，影响细胞死亡或存活。Mitsugumin 23 （MG23）是一种新发现的内质/肌浆网（ER/SR）跨膜蛋白，本质上被认为是低聚物。我们认为MG23的动态寡聚行为控制了它的功能作用。我们的最新数据表明，MG23在心脏纤维化，SR Ca2+泄漏和心力衰竭的早期阶段的作用。这使得MG23成为进一步深入研究的一个新的和及时的靶点，以揭示其报道的寡聚化所起的分子作用，并更好地了解其潜在的药物作用。虽然我们研究质膜整体蛋白寡聚化的能力在过去几年中有了很大的进步，但这些方法依赖于极端的光学切片，这是不适合内部细胞器的，如ER/SR。本博士项目的目的是利用共聚焦显微镜开发和应用时空分子亮度方法来解决MG23在病理生理条件下的寡聚化动力学问题。荧光波动光谱的使用为研究拥挤系统中的同质寡聚化提供了希望，在拥挤系统中，单个分子的可视化受到蛋白质密度和/或样品局部几何形状的挑战。结合分子生物学、电生理学、心力衰竭模型和先进的显微镜技术，我将可视化MG23在心脏细胞中的功能寡聚化，揭示心力衰竭条件下MG23的寡聚化，并将MG23寡聚化与心脏细胞病理，特别是凋亡和纤维化联系起来。该项目将开发一个工具箱来研究内质膜蛋白寡聚化，对心力衰竭中未折叠蛋白反应途径的更广泛研究产生影响。

Endoplasmic reticulum (ER) homeostasis is becoming an increasingly interesting target in heart failure, since accumulation of misfolded or unfolded proteins can easily lead to ER stress, which in turn can play an important role in the development of cardiovascular diseases. ER transmembrane proteins can play prominent signalling roles that influence cell death or survival. Mitsugumin 23 (MG23), is a newly discovered endoplasmic/sarcoplasmic reticulum (ER/SR) transmembrane protein thought to be oligomeric in nature. We suggest that the dynamic oligomerization behaviour of MG23 controls its functional role. Our latest data suggest a role for MG23 in cardiac fibrosis, SR Ca2+ leak and the early stages of heart failure. This makes MG23 a novel and timely target for further in-depth study to unravel the molecular role played by its reported oligomerization and provide a better understanding of its potential druggability. Although our ability to study oligomerization of plasma membrane integral proteins has greatly advanced over the last few years, these approaches rely on extreme optical sectioning, which is not amenable to internal organelles such as the ER/SR. The aim of this PhD project is to develop and apply spatial-temporal molecular brightness approaches using confocal microscopy to address the problem of the oligomerization dynamics of MG23 in pathophysiological conditions. The use of fluorescence fluctuation spectroscopy offers promise to study homo-oligomerization in crowded systems, where the visualization of individual molecules is challenged either by protein density and/or by the local geometry of the sample. Using a combination of molecular biology, electrophysiology, heart failure models and advanced microscopy techniques, I will visualize MG23 functional oligomerization in cardiac cells, unravel MG23 oligomerization in heart failure conditions, and correlate MG23 oligomerization to cardiac cell pathology, specifically apoptosis and fibrosis. This project will develop a toolbox to study ER membrane protein oligomerization, with ramifications to the broader study of the unfolded protein response pathway in heart failure.