The connection between ER-phagy, ER structure and hereditary spastic paraplegias

项目摘要 通过内质网相关降解作用降解内质网中的错误折叠蛋白 （ERAD）途径阻止潜在的毒性蛋白质进入分泌途径。然而， 不能清除ER中的所有蛋白质。例如，一些蛋白质，如易聚集蛋白质， 大的聚合物和纤维状蛋白质，对ERAD的降解具有抗性，必须通过 替代处置途径。由于易聚集蛋白质与神经退行性疾病有关， 疾病，了解这些替代处置途径如何发挥作用具有医学重要性。 内质网自噬（ER-phagy）是一种降解内质网结构域和聚集倾向的处理途径， proteins.在ER的连续网络上，特定的域是如何被定向为降级的， 未知我们已经发现了一种非规范形式的COPII涂层，包含SEC24C-SEC23， 与ER上的受体一起作用，靶向自噬的区域。ER上的ER-吞噬位点（ERPHS）是 与ER出口位点不同，在ER出口位点处，分泌物被装载到典型的COPII包被的囊泡中， 前往Golgi的交通我们的研究结果表明，ER结构可能是重要的ERPHS的形成。 此外，与遗传性痉挛性截瘫相关的几种内质网形成蛋白的突变， (HSP)导致内质网吞噬功能缺陷。这些发现表明ER-吞噬， ERPHS和HSP。 在本提案中，我描述了旨在解决ER结构所扮演的角色的几个目标 ERPHS的形成以及ERPHS的形成与HSP之间的联系。具体来说，我们将执行 活细胞成像和质谱实验，以表征ERPHS及其货物。错误折叠 将分析已知被ER-吞噬作用降解的蛋白质。迄今为止，六种ER自噬受体具有 被识别。我们的研究将解决SEC 24 C与自噬机制相互作用的时间，以及 与SEC24C相互作用。我们的生物化学研究可能会发现 在ER自噬过程中与SEC24C相互作用的新蛋白质。自噬报告者，成像分析和 生化研究将用于解决ER组织和ER成形蛋白在 ER-吞噬和ERPHS形成。我们将在Aim 2和Aim 3中分析的蛋白质与HSP相关 和热休克蛋白样神经病变总之，这些研究将阐明ER结构，ERPHS 形成和HSP。

Project Summary The degradation of misfolded proteins in the endoplasmic reticulum (ER) by the ER-associated degradation (ERAD) pathway prevents potentially toxic proteins from entering the secretory pathway. ERAD, however, cannot clear all proteins from the ER. For example, some proteins, such as aggregation-prone proteins, large polymers and fibrillar proteins, are resistant to degradation by ERAD and must be disposed of by alternate disposal pathways. As aggregation prone proteins have been to linked to neurodegenerative diseases, understanding how these alternate disposal pathways function is of medical importance. ER autophagy (ER-phagy) is a disposal pathway that degrades ER domains and aggregation-prone proteins. How specific domains, on the continuous network of the ER, are targeted for degradation is unknown. We have found that a non-canonical form of the COPII coat, that contains SEC24C-SEC23, works with receptors on the ER to target domains for autophagy. ER-phagy sites (ERPHS) on the ER are distinct from the ER exit sites where secretory cargo is loaded into canonical COPII coated vesicles that traffic to the Golgi. Our findings suggest that ER structure may be important for the formation of ERPHS. Additionally, mutations in several ER shaping proteins, associated with hereditary spastic paraplegias (HSP), lead to defects in ER-phagy. These findings suggest a link between ER-phagy, the formation of the ERPHS and HSP. In this proposal I describe several aims that are designed to address the role that ER structure plays in the formation of ERPHS and the link between ERPHS formation and HSP. Specifically, we will perform live cell imaging and mass spectroscopy experiments to characterize the ERPHS and their cargo. Misfolded proteins, known to be degraded by ER-phagy, will be analyzed. To date six ER autophagy receptors have been identified. Our studies will address when SEC24C interacts with the autophagy machinery and which of the six known receptors interact with SEC24C. Our biochemical studies may lead to the identification of new proteins that interact with SEC24C during ER autophagy. Autophagy reporters, imaging analysis and biochemical studies will be used to address the role that ER organization and ER shaping proteins play in ER-phagy and ERPHS formation. The proteins we will analyze in Aim 2 and Aim 3 are associated with HSP and HSP-like neuropathies. In total, these studies will shed light on the link between ER structure, ERPHS formation and HSP.