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

项目摘要 内质网错误折叠蛋白的内质网相关降解 (Erad)途径阻止潜在的有毒蛋白进入分泌途径。然而，埃拉德， 不能清除ER中的所有蛋白质。例如，一些蛋白质，如易于聚集的蛋白质， 大聚合物和纤维状蛋白质，可抵抗ERAD的降解，必须由 另一条处置路径。由于聚集倾向蛋白与神经退行性变有关 了解这些交替的处置途径如何发挥作用具有重要的医学意义。 ER自噬(ER-phagy)是一种降解ER结构域并易于聚集的处置途径 蛋白质。ER的连续网络上的特定域是如何针对降级的 未知。我们已经发现，含有SEC24C-SEC23的COPII涂层的非正则形式， 与内质网上的受体一起作用于自噬的靶域。ER上的内质网吞噬位点(ERPHS)是 与内质网出口部位不同，在内质网出口部位，分泌物被装载到标准的COPII涂层囊泡中，该囊泡 前往高尔基的车辆。我们的发现提示内质网结构可能在ERPHS的形成中起重要作用。 此外，几种内质网形成蛋白的突变与遗传性痉挛性截瘫相关 (HSP)，导致内质网吞噬缺陷。这些发现表明，内质网吞噬，形成的内质网 ERPHS和HSP。 在本提案中，我描述了几个旨在解决ER结构所扮演的角色的目标 ERPHS的形成以及ERPHS的形成与HSP之间的联系。具体地说，我们将表演 活体细胞成像和质谱学实验，以表征ERPHS及其货物。折叠错误 已知可被内质网吞噬降解的蛋白质将被分析。到目前为止，已经有6个ER自噬受体 已被确认身份。我们的研究将解决SEC24C何时与自噬机制相互作用以及 在已知的六种受体中，SEC24C与SEC24C相互作用。我们的生化研究可能会导致确定 内质网自噬过程中与SEC24C相互作用的新蛋白质。自噬记者，成像分析和 生化研究将被用来解决内质网组织和内质网形成蛋白在 ER-吞噬和ERPHS形成。我们将在目标2和目标3中分析的蛋白质与热休克蛋白有关 以及热休克蛋白样神经病。总之，这些研究将阐明ER结构和ERPHS之间的联系 形成和热休克蛋白。

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.