Characterization of the role of the NDRG3/GOSR2 complex in ER-to-Golgi transport

SNARE [可溶性NSF（N-乙基马来酰亚胺敏感因子）附着蛋白受体]蛋白通过介导囊泡与其靶膜的膜融合在真核细胞的膜运输中发挥重要作用。SNARE含有延伸的卷曲螺旋伸展（SNARE基序）并形成膜融合所必需的四元SNARE复合物。我们特别感兴趣的是内质网（ER）到高尔基体的运输和GOSR 2在这个过程中的作用。GOSR 2是一种Q-SNARE，我们已经确定了导致进行性肌阵挛癫痫（PME）和脊柱侧凸的突变。一些患者还存在先天性肌营养不良症（CMD），这表明GOSR 2功能的更严重的损害，并指出GOSR 2在骨骼肌中的另外的生理作用。在上一个资助期内，我们开发了一种基于酵母的实验方法来测试GOSR 2中导致PME的错义突变的功能。此外，我们进行了分子动力学（MD）模拟，以在分子水平上了解GOSR 2突变对SNARE复合物的影响。我们的数据表明，酵母和计算机实验的组合可以评估GOSR 2的功能障碍，从而可以预测疾病的过程。我们尝试产生GOSR 2基因敲除小鼠显示胚胎致死性，进一步表明GOSR 2功能的完全丧失是不相容的NDRG蛋白家族成员在调节膜运输。本项目将有助于我们了解NDRG 3和GOSR 2在常氧和缺氧条件下相互作用的生理功能，并将揭示这种相互作用是否对GOSR 2相关疾病有影响。此外，我们提出，调节SNARE复合物是NDRG蛋白家族成员的一种新的保守功能。与生活。此外，我们已经确定NDRG 3作为一个新的相互作用伙伴的GOSR 2。我们的初步结果表明，这种胞质蛋白控制ER-高尔基体运输缺氧。在本申请的框架内，我们打算在分子水平上表征NDRG 3/GOSR 2复合物，并旨在了解NDRG 3和GOSR 2相互作用的生理相关性。我们提出的实验，重点是NDRG 3在调节ER到高尔基体运输在常氧和缺氧条件下的作用，并希望了解GOSR 2/NDRG 3的相互作用是否有助于GOSR 2相关疾病的病理生理学。将分析以下基本方面：i）GOSR 2/NDRG 3复合物的分子表征ii）NDRG 3对ER至高尔基体转运的调节作用的表征和iii）NDRG蛋白家族成员在调节膜运输中的作用。该项目将有助于我们了解NDRG 3和GOSR 2在常氧和缺氧条件下相互作用的生理功能，并将揭示这种相互作用是否对GOSR 2相关疾病产生影响。此外，我们提出，调节SNARE复合物是NDRG蛋白家族成员的一种新的保守功能。

SNARE [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor] proteins play fundamental roles in Membrane trafficking in eukaryotic cells by mediating membrane fusion of vesicles with their target membrane. SNAREs contain an ex¬tended coiled-coil stretch (the SNARE motif) and form quaternary SNARE complexes that are necessary for membrane fusion. We are particular interested in Endoplasmic Reticulum (ER)-to-Golgi transport and the role of GOSR2 in this process. GOSR2 is a Q-SNARE in which we have identified mutations causing progressive myoclonus epilepsy (PME) and scoliosis. Some patients present also with congenital muscular dystrophy (CMD), suggesting a more severe impairment of GOSR2 function and point to an additional physiological role of GOSR2 in skeletal muscle. Within the last funding period, we developed a yeast-based experimental approach to test PME-causing missense mutations in GOSR2 for functionality. Furthermore, we performed molecular dynamics (MD) simulations to understand at a molecular level the impact of GOSR2 mutations on the SNARE complex. Our data suggest that a combination of yeast and in silico experiments can evaluate the functional impairment of GOSR2 thereby allowing to predict the course of disease. Our attempts to generate GOSR2 knockout mice showed embryonic lethality further suggesting that complete loss of function of GOSR2 is incompatible NDRG protein family members in regulating membrane trafficking.This project will contribute to our understanding of the physiological function of the interaction of NDRG3 and GOSR2 under normoxic and hypoxic conditions and will reveal if this interaction has an impact on GOSR2-related diseases. Furthermore, we propose that regulating SNARE complexes is a novel conserved function of NDRG Protein family members. with life. Furthermore, we have identified NDRG3 as a novel interaction partner of GOSR2. Our preliminary results suggest that this cytosolic protein controls ER-to-Golgi transport under hypoxia. Within the framework of this application, we intend to characterize the NDRG3/GOSR2 complex on a molecular level and aim to understand the physiological relevance of the interaction of NDRG3 and GOSR2. We are proposing experiments, which focus on the role of NDRG3 in modulating ER-to-Golgi transport under normoxic and hypoxic conditions and want to understand if GOSR2/NDRG3 interaction contributes to the pathophysiology of GOSR2-related disease. The following fundamental aspects will be analyzed: i) molecular characterization of the GOSR2/NDRG3 complex ii) characterization of the regulatory role of NDRG3 on ER-to-Golgi transport and iii) role of NDRG protein family members in regulating membrane trafficking. This project will contribute to our understanding of the physiological function of the interaction of NDRG3 and GOSR2 under normoxic and hypoxic conditions and will reveal if this interaction has an impact on GOSR2-related diseases. Furthermore, we propose that regulating SNARE complexes is a novel conserved function of NDRG Protein family members.