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)附着蛋白受体]蛋白通过介导囊泡与其靶膜的膜融合，在真核细胞的膜转运中发挥重要作用。圈套包含一个扩展的卷曲拉伸(圈套基序)，并形成膜融合所必需的第四层圈套复合体。我们特别感兴趣的是内质网(ER)到高尔基体的运输以及GOSR2在这一过程中的作用。GOSR2是一个Q-陷阱，我们已经在其中确定了导致进行性肌阵挛癫痫(PME)和脊柱侧弯的突变。一些患者还存在先天性肌营养不良症(CMD)，提示GOSR2功能受损更严重，并指出GOSR2在骨骼肌中具有额外的生理作用。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PME导致的GOSR2错义突变的功能性。此外，我们进行了分子动力学(MD)模拟，以在分子水平上了解GOSR2突变对SNARE复合体的影响。我们的数据表明，结合酵母菌和硅胶实验可以评估GOSR2的功能损害，从而能够预测疾病的进程。我们建立的GOSR2基因敲除小鼠的胚胎致死率进一步表明，GOSR2功能的完全丧失是调控膜转运的NDRG蛋白家族成员不相容的。本项目将有助于我们理解NDRG3和GOSR2在常氧和低氧条件下相互作用的生理功能，并揭示这种相互作用是否对GOSR2相关疾病产生影响。此外，我们认为调节SNARE复合体是NDRG蛋白家族成员的一种新的保守功能。用生命。此外，我们还确定NDRG3是GOSR2的一个新的相互作用伙伴。我们的初步结果表明，这种胞质蛋白在低氧条件下控制内质网到高尔基体的运输。在本申请的框架内，我们打算在分子水平上表征NDRG3/GOSR2复合体，并旨在了解NDRG3和GOSR2相互作用的生理相关性。我们建议进行实验，重点研究NDRG3在常氧和低氧条件下调节内质网到高尔基体运输的作用，并希望了解GOSR2/NDRG3相互作用是否有助于GOSR2相关疾病的病理生理。将分析以下基本方面：1)GOSR2/NDRG3复合体的分子特征；2)NDRG3对内质网到高尔基体转运的调控作用；3)NDRG蛋白家族成员在调控膜转运中的作用。本项目将有助于我们理解常氧和低氧条件下NDRG3和GOSR2相互作用的生理功能，并揭示这种相互作用是否对GOSR2相关疾病产生影响。此外，我们认为调节SNARE复合体是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.