Cartier Etienne, Garcia-Olivares Jennie, Janezic Eric, Viana Juan, Moore Michael, Lin Min Landon, Caplan Jeffrey L, Torres Gonzalo, Kim Yong-Hwan
Department of Biological Sciences, Delaware State University, Dover, DE, United States.
National Institute of Mental Health (NIMH), Bethesda, MD, United States.
Front Cell Neurosci. 2019 Feb 8;13:35. doi: 10.3389/fncel.2019.00035. eCollection 2019.
The dopamine transporter (DAT) is a plasma membrane protein responsible for the uptake of released dopamine back to the presynaptic terminal and ending dopamine neurotransmission. The DAT is the molecular target for cocaine and amphetamine as well as a number of pathological conditions including autism spectrum disorders, attention-deficit hyperactivity disorder (ADHD), dopamine transporter deficiency syndrome (DTDS), and Parkinson's disease. The DAT uptake capacity is dependent on its level in the plasma membrane. studies show that DAT functional expression is regulated by a balance of endocytosis, recycling, and lysosomal degradation. However, recent reports suggest that DAT regulation by endocytosis in neurons is less significant than previously reported. Therefore, additional mechanisms appear to determine DAT steady-state level and functional expression in the neuronal plasma membrane. Here, we hypothesize that the ubiquitin-like protein small ubiquitin-like modifier 1 (SUMO1) increases the DAT steady-state level in the plasma membrane. In confocal microscopy, fluorescent resonance energy transfer (FRET), and Western blot analyses, we demonstrate that DAT is associated with SUMO1 in the rat dopaminergic N27 and DAT overexpressing Human Embryonic Kidney cells (HEK)-293 cells. The overexpression of SUMO1 and the Ubc9 SUMO-conjugase induces DAT SUMOylation, reduces DAT ubiquitination and degradation, enhancing DAT steady-state level. In addition, the Ubc9 knock-down by interference RNA (RNAi) increases DAT degradation and reduces DAT steady-state level. Remarkably, the Ubc9-mediated SUMOylation increases the expression of DAT in the plasma membrane and dopamine uptake capacity. Our results strongly suggest that SUMOylation is a novel mechanism that plays a central role in regulating DAT proteostasis, dopamine uptake, and dopamine signaling in neurons. For that reason, the SUMO pathway including SUMO1, SUMO2, Ubc9, and DAT SUMOylation, can be critical therapeutic targets in regulating DAT stability and dopamine clearance in health and pathological states.
多巴胺转运体（DAT）是一种质膜蛋白，负责将释放的多巴胺摄取回突触前终末，从而终止多巴胺神经传递。DAT是可卡因、安非他明以及包括自闭症谱系障碍、注意力缺陷多动障碍（ADHD）、多巴胺转运体缺乏综合征（DTDS）和帕金森病在内的多种病理状况的分子靶点。DAT的摄取能力取决于其在质膜中的水平。研究表明，DAT的功能表达受内吞作用、再循环和溶酶体降解之间平衡的调节。然而，最近的报道表明，神经元中内吞作用对DAT的调节作用不如先前报道的那么显著。因此，似乎有其他机制决定了DAT在神经元质膜中的稳态水平和功能表达。在此，我们假设泛素样蛋白小泛素样修饰物1（SUMO1）可提高DAT在质膜中的稳态水平。在共聚焦显微镜、荧光共振能量转移（FRET）和蛋白质印迹分析中，我们证明在大鼠多巴胺能N27细胞和过表达DAT的人胚肾细胞（HEK）-293细胞中，DAT与SUMO1相关联。SUMO1和Ubc9 SUMO连接酶的过表达诱导DAT的SUMO化，减少DAT的泛素化和降解，提高DAT的稳态水平。此外，通过干扰RNA（RNAi）敲低Ubc9会增加DAT的降解并降低DAT的稳态水平。值得注意的是，Ubc9介导的SUMO化增加了质膜中DAT的表达以及多巴胺摄取能力。我们的结果有力地表明，SUMO化是一种在调节神经元中DAT蛋白质稳态、多巴胺摄取和多巴胺信号传导方面起核心作用的新机制。因此，包括SUMO1、SUMO2、Ubc9和DAT SUMO化在内的SUMO途径可能是调节健康和病理状态下DAT稳定性和多巴胺清除的关键治疗靶点。
