Comprehensive search of Treatment-resistant depression associated protein by time-controlled transcardiac perfusion cross-linking technique

血清素转运体（SERT）功能的改变可能与难治性抑郁症有关。抗抑郁，血清素选择性再摄取抑制剂（SSRI）作用于存在于细胞表面的SERT，其受多种细胞机制的调节，包括与其他蛋白质的相互作用。在本研究中，我们通过时间控制的经心灌注交联和下拉系统寻找新的SERT结合蛋白，并研究其中一种蛋白的表达是否抑制SSRI对血清素能神经元的作用。n -乙基马来酰亚胺敏感因子（NSF）被下拉系统鉴定为一种新的SERT结合蛋白。在HEK293-hSERT细胞中，研究了敲低新的SERT结合蛋白对SERT功能和膜表达的影响。NSF与SERT在质膜共定位，NSF敲低导致SERT在细胞膜上的表达和摄取功能降低。NSF内源性与SERT共定位，并与SERT相互作用。时间控制经心脏灌注交联技术已筛选出85个候选蛋白。然后，用散弹枪分析法从候选蛋白中鉴定出14个蛋白。在小鼠原代培养的中缝神经元中，研究了SERT与14种蛋白、NSF和Syntaxin 1A的内源性相互作用。蛋白质和难治性抑郁症之间的关系在这一资助期限内尚未得到评估。

Change in serotonin transporter (SERT) function may be implicated in treatment-resistant depression. Anti-depressant, serotonin selective reuptake inhibitor (SSRI) is acting on SERT present at the cell surface, which is regulated by various cellular mechanisms including interactions with other proteins. In this study, we have searched for novel SERT -binding proteins by time-controlled transcardiac perfusion cross-linking and a pull-down system, and investigated whether the expression of one such protein inhibited the effect of SSRI on serotonergic neuron.N-ethylmaleimide-sensitive factor (NSF) was identified by a pull-down system as a novel SERT -binding protein. Alterations of SERT function and membrane expression upon knockdown of the novel SERT -binding protein were studied in HEK293-hSERT cells. NSF co-localized with SERT at the plasma membrane, and NSF knockdown resulted in decreased SERT expression at the cell membranes and its uptake function. NSF endogenously co-localized with SERT and interacted with SERT. Time-controlled transcardiac perfusion cross-linking technique has screened 85 candidate proteins. Then, 14 proteins have identified from the candidates by shot-gun analysis score. Endogenous interaction of SERT with 14 proteins, NSF and Syntaxin 1A wereevaluated in mouse primary cultured raphe neurons. The association between proteinsand treatment-resistant depression have not evaluated within this term of grant-in-aid.