Xi Quansheng, Pauer Gayle J T, Ball Sherry L, Rayborn Mary, Hollyfield Joe G, Peachey Neal S, Crabb John W, Hagstrom Stephanie A
Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Invest Ophthalmol Vis Sci. 2007 Jun;48(6):2837-44. doi: 10.1167/iovs.06-0059.
Tubby-like proteins (TULPs) are a family of four proteins, two of which have been linked to neurosensory disease phenotypes. TULP1 is a photoreceptor-specific protein that is mutated in retinitis pigmentosa, an inherited retinal disease characterized by the degeneration of rod and cone photoreceptor cells. To investigate the function of TULP1 in maintaining the health of photoreceptors, the authors sought the identification of interacting proteins.
Immunoprecipitation from retinal lysates, followed by liquid chromatography tandem mass spectrometry and in vitro binding assays, were used to identify TULP1 binding partners. RT-PCR was performed on total RNA from wild-type mouse retina to identify the Dynamin-1 isoform expressed in the retina. Immunocytochemistry was used to determine the localization of TULP1 and Dynamin-1 in photoreceptor cells. Electroretinography (ERG) and light microscopy were used to phenotype tulp1-/- mice at a young age.
Immunoprecipitation from retinal lysate identified Dynamin-1 as a possible TULP1 binding partner. GST pull-down assays further supported an interaction between TULP1 and Dynamin-1. In photoreceptor cells, Dynamin-1 and TULP1 colocalized primarily to the outer plexiform layer, where photoreceptor terminals synapse on second-order neurons and, to a lesser extent, to the inner segments, where polarized protein translocation occurs. ERG analyses in young tulp1-/- mice indicated a decreased b-wave at ages when the retina retained a full complement of photoreceptor cells.
These data indicated that TULP1 interacts with Dynamin-1 and suggested that TULP1 is involved in the vesicular trafficking of photoreceptor proteins, both at the nerve terminal during synaptic transmission and at the inner segment during protein translocation to the outer segment. These results also raised the possibility that normal synaptic function requires TULP1, and they motivate a closer look at synaptic architecture in the developing tulp1-/- retina.
类Tubby蛋白（TULPs）是一个由四种蛋白质组成的家族，其中两种已与神经感觉疾病表型相关联。TULP1是一种光感受器特异性蛋白，在色素性视网膜炎中发生突变，色素性视网膜炎是一种遗传性视网膜疾病，其特征是视杆和视锥光感受器细胞退化。为了研究TULP1在维持光感受器健康中的功能，作者试图鉴定相互作用蛋白。
从视网膜裂解物中进行免疫沉淀，随后进行液相色谱串联质谱分析和体外结合试验，以鉴定TULP1结合伴侣。对野生型小鼠视网膜的总RNA进行逆转录聚合酶链反应（RT-PCR），以鉴定在视网膜中表达的发动蛋白-1同工型。免疫细胞化学用于确定TULP1和发动蛋白-1在光感受器细胞中的定位。视网膜电图（ERG）和光学显微镜用于对幼年tulp1-/-小鼠进行表型分析。
从视网膜裂解物中进行免疫沉淀鉴定出发动蛋白-1是一种可能的TULP1结合伴侣。谷胱甘肽S-转移酶（GST）下拉试验进一步支持了TULP1与发动蛋白-1之间的相互作用。在光感受器细胞中，发动蛋白-1和TULP1主要共定位于外网状层，光感受器终末在此与二级神经元形成突触，在较小程度上共定位于内节，极化蛋白易位在此发生。对幼年tulp1-/-小鼠的ERG分析表明，在视网膜保留完整光感受器细胞的年龄阶段，b波降低。
这些数据表明TULP1与发动蛋白-1相互作用，并提示TULP1参与光感受器蛋白的囊泡运输，在突触传递过程中于神经终末以及在蛋白易位至外节过程中于内节均发挥作用。这些结果还提出了正常突触功能需要TULP1的可能性，并促使人们更仔细地观察发育中的tulp1-/-视网膜中的突触结构。
