探究TAPT1参与原生纤毛生成的分子机制及对骨发育的影响

The evolutionarily conserved transmembrane anterior posterior transformation 1 protein (TAPT1, also named POD1 in Arabidopsis thaliana) has been reported to be associated with endoplasmic reticulum (ER) membrane via its interaction with CALRETICULIN3 (CRT3), and acts as a chaperone involved in ER quality control for pollen tube guidance in Arabidopsis, and its mutations in human result in osteochondrodysplasia. .We have found that TAPT1, required for primary cilium formation, localized to the ciliary transition zone and along the cilia. TAPT1 binds to the transition zone protein TCTN3. Transmission electron microscopy (TEM) displayed several vesicles inside a ciliary vesicle (CV) of a TAPT1-null cell. Subcellular fractionation of HeLa cells revealed that TAPT1, behaving as peripheral membrane protein, was mainly associated with membranes, from which they could be extracted by buffers containing a high salt concentration. .The aforementioned findings promote us to address the following questions. (1) Is the distribution of proteins localizing to the transition zone and cilia affected in Tapt1-knockout MEFs and TAPT1-depleted RPE-1 cells? (2) Is TAPT1 a novel membrane-tethering factor responsible for the ciliary receptor-containing vesicles tethered to the ciliary transition zone? (3) Does Osx1-Cre-mediated conditional deletion of Tapt1 in the osteoblast lineage result in defective cilium formation in mouse tibia bone? (4) Does the defective ciliogenesis contribute to skeletal abnormalities in osteoblast-specific Tapt1 knockout mice?.Results from the research will provide key mechanistic insights to explicate function of a transition zone protein in primary cilium formation and its involvement in ciliopathy, and enhance our understanding of ciliary proteins functionally conserved across species.

进化保守的TAPT1蛋白，其同源蛋白在拟南芥中称为POD1，可作为参与内质网质量控制的分子伴侣指导拟南芥中花粉管导向。人TAPT1的突变导致骨软骨发育不良。我们发现，TAPT1定位于纤毛及其过渡区；TAPT1与膜相关，且与过渡区蛋白TCTN3相互作用；敲除TAPT1影响原生纤毛生成，并且在纤毛囊泡内出现小囊泡的积累。基于上述发现，本课题将利用敲除TAPT1的小鼠胚胎成纤维细胞和人视网膜上皮细胞，阐明TAPT1是否是一种新的膜栓系因子，研究TAPT1影响原生纤毛生成的分子机制；利用Osx1-Cre介导的成骨细胞特异性Tapt1基因敲除小鼠模型，在细胞和整体水平探究TAPT1与骨细胞纤毛形成及骨发育的关系。本项目的完成将为阐明纤毛过渡区蛋白参与原生纤毛生成并影响骨发育的分子机制提供理论依据，同时有利于对理解与膜相关的纤毛蛋白在不同物种间的功能相似性提供科学思路。

我们首先构建了Tapt1 conventional knockout (Tapt1 KO)小鼠和Tapt1flox/flox小鼠，并且制备了TAPT1抗体，经过验证，抗体特异性良好。发现TAPT1定位在内质网。.因为肝脏内质网丰富，所以我们构建了肝组织特异性Tapt1敲除(Tapt1flox/flox；Alb-Cre, 即Tapt1 cKO)小鼠。发现30天Tapt1 cKO小鼠呈现明显脂肪肝表型。通过肝组织切片的苏木精-伊红染色、油红O染色，分离原代肝细胞Bodipy493染色，发现Tapt1 cKO小鼠肝脏存在大量脂滴。测量生化指标表明敲除Tapt1肝脏会出现甘油三酯和胆固醇累积。通过分析极低密度脂蛋白(Very low density lipoprotein，VLDL)的分泌速率结果，以及肝脏组织电镜和血清VLDL电镜的结果表明：肝脏缺失TAPT1通过影响VLDL的脂化导致非酒精脂肪肝的发生。.我们成功构建了Tapt1全身性基因敲除小鼠，Tapt1 KO小鼠出生后出现呼吸窘迫，均于出生后2小时内死亡。苏木精-伊红染色结果表明Tapt1 KO小鼠肺泡扩张不全和肺泡壁增厚。进一步实验表明Tapt1 KO小鼠肺中的表面活性蛋白B前体表面活性蛋白C前体明显要比野生型小鼠的多，说明Tapt1 KO小鼠的肺二型上皮细胞不能将表面活性蛋白前体加工修饰形成成熟的表面活性蛋白分泌到肺泡表面从而导致肺不能正常的扩张，以至于在出生后呼吸困难，迅速死亡 。.总之，TAPT1在VLDL脂化和肺表面活性蛋白成熟方面扮演了重要角色，它的缺失将导致肝脏脂质代谢紊乱及肺不张引起的呼吸窘迫综合症。本研究为进一步阐明非酒精性脂肪肝的致病机制，以及后期的临床干预提供了新思路；肺二型上皮细胞将表面活性蛋白前体进行翻译后加工修饰形成成熟的表面活性蛋白并分泌到细胞表面维持肺泡表面张力从而完成肺的正常扩张，Tapt1缺失影响肺表面活性蛋白的成熟，可能为新生儿呼吸窘迫综合征的致病基因。