【作者】 吴斌；

【导师】 裴端卿； 舒晓东；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2011， 博士

【摘要】 内涵体转运是真核调节蛋白质功能的一个重要手段。蛋白需要通过内涵体转运在适当的时间定位到适当的区域以发挥它正常的生理功能。同时内涵体转运也可以通过修饰和降解等方法调节蛋白的活性。Sorting Nexins(SNXs)是一个新的与内涵体分选相关蛋白家族。它们以PX结构域为特征结构。PX结构域是个磷酸肌醇结合结构域,SNXs通过PX与磷酸肌醇的结合定位到内涵体膜上。由于SNXs在除PX结构域外的蛋白结构上较大的差异,这些成员在多样的内涵体转运通路上发挥功能。SNX10,SNX11是SNXs家族结构最简单的一类蛋白,除了PX结构域外不含有其它已知的功能性结构域。这两个蛋白在氨基酸序列具有很高的相似性,在进化上也最为接近。但它们的功能完全未知。我们实验室第一次发现在细胞中高表达SNX10可以诱导出巨大的空泡结构,但对这个过程的分子机制并不清楚。我们发现SNX10主要定位在Rab5阳性的早期内涵体上,而诱导出的空泡则显示出晚期内涵体标记,说明SNX10可能开启了一条通路,加速了由早期内涵体向晚期内涵体的转变。而在这个过程中抑制Rab7可以起到抑制SNX10表型的作用,而Rab5抑制则不产生影响,说明这条通路不依赖于Rab5而依赖于Rab7。同时我们发现SNX11可以特异性的抑制SNX10的表型。SNX11在早期和晚期内涵体上都有部分定位。当SNX11与SNX10一起转染入细胞时,SNX11与SNX10共定位在早期内涵体上,并抑制Rab7标记的晚期内涵体的扩大。进一步的研究发现,V-ATPase参与了SNX10诱导的空泡化。SNX10与V-ATPase的V1D亚基相互作用,由此V-ATPase被异常激活可能是增强的内涵体膜融合和酸化的原因。SNX11也与V1D亚基相互作用。SNX11,SNX10对V1D的结合具有竞争关系,并且SNX11表现出更强的亲和力。V-ATPase的异常活性也是VacA诱导细胞空泡的原因,我们发现SNX11也可以抑制VacA诱导的空泡的形成。以上这些结果说明了SNX10,SNX11通过V-ATPase调节内涵体。我们进一步在斑马鱼中研究SNX10的生理功能。用morpholino的方法敲降SNX10后,可以破坏斑马鱼的左右不对称发育。KV(Kupffer’s Vesicle)是决定斑马鱼作用不对称发育的重要器官,而KV中纤毛在其发挥功能的关键。我们发现在注射SNX10 morpholino的胚胎中,KV的纤毛形成受到抑制。体外实验的结果表明SNX10调节细胞的纤毛发生。SNX10定位在纤毛基体的周围。负责高尔基体与基体之间膜泡运输的Rab11与SNX10表现出部分功定位。与纤毛膜泡转运相关的蛋白BBS4,IFT88也表现出与SNX10的共定位。而用siRNA敲降SNX10可以阻碍细胞中的纤毛形成。因为SNX10/V-ATPase一起调节内涵体形态,所以V-ATPase也可能参与对纤毛膜泡运输的调控。用V1D和Vod1的GFP融合蛋白我们发现V-ATPase定位在基体上,与纤毛运输相关的蛋白表现出共定位。在细胞中敲降V-ATPase可以抑制纤毛的形成,而在斑马鱼中敲降V-ATPase则在KV,原肾管,侧线等多个器官中造成纤毛缺陷。而且在体内和体外实验中,SNX10和V-ATPase对纤毛的调节都表现出协同作用。这些结果支持SNX10/V-ATPase通过膜泡转运调节纤毛发生的假设。在进一步的研究中,我们发现V-ATPase在中心体上定位依赖于SNX10,在敲降SNX10的细胞中,V-ATPase在中心体上定位的比例明显降低。这说明了在这过程中SNX10对V-ATPase的调节方式。同时我们也发现SNX10,V-ATPase的siRNA可以阻止Rab8向纤毛的转运,这一结果从另一侧面说明在纤毛发生过程中SNX10/V-ATPase对膜泡转运的调节。综上所述,我们发现:1, SNX10,SNX11调节早期内涵体向晚期内涵体的转变,这个调节是通过V-ATPase来实现的。SNX10和SNX11都与V-ATPase的V1D亚基相互作用,2, SNX11通过竞争性的与V1D结合来抑制SNX10的功能。3, SNX10/V-ATPase途径调节纤毛的发生。SNX10调节V-ATPase在中心体的定位。它们都是向纤毛方向的膜泡转运所需要的。更多还原

【Abstract】 In eukaryotic cells, endosome trafficking is an important method to regulate the function of proteins. The proteins localized to the proper position at the proper time by trafficking. Endosome trafficking also regulated proteins activity by various ways such as modification and degradation. Sorting Nexins (SNXs) are new protein family associated with endosome sorting. They are characterized by phox-homolog (PX) domain. PX domain is a phosphatidylinositol(PIs)-binding domain. Via the binding of PX domain and PIs, SNXs localize to the membrane of endosome. Due to the difference domains in their structure beside PX domain, the members show different function in various endosome trafficking pathway.SNX10 and SNX11 are members with simple structure in the family. They have only PX domain. The two proteins show high similarity in sequence and are close in evolution. But their functions are completely unknown. Our lab first discovered the overexpression of SNX10 would induce giant vacuoles in cells, but the molecular mechanism was unclear. We found SNX10 mainly localized on early endosome and show co-localization with Rab5, but the giant vacuoles induced by SNX10 showed Rab7 positive which indicated late endosome. Rab7 also showed partly co-localization with SNX10 on some vesicles. As SNX10 was absence on the giant vacuoles’membrane, it seemed a pathway that accelerated the transformation from early endosome to late endosome was switched on by SNX10. The pathway showed Rab5 independent and Rab7 dependent as co-expression SNX10 and a dominant negative mutant of Rab5 didn’t show any reduction of vacuolation rate while Rab7’s dominant mutant could inhibit the effect of SNX10. On the other hand, SNX11 was able to inhibit the vacuolation induced by SNX10. SNX11 showed partly co-localization with Rab5 and Rab7. When SNX10 and SNX11 were co-tranfected into cells, SNX11 could block the extra enlargement of late endosome and co-localized with SNX10 on early endosome. Further investigation showed V-ATPase participated in the vacuolation. SNX10 interacted with the D subunit (V1D) of V-ATPase and the enhancement of membrane fusion and acidification may due to the extra V-ATPase activity induced by SNX10. SNX11 also interacted with D subunit. There were competition between SNX10 and SNX11 in V1D interaction, and SNX11 showed higher affinity. This could explain the inhibition of SNX11 on SNX10. Furthermore, SNX11 could inhibit the vacuolation induced by VacA, which also due to the extra activity of V-ATPase. All these results showed SNX10, SNX11 regulated the endosome trafficking via V-ATPase.We also investigated the physiological function of SNX10 in zebrafish (Danio rerio). Knockdown SNX10 by morpholino in zebrafish would disrupt the Left-Right patterning. Kupffer’s Vesicle (KV) is an important organ in L-R patterning, and the cilia in KV play a critical role. We found there was defection of ciliogenesis in KV in the SNX10 morphant. The in vitro experiment also showed the regulation of SNX10 on ciliogenesis in cells. SNX10 localized around the basal body, and partly co-localized with Rab11, which regulated the trafficking between Golgi and basal body. The cilium vesicles trafficking proteins BBS4 and IFT88 also showed co-localized with SXN10. Knockdown SNX10 by siRNA would reduce the cilia formation. As the regulation of SNX10/V-ATPase on endosome trafficking, we doubled the participation of V-ATPase in ciliogenensis. Using GFP-fused V1D and Vod1 subunit, we found V-ATPase localized on the basal body, and co-localized with the proteins involved in the cilia trafficking. siRNA knockdown of V-ATPase in cells showed the reduction of ciliogenesis and knockdown of V-ATPase in zebrafish would cause the defect of cilia in multi organs, such as KV, renal tube and lateral line. SNX10 and V-ATPase also showed co-operation in ciliogenesis regulation in vitro and in vivo. These result confirmed the hypothesis that SNX10/V-ATPase regulate ciliogenesis via vesicles trafficking. In further investigation, we found the centrosome localization of V-ATPase was SNX10 dependent. The ratio of V-ATPase localized on centrosome was reduced in cells treated by SNX10 siRNA. This explained the regulation of SNX10 on V-ATPase. We also found the trafficking of Rab8 into cilia was blocked by SNX10 and V-ATPase siRNA which could reveal the vesicles trafficking regulation by SNX10/V-ATPase in ciliogenesis in another means.In summary, we found:1. SNX10 and SNX11 regulated the trafficking from early endosome to late endosome, and the regulation was V-ATPase dependent. SNX10 and SNX11 both interacted with V1D subunit, SNX11 inhibited SNX10’s function via binding to V1D competitively.2. SNX10/V-ATPase regulated ciliogenesis in vivo and in vitro. SNX10 regulated the centrosome localization of V-ATPase,and they were required for the trafficking of vesicles into cilia.更多还原