氧化固醇结合蛋白ORP7与Gate16相互作用调节GS28蛋白稳定性
摘要
氧化固醇结合蛋白家族(Oxysterol Bingding Proteins,简称ORPs)是具有氧化固醇结合域的一类蛋白。ORP7是氧化固醇结合蛋白家族中的一员,其功能尚未阐明。在本文研究中,我们构建了人类ORP7基因的诱饵载体,通过酵母双杂交系统筛选Universal Human cDNA Library,发现ORP7与Golgi-associated ATPase enhancer of 16 kDa (Gate16)蛋白能够相互作用。Gate-16具有调节高尔基体蛋白Golgi SNARE of 28 kDa (GS28)稳定性的功能,并且在自吞噬形成中发挥作用。我们通过双分子荧光互补实验进一步证实了ORP7-Gate16在活细胞内的相互作用,酵母双杂交实验进一步证实ORP7的第1-142个氨基酸和Gate16(?)的第30-117个氨基酸是它们发生相互作用的关键区域。在293A细胞中,shRNA介导的ORP7基因沉默使GS28蛋白的含量上升40%,而过表达ORP7得到相反的结果(GS28蛋白水平下降25%)。ORP7对GS28蛋白水平的调节并不是发生在转录水平,而是调节GS28蛋白在蛋白酶体途径中的降解实现的。过表达缺失Gate-16结合结构域的ORP7cDNA未发现GS28水平的变化,证明Gate-16介导了ORP7对GS28的调节。进一步研究发现,ORP7过表达引起反面高尔基体结构的碎裂。ORP7是氧化固醇在细胞内的传感器之一,25-OH同样可以引起GS28通过蛋白酶体途径的降解,并且这种降解是由ORP7介导的。另外,过表达ORP7导致多数细胞内的自吞噬标记蛋白LC3聚集形成泡状结构,Gate-16也存在于ORP7阳性的泡状结构中,提示过表达的ORP7蛋白将Gate-16“捕获”到自噬体内。总之,我们的结果证明ORP7通过与Gate-16的相互作用调节GS28蛋白的稳定性,并且可能介导了氧化固醇25-OH对GS28和高尔基体功能的影响。
ORP7 is a member of oxysterol-binding protein (OSBP) family, the function of which has remained obscure. In this study, we identified by yeast two-hybrid screening an interaction partner of ORP7, Golgi-associated ATPase enhancer of 16 kDa (GATE-16), which (i) regulates Golgi SNARE of 28 kDa (GS28) function and stability, and (ii) plays a role in autophagosome biogenesis. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay in living cells. The interacting regions were delineated within aa 1-142 of ORP7 and aa 30-117 of GATE-16. ORP7 knocking down in 293A cells resulted in a 40% increase of GS28 protein while ORP7 overexpression had the opposite effect (25% decrease of GS28). We show evidence that the regulation of GS28 by ORP7 does not occur at the level of transcription, but involves degradation of GS28 on proteasomes, Truncated ORP7 that lacks the GATE-16 binding region failed to affect GS28 stability, evidencing for specificity of the observed effect. Similar to ORP7 overexpression, treatment of cells with 25-hydroxycholesterol (25-OH) resulted GS28 destabilization, which was potentiated by excess ORP7. Overexpression of ORP7 led in most cells to formation of vacuolar structures positive for RFP-LC3, thus representing autophagic elements. Also GATE-16 was found in the vacuolar ORP7-positive elements, suggesting that excess ORP7 increases entrapment of GATE-16 in autophagosomes. Taken together, our results suggest that ORP7 negatively regulates GS28 protein stability via sequestration of GATE-16, and may mediate the effect of 25-OH on GS28 and Golgi function.
ORP7 is a member of oxysterol-binding protein (OSBP) family, the function of which has remained obscure. In this study, we identified by yeast two-hybrid screening an interaction partner of ORP7, Golgi-associated ATPase enhancer of 16 kDa (GATE-16), which (i) regulates Golgi SNARE of 28 kDa (GS28) function and stability, and (ii) plays a role in autophagosome biogenesis. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay in living cells. The interacting regions were delineated within aa 1-142 of ORP7 and aa 30-117 of GATE-16. ORP7 knocking down in 293A cells resulted in a 40% increase of GS28 protein while ORP7 overexpression had the opposite effect (25% decrease of GS28). We show evidence that the regulation of GS28 by ORP7 does not occur at the level of transcription, but involves degradation of GS28 on proteasomes, Truncated ORP7 that lacks the GATE-16 binding region failed to affect GS28 stability, evidencing for specificity of the observed effect. Similar to ORP7 overexpression, treatment of cells with 25-hydroxycholesterol (25-OH) resulted GS28 destabilization, which was potentiated by excess ORP7. Overexpression of ORP7 led in most cells to formation of vacuolar structures positive for RFP-LC3, thus representing autophagic elements. Also GATE-16 was found in the vacuolar ORP7-positive elements, suggesting that excess ORP7 increases entrapment of GATE-16 in autophagosomes. Taken together, our results suggest that ORP7 negatively regulates GS28 protein stability via sequestration of GATE-16, and may mediate the effect of 25-OH on GS28 and Golgi function.
引文
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