The G32E functional variant reduces activity of PPARD by nuclear export and post-translational modification in pigs.
The G32E functional variant reduces activity of PPARD by nuclear export and post-translational modification in pigs.
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DOI：
10.1371/journal.pone.0075925
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Huang L
中科院分区：
文献类型：
--
作者：
Duan Y;Brenig B;Wu X;Ren J;Huang L
过氧化物酶体增殖物激活受体β/δ（PPARD）是多种生物学功能的关键和多方面的决定因素，包括脂质代谢、胚胎发育、炎症反应、伤口愈合和癌症。最近，我们提出了一个新的功能，猪PPARD（sPPARD）在外耳发育。一个错义突变（G32 E）在进化保守结构域的sPPARD显着增加外耳大小的猪。在这里，我们研究了在细胞水平上的因果突变的潜在分子机制。使用荧光素酶报告系统，我们表明G32 E取代以配体依赖的方式降低了sPPARD的转录活性。通过比较野生型和突变型sPPARD在PK-15细胞和耳廓软骨衍生的原代软骨细胞中的亚细胞定位，我们发现G32 E取代促进CRM-1介导的sPPARD核输出。利用表面等离子体共振技术，我们进一步揭示了G32 E取代对其配体结合亲和力的影响可以忽略不计。最后，我们使用免疫共沉淀和荧光素酶报告基因分析表明，G32 E取代通过阻断关键的A/B结构域的泛素化而大大降低了泛素化水平，从而降低了sPPARD的转录活性。综上所述，我们的研究结果强烈支持G32 E是一种在sPPARD生物学活性中起关键作用的功能变体，这推进了我们对sPPARD G32 E对猪耳大小的潜在机制的理解。
Peroxisome proliferator-activated receptor beta/delta (PPARD) is a crucial and multifaceted determinant of diverse biological functions including lipid metabolism, embryonic development, inflammatory response, wound healing and cancer. Recently, we proposed a novel function of porcine PPARD (sPPARD) in external ear development. A missense mutation (G32E) in an evolutionary conservative domain of sPPARD remarkably increases external ear size in pigs. Here, we investigated the underlying molecular mechanism of the causal mutation at the cellular level. Using a luciferase reporter system, we showed that the G32E substitution reduced transcription activity of sPPARD in a ligand-dependent manner. By comparison of the subcellular localization of wild-type and mutated sPPARD in both PK-15 cells and pinna cartilage-derived primary chondrocytes, we found that the G32E substitution promoted CRM-1 mediated nuclear exportation of sPPARD. With the surface plasmon resonance technology, we further revealed that the G32E substitution had negligible effect on its ligand binding affinity. Finally, we used co-immunoprecipitation and luciferase reporter assays to show that the G32E substitution greatly reduced ubiquitination level by blocking ubiquitination of the crucial A/B domain and consequently decreased transcription activity of sPPARD. Taken together, our findings strongly support that G32E is a functional variant that plays a key role in biological activity of sPPARD, which advances our understanding of the underlying mechanism of sPPARD G32E for ear size in pigs.
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影响因子：
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作者：
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10.1073/pnas.0308518100
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发表时间：
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影响因子：
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